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Among hematologic malignancies, the classic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered a model of inflammation-related cancer development. In this context, the use of immune-modulating agents has recently expanded the MPN therapeutic scenario. Cytokines are key mediators of an auto-amplifying, detrimental cross-talk between the MPN clone and the tumor microenvironment represented by immune, stromal, and endothelial cells. This review focuses on recent advances in cytokine-profiling of MPN patients, analyzing different expression patterns among the three main Philadelphia-negative (Ph-negative) MPNs, as well as correlations with disease molecular profile, phenotype, progression, and outcome. The role of the megakaryocytic clone as the main source of cytokines, particularly in myelofibrosis, is also reviewed. Finally, we report emerging intriguing evidence on the contribution of host genetic variants to the chronic pro-inflammatory state that typifies MPNs.

A small library of 3-thia-7,9-diazabicyclo[3.3.1]nonanes was synthesized and their opioid receptors affinity and selectivity evaluated. Among these novel sulfur-bridged compounds, the (E) 9-[3'-(3-chlorophenyl)-but-2'-en-1'-yl]-7-propionyl-3-thia-7,9-diazabicyclo[3.3.1]nonane 2i emerged as the derivative with the highest μ receptor affinity (K i  = 85 nM) and selectivity (K i μ/δ = 58.8, K i μ/κ > 117.6). The antinociceptive activity of 2i was also evaluated in acute thermal pain. Docking studies disclosed the specific pattern of interactions of these derivatives., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Glutamate cytotoxicity is implicated in neuronal death in different neurological disorders including stroke, traumatic brain injury, and neurodegenerative diseases. Adenosine is a nucleoside that plays an important role in modulating neuronal activity and its receptors have been identified as promising therapeutic targets for glutamate cytotoxicity. The purpose of this study is to elucidate the role of adenosine and its receptors on glutamate-induced injury in PC12 cells and to verify the protective effect of the novel A 1 adenosine receptor positive allosteric modulator, TRR469. Flow cytometry experiments to detect apoptosis revealed that adenosine has a dual role in glutamate cytotoxicity, with A 2A and A 2B adenosine receptor (AR) activation exacerbating and A 1 AR activation improving glutamate-induced cell injury. The overall effect of endogenous adenosine in PC12 cells resulted in a facilitating action on glutamate cytotoxicity, as demonstrated by the use of adenosine deaminase and selective antagonists. However, enhancing the action of endogenous adenosine on A 1 ARs by TRR469 completely abrogated glutamate-mediated cell death, caspase 3/7 activation, ROS production, and mitochondrial membrane potential loss. Our results indicate a novel potential therapeutic strategy against glutamate cytotoxicity based on the positive allosteric modulation of A 1 ARs.

Adenosine modulates many physiological processes through the interaction with adenosine receptors (ARs) named as A 1 , A 2A , A 2B, and A 3 ARs. During ischemic stroke, adenosine mediates neuroprotective and anti-inflammatory effects through ARs activation. One of the dominant pathways generating extracellular adenosine involves the dephosphorylation of ATP by ecto-nucleotidases CD39 and CD73, which efficiently hydrolyze extracellular ATP to adenosine. The aim of the study is to assess the presence of ARs in lymphocytes from ischemic stroke patients compared to healthy subjects and to analyze changes in CD39 and CD73 expression in CD4 + and CD8 + lymphocytes. Saturation binding experiments revealed that A 2A ARs affinity and density were significantly increased in ischemic stroke patients whilst no differences were found in A 1 , A 2B, and A 3 ARs. These results were also confirmed in reverse transcription (RT)-polymerase chain reaction (PCR) assays where A 2A AR mRNA levels of ischemic stroke patients were higher than in control subjects. In flow cytometry experiments, the percentage of CD73 + cells was significantly decreased in lymphocytes and in T-lymphocyte subclasses CD4 + and CD8 + obtained from ischemic stroke patients in comparison with healthy individuals. These data corroborate the importance of the adenosinergic system in ischemic stroke and could open the way to more targeted therapeutic approaches and biomarker development for ischemic stroke., Competing Interests: The authors declare no conflict of interest.

Literature studies suggest important protective effects of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) on inflammatory pathways affecting joint and cerebral diseases. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. Therefore the aim of this study was to identify the molecular targets of PEMFs anti-neuroinflammatory action. The effects of PEMF exposure in cytokine production by lipopolysaccharide (LPS)-activated N9 microglial cells as well as the pathways involved, including adenylyl cyclase (AC), phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and delta (PKC-δ), p38, ERK1/2, JNK1/2 mitogen activated protein kinases (MAPK), Akt and caspase 1, were investigated. In addition, the ability of PEMFs to modulate ROS generation, cell invasion and phagocytosis, was addressed. PEMFs reduced the LPS-increased production of TNF-α and IL-1β in N9 cells, through a pathway involving JNK1/2. Furthermore, they decreased the LPS-induced release of IL-6, by a mechanism not dependent on AC, PLC, PKC-ε, PKC-δ, p38, ERK1/2, JNK1/2, Akt and caspase 1. Importantly, a significant effect of PEMFs in the reduction of crucial cell functions specific of microglia like ROS generation, cell invasion and phagocytosis was found. PEMFs inhibit neuroinflammation in N9 cells through a mechanism involving, at least in part, the activation of JNK MAPK signalling pathway and may be relevant to treat a variety of diseases characterized by neuroinflammation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Low-energy low-frequency pulsed electromagnetic fields (PEMFs) exert several protective effects, such as the regulation of kinases, transcription factors as well as cell viability in both central and peripheral biological systems. However, it is not clear on which bases they affect neuroprotection and the mechanism responsible is yet unknown. In this study, we have characterized in nerve growth factor-differentiated pheochromocytoma PC12 cells injured with hypoxia: (i) the effects of PEMF exposure on cell vitality; (ii) the protective pathways activated by PEMFs to relief neuronal cell death, including adenylyl cyclase, phospholipase C, protein kinase C epsilon and delta, p38, ERK1/2, JNK1/2 mitogen-activated protein kinases, Akt and caspase-3; (iii) the regulation by PEMFs of prosurvival heat-shock proteins of 70 (HSP70), cAMP response element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), and Bcl-2 family proteins. The results obtained in this study show a protective effect of PEMFs that are able to reduce neuronal cell death induced by hypoxia by modulating p38, HSP70, CREB, BDNF, and Bcl-2 family proteins. Specifically, we found a rapid activation (30 min) of p38 kinase cascade, which in turns enrolles HSP70 survival chaperone molecule, resulting in a significant CREB phosphorylation increase (24 hr). In this cascade, later (48 hr), BDNF and the antiapoptotic pathway regulated by the Bcl-2 family of proteins are recruited by PEMFs to enhance neuronal survival. This study paves the way to elucidate the mechanisms triggered by PEMFs to act as a new neuroprotective approach to treat cerebral ischemia by reducing neuronal cell death., (© 2019 Wiley Periodicals, Inc.)

Introduction : There is a vicious cycle of tumor hypoxia, high adenosine levels, immune suppression and cancer growth that involves the use of adenosine receptor ligands in tumors. After several years of research, the candidates emerging as promising new anticancer drugs are A 3 adenosine receptor agonists and A 2A receptor antagonists. Areas covered : The authors give an updated overview of the field related to A 3 receptor agonists and A 2A receptor antagonists in cancer and propose their perspectives on the status of these compounds in oncology. The rationale for the modulation of adenosine receptors in cancer is addressed, starting from the first in vitro evidence of their efficacy up to the animal and clinical studies. Expert opinion : A 3 and A 2A receptors are attractive targets in oncologic therapy due to their involvement in cancer progression and immune-resistance. Of relevance, the A 3 subtype is also a tumor marker to be used in a personalized drug treatment program while the A 2A receptor, playing a non-redundant role in immunomodulation, may be blocked in combination with checkpoint inhibitors to improve their efficacy. The future will reveal how successful this approach is in the fight against cancer.

The A 3 adenosine receptor (A 3 AR) subtype is a novel, promising therapeutic target for inflammatory diseases, such as rheumatoid arthritis (RA) and psoriasis, as well as liver cancer. A 3 AR is coupled to inhibition of adenylyl cyclase and regulation of mitogen-activated protein kinase (MAPK) pathways, leading to modulation of transcription. Furthermore, A 3 AR affects functions of almost all immune cells and the proliferation of cancer cells. Numerous A 3 AR agonists, partial agonists, antagonists, and allosteric modulators have been reported, and their structure-activity relationships (SARs) have been studied culminating in the development of potent and selective molecules with drug-like characteristics. The efficacy of nucleoside agonists may be suppressed to produce antagonists, by structural modification of the ribose moiety. Diverse classes of heterocycles have been discovered as selective A 3 AR blockers, although with large species differences. Thus, as a result of intense basic research efforts, the outlook for development of A 3 AR modulators for human therapeutics is encouraging. Two prototypical selective agonists, N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA; CF101) and 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA; CF102), have progressed to advanced clinical trials. They were found safe and well tolerated in all preclinical and human clinical studies and showed promising results, particularly in psoriasis and RA, where the A 3 AR is both a promising therapeutic target and a biologically predictive marker, suggesting a personalized medicine approach. Targeting the A 3 AR may pave the way for safe and efficacious treatments for patient populations affected by inflammatory diseases, cancer, and other conditions., (© 2017 Wiley Periodicals, Inc.)

Adenosine is a ubiquitous endogenous autacoid whose effects are triggered through the enrollment of four G protein-coupled receptors: A 1 , A 2A , A 2B , and A 3 . Due to the rapid generation of adenosine from cellular metabolism, and the widespread distribution of its receptor subtypes in almost all organs and tissues, this nucleoside induces a multitude of physiopathological effects, regulating central nervous, cardiovascular, peripheral, and immune systems. It is becoming clear that the expression patterns of adenosine receptors vary among cell types, lending weight to the idea that they may be both markers of pathologies and useful targets for novel drugs. This review offers an overview of current knowledge on adenosine receptors, including their characteristic structural features, molecular interactions and cellular functions, as well as their essential roles in pain, cancer, and neurodegenerative, inflammatory, and autoimmune diseases. Finally, we highlight the latest findings on molecules capable of targeting adenosine receptors and report which stage of drug development they have reached.

In recent years, cannabinoid type 2 receptors (CB 2 R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB 2 R are devoid of the psychoactive effects typically observed for CB 1 R ligands. Based on our recent studies on a class of pyridazinone 4-carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure-activity relationships for this promising scaffold with the aim to develop potent CB 2 R ligands. In binding assays, two of the new synthesized compounds [6-(3,4-dichlorophenyl)-2-(4-fluorobenzyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2,3-dihydropyridazine-4-carboxamide (2) and 6-(4-chloro-3-methylphenyl)-cis-N-(4-methylcyclohexyl)-3-oxo-2-pentyl-2,3-dihydropyridazine-4-carboxamide (22)] showed high CB 2 R affinity, with K i values of 2.1 and 1.6 nm, respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB 2 R inverse agonists. Compound 22 displayed the highest CB 2 R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle-switch residue, W6.48., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Adenosine receptors (AR) are a family of G-protein coupled receptors, comprised of four members, named A 1 , A 2A , A 2B , and A 3 receptors, found widely distributed in almost all human body tissues and organs. To date, they are known to participate in a large variety of physiopathological responses, which include vasodilation, pain, and inflammation. In particular, in the central nervous system (CNS), adenosine acts as a neuromodulator, exerting different functions depending on the type of AR and consequent cellular signaling involved. In terms of molecular pathways and second messengers involved, A 1 and A 3 receptors inhibit adenylyl cyclase (AC), through G i/o proteins, while A 2A and A 2B receptors stimulate it through G s proteins. In the CNS, A 1 receptors are widely distributed in the cortex, hippocampus, and cerebellum, A 2A receptors are localized mainly in the striatum and olfactory bulb, while A 2B and A 3 receptors are found at low levels of expression. In addition, AR are able to form heteromers, both among themselves (e.g., A 1 /A 2A ), as well as with other subtypes (e.g., A 2A /D 2 ), opening a whole range of possibilities in the field of the pharmacology of AR. Nowadays, we know that adenosine, by acting on adenosine A 1 and A 2A receptors, is known to antagonistically modulate dopaminergic neurotransmission and therefore reward systems, being A 1 receptors colocalized in heteromeric complexes with D 1 receptors, and A 2A receptors with D 2 receptors. This review documents the present state of knowledge of the contribution of AR, particularly A 1 and A 2A , to psychostimulants-mediated effects, including locomotor activity, discrimination, seeking and reward, and discuss their therapeutic relevance to psychostimulant addiction. Studies presented in this review reinforce the potential of A 1 agonists as an effective strategy to counteract psychostimulant-induced effects. Furthermore, different experimental data support the hypothesis that A 2A /D 2 heterodimers are partly responsible for the psychomotor and reinforcing effects of psychostimulant drugs, such as cocaine and amphetamine, and the stimulation of A 2A receptor is proposed as a potential therapeutic target for the treatment of drug addiction. The overall analysis of presented data provide evidence that excitatory modulation of A 1 and A 2A receptors constitute promising tools to counteract psychostimulants addiction.

Several evidences indicate that the ubiquitous nucleoside adenosine, acting through A 1 , A 2A , A 2B , and A 3 receptor (AR) subtypes, plays crucial roles in tumor development. Adenosine has contrasting effects on cell proliferation depending on the engagement of different receptor subtypes in various tumors. The involvement of A 2A ARs in human A375 melanoma, as well as in human A549 lung and rat MRMT1 breast carcinoma proliferation has been evaluated in view of the availability of a novel A 2A AR antagonist, with high affinity and selectivity, named as 2-(2-furanyl)-N 5 -(2-methoxybenzyl)[1,3]thiazolo[5,4-d]pyrimidine-5,7-diammine (TP455). Specifically, the signaling pathways triggered in the cancer cells of different origin and the antagonist effect of TP455 were investigated. The A 2A AR protein expression was evaluated through receptor binding assays. Furthermore, the effect of A 2A AR activation on cell proliferation at 24, 48 and 72 hours was studied. The selective A 2A AR agonist 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine hydrochloride (CGS21680), concentration-dependently induced cell proliferation in A375, A549, and MRMT1 cancer cells and the effect was potently antagonized by the A 2A AR antagonist TP455, as well as by the reference A 2A AR blocker 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM241385). As for the signaling pathway recruited in this response we demonstrated that, by using the specific inhibitors of signal transduction pathways, the effect of A 2A AR stimulation was induced through phospholipase C (PLC) and protein kinase C-delta (PKC-δ). In addition, we evaluated, through the AlphaScreen SureFire phospho(p) protein assay, the kinases enrolled by A 2A AR to stimulate cell proliferation and we found the involvement of protein kinase B (AKT), extracellular regulated kinases (ERK1/2), and c-Jun N-terminal kinases (JNKs). Indeed, we demonstrated that the CGS21680 stimulatory effect on kinases was strongly reduced in the presence of the new potent compound TP455, as well as by ZM241385, confirming the role of the A 2A AR. In conclusion, the A 2A AR activation stimulates proliferation of A375, A549, and MRMT1 cancer cells and importantly TP455 reveals its capability to counteract this effect, suggesting selective A 2A AR antagonists as potential new therapeutics.

Adenosine is an endogenous ubiquitous purine nucleoside, which is increased by hypoxia, ischaemia and tissue damage and mediates a number of physiopathological effects by interacting with four GPCRs, identified as A 1 , A 2A , A 2B and A 3 . Physiological and acutely increased adenosine is mostly associated with beneficial effects that include vasodilatation and a decrease in inflammation. In contrast, chronic overproduction of adenosine occurs in important pathological states, where long-lasting increases in the nucleoside levels are responsible for the bad side of adenosine associated with chronic inflammation, fibrosis and organ damage. In this review, we describe and critically discuss the pathological overproduction of adenosine and analyse when, where and how adenosine exerts its detrimental effects throughout the body., (© 2017 The British Pharmacological Society.)

Transient receptor potential vanilloid 1 (TRPV1) is an ion channel expressed on sensory neurons triggering an influx of cations. TRPV1 receptors function as homotetramers responsive to heat, proinflammatory substances, lipoxygenase products, resiniferatoxin, endocannabinoids, protons, and peptide toxins. Its phosphorylation increases sensitivity to both chemical and thermal stimuli, while desensitization involves a calcium-dependent mechanism resulting in receptor dephosphorylation. TRPV1 functions as a sensor of noxious stimuli and may represent a target to avoid pain and injury. TRPV1 activation has been associated to chronic inflammatory pain and peripheral neuropathy. Its expression is also detected in nonneuronal areas such as bladder, lungs, and cochlea where TRPV1 activation is responsible for pathology development of cystitis, asthma, and hearing loss. This review offers a comprehensive overview about TRPV1 receptor in the pathophysiology of chronic pain, epilepsy, cough, bladder disorders, diabetes, obesity, and hearing loss, highlighting how drug development targeting this channel could have a clinical therapeutic potential. Furthermore, it summarizes the advances of medicinal chemistry research leading to the identification of highly selective TRPV1 antagonists and their analysis of structure-activity relationships (SARs) focusing on new strategies to target this channel., (© 2016 Wiley Periodicals, Inc.)

In the present study, the effect of low-frequency, low-energy pulsed electromagnetic fields (PEMFs) has been investigated by using different cell lines derived from neuron-like cells and microglial cells. In particular, the primary aim was to evaluate the effect of PEMF exposure in inflammation- and hypoxia-induced injury in two different neuronal cell models, the human neuroblastoma-derived SH-SY5Y cells and rat pheochromocytoma PC12 cells and in N9 microglial cells. In neuron-like cells, live/dead and apoptosis assays were performed in hypoxia conditions from 2 to 48 h. Interestingly, PEMF exposure counteracted hypoxia damage significantly reducing cell death and apoptosis. In the same cell lines, PEMFs inhibited the activation of the hypoxia-inducible factor 1α (HIF-1α), the master transcriptional regulator of cellular response to hypoxia. The effect of PEMF exposure on reactive oxygen species (ROS) production in both neuron-like and microglial cells was investigated considering their key role in ischemic injury. PEMFs significantly decreased hypoxia-induced ROS generation in PC12, SH-SY5Y, and N9 cells after 24 or 48 h of incubation. Moreover, PEMFs were able to reduce some of the most well-known pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 release in N9 microglial cells stimulated with different concentrations of LPS for 24 or 48 h of incubation time. These results show a protective effect of PEMFs on hypoxia damage in neuron-like cells and an anti-inflammatory effect in microglial cells suggesting that PEMFs could represent a potential therapeutic approach in cerebral ischemic conditions. J. Cell. Physiol. 232: 1200-1208, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A 2A and A₃AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A 2A and A₃ARs in lymphocytes obtained from patients when compared with healthy subjects. A 2A and A₃AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A 2A and A₃AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A 2A and A₃ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

The hallmark of neuroinflammation is the activation of microglia, the immunocompetent cells of the CNS, releasing a number of proinflammatory mediators implicated in the pathogenesis of neuronal diseases. Adenosine is an ubiquitous autacoid regulating several microglia functions through four receptor subtypes named A 1 , A 2A , A 2B and A 3 (ARs), that represent good targets to suppress inflammation occurring in CNS. Here we investigated the potential role of ARs in the modulation of IL-6 secretion and cell proliferation in primary microglial cells. The A 2B AR agonist 2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2-pyridinyl]thio]-acetamide (BAY60-6583) stimulated IL-6 increase under normoxia and hypoxia, in a dose- and time-dependent way. In cells incubated with the blockers of phospholipase C (PLC), protein kinase C epsilon (PKC-ε) and PKC delta (PKC-δ) the IL-6 increase due to A 2B AR activation was strongly reduced, whilst it was not affected by the inhibitor of adenylyl cyclase (AC). Investigation of cellular signalling involved in the A 2B AR effect revealed that only the inhibitor of p38 mitogen activated protein kinase (MAPK) was able to block the agonist's effect on IL-6 secretion, whilst inhibitors of pERK1/2, JNK1/2 MAPKs and Akt were not. Stimulation of p38 by BAY60-6583 was A 2B AR-dependent, through a pathway affecting PLC, PKC-ε and PKC-δ but not AC, in both normoxia and hypoxia. Finally, BAY60-6583 increased microglial cell proliferation involving A 2B AR, PLC, PKC-ε, PKC-δ and p38 signalling. In conclusion, A 2B ARs activation increased IL-6 secretion and cell proliferation in murine primary microglial cells, through PLC, PKC-ε, PKC-δ and p38 pathways, thus suggesting their involvement in microglial activation and neuroinflammation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Adenosine, the purine nucleoside, mediates its effects through activation of four G-protein coupled adenosine receptors (ARs) named as A 1 , A 2A , A 2B and A 3 . In particular, A 1 ARs are distributed through the body, primarily inhibitory in the regulation of adenylyl cyclase activity and able to reduce the cyclic AMP levels. Considerable advances have been made in the pharmacological and molecular characterization of A 1 ARs, which had been proposed as targets for the discovery and drug design of antagonists, agonists and allosteric enhancers. Several lines of evidence indicate that adenosine interacting with A 1 ARs may be an endogenous protective agent in the human body since it prevents the damage caused by various pathological conditions, such as in ischemia/hypoxia, epileptic seizures, excitotoxic neuronal injury and cardiac arrhythmias in cardiovascular system. It has also been reported that one of the most promising targets for the development of new anxiolytic drugs could be A 1 ARs, and that their activation may reduce pain signaling in the spinal cord. A 1 AR antagonists induce diuresis and natriuresis in various experimental models, mediating the inhibition of A 1 ARs in the proximal tubule which is primarily responsible for reabsorption and fluid uptake. In addition, the results of various studies indicate that adenosine is present within pancreatic islets and is implicated through A 1 ARs in the regulation of insulin secretion and in glucose concentrations. In the present paper it will become apparent that A 1 ARs could be implicated in the pharmacological treatment of several pathologies with an important influence on human health.

Purinergic signaling is involved in psoriasis, a chronic skin disease characterized by increased epidermis cell growth. In particular, Andrés et al. focus on the keratinocyte biology modulated by adenosine receptors providing evidence that the A 2B subtype plays a prominent role in the reduction of keratinocyte proliferation whereas A 2A and A 2B agonists have antiinflammatory effects independent of adenosine receptors. The authors report that psoriatic epidermis presents a deregulated adenosine receptor expression profile with reduced A 2B and increased A 2A ., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Several studies explored the biological effects of low frequency low energy pulsed electromagnetic fields (PEMFs) on human body reporting different functional changes. Much research activity has focused on the mechanisms of interaction between PEMFs and membrane receptors such as the involvement of adenosine receptors (ARs). In particular, PEMF exposure mediates a significant upregulation of A 2A and A 3 ARs expressed in various cells or tissues involving a reduction in most of the proinflammatory cytokines. Of particular interest is the observation that PEMFs, acting as modulators of adenosine, are able to increase the functionality of the endogenous agonist. By reviewing the scientific literature on joint cells, a double role for PEMFs could be hypothesized in vitro by stimulating cell proliferation, colonization of the scaffold, and production of tissue matrix. Another effect could be obtained in vivo after surgical implantation of the construct by favoring the anabolic activities of the implanted cells and surrounding tissues and protecting the construct from the catabolic effects of the inflammatory status. Moreover, a protective involvement of PEMFs on hypoxia damage in neuron-like cells and an anti-inflammatory effect in microglial cells have suggested the hypothesis of a positive impact of this noninvasive biophysical stimulus., Competing Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.

Activation of A 1 adenosine receptors (ARs) has been associated with anxiolytic-like effects in different behavioral tests, but development of A 1 AR agonists for therapeutic use has been hampered, most likely due to the presence of side effects. With the aim to identify a safer approach for the treatment of anxiety, we investigated, in mice, the anxiolytic-like properties of a novel A 1 AR positive allosteric modulator, TRR469. Acute administration of TRR469 (0.3-3 mg/kg) resulted in robust anxiolytic-like effects in the elevated plus maze, the dark/light box, the open field and the marble burying tests. The magnitude of the anxiolytic action of TRR469 was comparable to that obtained with benzodiazepine diazepam (1 mg/kg). The use of the A 1 AR antagonist DPCPX (3 mg/kg) suggested that the effects of TRR469 were mediated by this receptor subtype. In contrast to diazepam, the novel positive allosteric modulator did not potentiate the sedative effect of ethanol (3.5 g/kg) evaluated by the loss of righting reflex. While diazepam produced motor coordination impairment in the rotarod test, this effect being enhanced by the presence of ethanol (1.5 g/kg), TRR469 did not elicit locomotor disturbances either when administered alone or in the presence of ethanol. In vitro, TRR469 was able to increase the number of A 1 AR recognizable by the agonist radioligand [ 3 H]-CCPA in mouse brain regions involved in emotional processes. TRR469 markedly increased the affinity of the agonist CCPA, suggesting the capability, in vivo, to increase the affinity of endogenous adenosine. Taken together, these findings indicate that the positive allosteric modulation of A 1 AR may represent a promising approach for the treatment of anxiety-related disorders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Pyrazolo[5,1-f][1,6]naphthyridine-carboxamide derivatives were synthesized and evaluated for the affinity at CB 1 and CB 2 receptors. Based on the AgOTf and proline-cocatalyzed multicomponent methodology, the ethyl 5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (12) and ethyl 5-(2,4-dichlorophenyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxylate (13) intermediates were synthesized from the appropriate o-alkynylaldehydes, p-toluenesulfonyl hydrazide and ethyl pyruvate. Most of the novel compounds feature a p-tolyl (8a-i) or a 2,4-dichlorophenyl (8j) motif at the C 5 -position of the tricyclic pyrazolo[5,1-f][1,6]naphthyridine scaffold. Structural variation on the carboxamide moiety at the C 2 -position includes basic monocyclic, terpenoid and adamantine-based amines. Among these derivatives, compound 8h (N-adamant-1-yl-5-(p-tolyl)pyrazolo[5,1-f][1,6]naphthyridine-2-carboxamide) exhibited the highest CB 2 receptor affinity (K i =33nM) and a high degree of selectivity (K i CB 1 /K i CB 2 =173:1), whereas a similar trend in the near nM range was seen for the bornyl analogue (compound 8f, K i =53nM) and the myrtanyl derivative 8j (K i =67nM). Effects of 8h, 8f and 8j on forskolin-stimulated cAMP levels were determined, showing antagonist/inverse agonist properties for such compounds. Docking studies conducted for these derivatives and the reference antagonist/inverse agonist compound 4 (SR144528) disclosed the specific pattern of interactions probably related to the pyrazolo[5,1-f][1,6]naphthyridine scaffold as CB 2 inverse agonists., (Copyright © 2016. Published by Elsevier Ltd.)

The importance of adenosine for human health cannot be overstated. Indeed, this ubiquitous nucleoside is an integral component of ATP, and regulates the function of every tissue and organ in the body. Acting via receptor-dependent and -independent mechanisms [the former mediated via four G-protein-coupled receptors (GPCRs), A1, A2A, A2B, and A3,], it has a significant role in protecting against cell damage in areas of increased tissue metabolism, and combating organ dysfunction in numerous pathological states. Accordingly, raised levels of adenosine have been demonstrated in epilepsy, ischaemia, pain, inflammation, and cancer, in which its behaviour can be likened to that of a guardian angel, even though there are instances in which overproduction of adenosine is pathological. In this review, we condense the current body of knowledge on the issue, highlighting when, where, and how adenosine exerts its protective effects in both the brain and the periphery., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Microglia are central nervous system (CNS)-resident immune cells, that play a crucial role in neuroinflammation. Hypoxia-inducible factor-1 (HIF-1), the main transcription factor of hypoxia-inducible genes, is also involved in the immune response, being regulated in normoxia by inflammatory mediators. Adenosine is an ubiquitous nucleoside that has an influence on many immune properties of microglia through interaction with four receptor subtypes. The aim of this study was to investigate whether adenosine may affect microglia functions by acting on HIF-1α modulation. Primary murine microglia were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists and antagonists and HIF-1α accumulation and downstream genes regulation were determined. Adenosine increased LPS-induced HIF-1α accumulation leading to an increase in HIF-1α target genes involved in cell metabolism [glucose transporter-1 (GLUT-1)] and pathogens killing [inducible nitric-oxide synthase (iNOS)] but did not induce HIF-1α dependent genes related to angiogenesis [vascular endothelial growth factor (VEGF)] and inflammation [tumor necrosis factor-α (TNF-α)]. The stimulatory effect of adenosine on HIF-1α and its target genes was essentially exerted by activation of A 2A through p44/42 and A 2B subtypes via p38 mitogen-activated protein kinases (MAPKs) and Akt phosphorylation. Furthermore the nucleoside raised VEGF and decreased TNF-α levels, by activating A 2B subtypes. In conclusion adenosine increases GLUT-1 and iNOS gene expression in a HIF-1α-dependent way, through A 2A and A 2B receptors, suggesting their role in the regulation of microglial cells function following injury. However, inhibition of TNF-α adds an important anti-inflammatory effect only for the A 2B subtype. GLIA 2015;63:1933-1952., (© 2015 Wiley Periodicals, Inc.)

Over the last two decades, diabetes mellitus has become one of the most challenging health problems worldwide. Diabetes mellitus, classified as type I and II, is a pathology concerning blood glucose level in the body. The nucleoside adenosine has long been known to affect insulin secretion, glucose homeostasis and lipid metabolism, through activation of four G protein coupled adenosine receptors (ARs), named A1, A2A, A2B and A3. Currently, the novel promising subtype to develop new drugs for diabetes treatment is the A2BAR subtype. The use of selective agonists and antagonists for A2BAR subtype in various diabetic animal models allowed us to identify several effects of A2BAR signaling in cell metabolism. In particular, the focus of this review is to summarize the studies on purinergic signaling associated with diabetes through A2BARs modulation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

By general consensus, the omnipresent purine nucleoside adenosine is considered a major regulator of local tissue function, especially when energy supply fails to meet cellular energy demand. Adenosine mediation involves activation of a family of four G protein-coupled adenosine receptors (ARs): A(1), A(2)A, A(2)B, and A(3). The A(3) adenosine receptor (A(3)AR) is the only adenosine subtype to be overexpressed in inflammatory and cancer cells, thus making it a potential target for therapy. Originally isolated as an orphan receptor, A(3)AR presented a twofold nature under different pathophysiologic conditions: it appeared to be protective/harmful under ischemic conditions, pro/anti-inflammatory, and pro/antitumoral depending on the systems investigated. Until recently, the greatest and most intriguing challenge has been to understand whether, and in which cases, selective A(3) agonists or antagonists would be the best choice. Today, the choice has been made and A(3)AR agonists are now under clinical development for some disorders including rheumatoid arthritis, psoriasis, glaucoma, and hepatocellular carcinoma. More specifically, the interest and relevance of these new agents derives from clinical data demonstrating that A(3)AR agonists are both effective and safe. Thus, it will become apparent in the present review that purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

A series of 1,3,6-triphenylpyrazolo[3,4-b]pyridin-4-one derivatives was designed, synthesized and evaluated for cytotoxic activity in A375 human melanoma and human erythroleukemia (HEL) cells. The new pyrazolopyridones displayed comparable activities to the antitumor compound etoposide. The inhibitory effect of compounds 17, 18, 27 and 32 against topoisomerase II-mediated cleavage activities was measured finding good correlation with the results obtained from MTS assay. Docking studies into bacterial topoisomerase II (DNA Gyrase), topoisomerase IIα and topoisomerase IIβ binding sites in the DNA binding interface were performed.

Adenosine mediates its effects through activation of a family of four G-protein-coupled receptors, named A1 , A2A , A2B and A3 . This nucleoside plays an important role in immunity and inflammation, and the A2A adenosine receptor subtype has a key role in the inhibition of inflammatory processes besides promoting wound healing. In this issue of Experimental Dermatology, Arasa et al. show that the topical application of a selective A2A agonist, CGS 21680, to mouse skin reduced epidermal hyperplasia as well as skin inflammation, similarly to topical corticoids, without side effects like skin atrophy. Rigorously following up this work is important for the development of novel treatment strategies for chronic hyperproliferative inflammatory dermatoses, such as targeting the A2A adenosine receptor family., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

A(1) adenosine receptors (ARs) have been identified as a potential target for the development of anti-nociceptive compounds. The present study explores the analgesic effects of a novel A(1)AR positive allosteric modulator, TRR469, in different models of acute and chronic pain in mice. To evaluate the allosteric enhancement, in vitro binding experiments were performed. The anti-nociceptive properties were investigated in formalin and writhing tests, and in the streptozotocin-induced diabetic neuropathic pain model. Rotarod and catalepsy tests were used to identify potential side effects, while the functional effect of TRR469 was studied using [(3)H]-d-aspartate release from synaptosomes. TRR469 effectively inhibited nociceptive responses in the formalin and writhing tests, with effects comparable to those of the reference analgesic morphine. Isobolographic analysis of the combination of TRR469 and morphine revealed an additive interaction. TRR469 was anti-allodynic in the neuropathic pain model and did not display locomotor or cataleptic side effects. TRR469 enhanced the binding of the agonist radioligand [(3)H]-CCPA and induced a 33-fold increase of adenosine affinity in spinal cord membranes. In mouse spinal cord synaptosomes, TRR469 enhanced the inhibitory effect of A(1)AR activation on [(3)H]-d-aspartate release, a non-metabolizable analogue of glutamate. In conclusion, this research demonstrates the anti-nociceptive effect of the novel compound TRR469, one of the most potent and effective A(1)AR positive allosteric modulators so far synthesized. The use of TRR469 allows for the possibility of exploiting analgesic properties of endogenous adenosine, with a minor potential to develop the various side effects often associated with the use of direct receptor agonists., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes A1, A2A, A2B and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia and inflammation and plays an important role in brain injury. HIF-1 is expressed by astrocytes, however the regulatory role played by Ado on HIF-1α modulation induced by inflammatory and hypoxic conditions has not been investigated. Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without Ado, Ado receptor agonists, antagonists and receptor silencing, before exposure to normoxia or hypoxia. HIF-1α accumulation and downstream genes regulation were determined. Ado inhibited LPS-increased HIF-1α accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with the blockers of p44/42 MAPK and Akt, LPS-induced HIF-1α accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect and Ado inhibited kinases phosphorylation. A series of angiogenesis and metabolism related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, with the exception of GLUT-1 and hexochinase II that were elevated by LPS only in normoxia and inhibited by Ado receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia and inhibited by Ado, through A1 and A3 receptor subtypes. In conclusion A1 and A3 receptors reduce the LPS-mediated HIF-1α accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, like iNOS and A2B receptors, in both normoxic and hypoxic conditions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Anti-nociceptive tolerance to opioids severely limits their clinical efficacy for the treatment of chronic pain syndromes. Glia has a central role in the development of morphine tolerance. Here, we characterized the receptor-proximal signaling events that link μ-opioid receptors to activation of Akt and ERKs in lipopolysaccharide (LPS)-stimulated murine microglial cells with the aim to define the molecular mechanism contributing to the ability of morphine to increase inflammatory mediators such as nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in activated microglial cells. In particular, the role of PKCɛ isoform in μ-opioid-induced inflammatory response in microglia was investigated. The results indicate that morphine increases the LPS-induced expression and activation of PKCɛ and stimulates Akt pathway upstream of ERK1/2 and iNOS. Furthermore, we found that morphine enhanced the release of IL-1β, TNF-α, IL-6, and of NO via μ-opioid receptor-PKCɛ signaling pathway in activated microglial cells, mediating a proinflammatory phenotype in mouse microglial cells. Together, these data suggest that the modulation of μ-opioid receptor signaling on microglia through PKCɛ selective inhibition may provide a means to attenuate glial activation and, as a consequence, to treat opioid development of tolerance and dependence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is an autoimmune-mediated inflammatory disease characterized by multifocal areas of demyelination. Experimental evidence indicates that A2A adenosine receptors (ARs) play a pivotal role in the inhibition of inflammatory processes. The aim of this study was to investigate the contribution of A2A ARs in the inhibition of key pro-inflammatory mediators for the pathogenesis of MS. In lymphocytes from MS patients, A1, A2A, A2B, and A3 ARs were analyzed by using RT-PCR, Western blotting, immunofluorescence, and binding assays. Moreover the effect of A2A AR stimulation on proinflammatory cytokine release such as TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and on lymphocyte proliferation was evaluated. The capability of an A2A AR agonist on the modulation of very late antigen (VLA)-4 expression and NF-κB was also explored. A2A AR upregulation was observed in lymphocytes from MS patients in comparison with healthy subjects. The stimulation of these receptors mediated a significant inhibition of TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and cell proliferation as well as VLA-4 expression and NF-κB activation. This new evidence highlights that A2A AR agonists could represent a novel therapeutic tool for MS treatment as suggested by the antiinflammatory role of A2A ARs in lymphocytes from MS patients., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cannabinoid CB(2) receptor activation by selective agonists has been shown to produce analgesic effects in preclinical models of inflammatory, neuropathic, and bone cancer pain. In this study the effect of a novel CB(2)agonist (MT178) was evaluated in different animal models of pain. First of all, in vitro competition binding experiments performed on rat, mouse, or human CB receptors revealed a high affinity, selectivity, and potency of MT178. The analgesic properties of the novel CB(2) agonist were evaluated in various in vivo experiments, such as writhing and formalin assays, showing a good efficacy comparable with that produced by the nonselective CB agonist WIN 55,212-2. A dose-dependent antiallodynic effect of the novel CB(2) compound in the streptozotocin-induced diabetic neuropathy was found. In a bone cancer pain model and in the acid-induced muscle pain model, MT178 was able to significantly reduce mechanical hyperalgesia in a dose-related manner. Notably, MT178 failed to provoke locomotor disturbance and catalepsy, which were observed following the administration of WIN 55,212-2. CB(2) receptor mechanism of action was investigated in dorsal root ganglia where MT178 mediated a reduction of [(3)H]-d-aspartate release. MT178 was also able to inhibit capsaicin-induced substance P release and NF-κB activation. These results demonstrate that systemic administration of MT178 produced a robust analgesia in different pain models via CB(2) receptors, providing an interesting approach to analgesic therapy in inflammatory and chronic pain without CB(1)-mediated central side effects., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Adenosine receptors (ARs) have an important role in the regulation of inflammation and their activation is involved in the inhibition of pro-inflammatory cytokine release. The effects of pulsed electromagnetic fields (PEMFs) on inflammation have been reported and we have demonstrated that PEMFs increased A2A and A3AR density and functionality in different cell lines. Chondrocytes and osteoblasts are two key cell types in the skeletal system that play important role in cartilage and bone metabolism representing an interesting target to study the effect of PEMFs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-inflammatory effect of A2A and/or A3ARs in T/C-28a2 chondrocytes and hFOB 1.19 osteoblasts. Immunofluorescence, mRNA analysis and saturation binding assays revealed that PEMF exposure up-regulated A2A and A3AR expression. A2A and A3ARs were able to modulate cAMP production and cell proliferation. The activation of A2A and A3ARs resulted in the decrease of some of the most relevant pro-inflammatory cytokine release such as interleukin (IL)-6 and IL-8, following the treatment with IL-1β as an inflammatory stimuli. In human chondrocyte and osteoblast cell lines, the inhibitory effect of A2A and A3AR stimulation on the release of prostaglandin E2 (PGE2), an important lipid inflammatory mediator, was observed. In addition, in T/C-28a2 cells, the activation of A2A or A3ARs elicited an inhibition of vascular endothelial growth factor (VEGF) secretion. In hFOB 1.19 osteoblasts, PEMF exposure determined an increase of osteoprotegerin (OPG) production. The effect of the A2A or A3AR agonists in the examined cells was enhanced in the presence of PEMFs and completely blocked by using well-known selective antagonists. These results demonstrated that PEMF exposure significantly increase the anti-inflammatory effect of A2A or A3ARs suggesting their potential therapeutic use in the therapy of inflammatory bone and joint disorders.

A(2A) adenosine receptors (ARs) play a key role in the inhibition of the inflammatory process. The purpose of this study was to evaluate the modulation of A(2A)ARs in rheumatoid arthritis (RA) patients after different pharmacological treatments and to investigate the effect of A(2A)AR stimulation in a rat model of arthritis. We investigated A(2A)AR density and functionality in RA progression by using a longitudinal study in RA patients before and after methotrexate (MTX), anti-TNFα agents or rituximab treatments. A(2A)ARs were analyzed by saturation binding assays in lymphocytes from RA patients throughout the 24-month study timeframe. In an adjuvant-induced arthritis model in rats we showed the efficacy of the A(2A)AR agonist, CGS 21680 in comparison with standard therapies by means of paw volume assessment, radiographic and ultrasonographic imaging. Arthritic-associated pain was investigated in mechanical allodynia and thermal hyperalgesia tests. IL-10 release following A(2A)AR stimulation in lymphocytes from RA patients and in serum from arthritic rats was measured. In lymphocytes obtained from RA patients, the A(2A)AR up-regulation was gradually reduced in function of the treatment time and the stimulation of these receptors mediated a significant increase of IL-10 production. In the same cells, CGS 21680 did not affected cell viability and did not produced cytotoxic effects. The A(2A)AR agonist CGS 21680 was highly effective, as suggested by the marked reduction of clinical signs, in rat adjuvant-induced arthritis and associated pain. This study highlighted that A(2A)AR agonists represent a physiological-like therapeutic alternative for RA treatment as suggested by the anti-inflammatory role of A(2A)ARs in lymphocytes from RA patients. The effectiveness of A(2A)AR stimulation in a rat model of arthritis supported the role of A(2A)AR agonists as potential pharmacological treatment for RA.

Hydrogen sulfide (H(2)S) is a novel signaling molecule with both pro- or anti-inflammatory effect. The present study aimed to: (i) characterize the in vitro effects of H(2)S on human keratinocyte's proliferation and death; (ii) investigate the ability of H(2)S to modulate VEGF and NO production; (iii) examine the intracellular signaling pathways involved in VEGF and NO modulatory effect. We found that exogenous application of H(2)S (NaHS and GYY4137 as H(2)S donors) significantly enhances NO through increase of iNOS, in a manner Akt-dependent. The increment in NO down-regulates ERK1/2 activation thereby resulting in the decrease of VEGF release. We suggest that H(2)S-releasing agents may be promising therapeutics for chronic inflammatory disorders of the skin, i.e. psoriasis, in which NO increases as well as anti-VEGF treatments have been suggested to be novel effective approaches., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Human reproduction is complex and prone to failure. Though causes of miscarriage remain unclear, adenosine, a proangiogenic nucleoside, may help determine pregnancy outcome. Although adenosine receptor (AR) expression has been characterized in euploid pregnancies, no information is available for aneuploidies, which, as prone to spontaneous abortion (SA), are a potential model for shedding light on the mechanism regulating this event. AR expression was investigated in 71 first-trimester chorionic villi (CV) samples and cultured mesenchymal cells (MC) from euploid and TR21 pregnancies, one of the most frequent autosomal aneuploidy, with a view to elucidating their potential role in the modulation of vascular endothelial growth factor (VEGF) and nitric oxide (NO). Compared to euploid cells, reduced A(1) and A(2B) expression was revealed in TR21 CV and MCs. The non-selective adenosine agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased NO, by activating, predominantly, A(1)AR and A(2A)AR through a molecular pathway involving hypoxia-inducible-factor-1 (HIF-1α), and increased VEGF, mainly through A(2B). In conclusion the adenosine transduction cascade appears to be disturbed in TR21 through reduced expression of A(2B) and A(1)ARs. These anomalies may be implicated in complications such as fetal growth restriction, malformation and/or SA, well known features of aneuploid pregnancies. Therefore A(1) and A(2B)ARs could be potential biomarkers able to provide an early indication of SA risk and their stimulation may turn out to improve fetoplacental perfusion by increasing NO and VEGF., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background and Purpose: Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs in chronic immune inflammatory diseases. In the present study, we characterized the signal transduction pathways affected by CB(2) receptors in quiescent and lipopolysaccharide (LPS)-stimulated murine microglia., Experimental Approach: We examined the effects of the synthetic CB(2) receptor ligand, JWH-015, on phosphorylation of MAPKs and NO production., Key Results: Stimulation of CB(2) receptors by JWH-015 activated JNK-1/2 and ERK-1/2 in quiescent murine microglial cells. Furthermore, CB(2) receptor activation increased p-ERK-1/2 at 15 min in LPS-stimulated microglia. Surprisingly, this was reduced after 30 min in the presence of both LPS and JWH-015. The NOS inhibitor L-NAME blocked the ability of JWH-015 to down-regulate the LPS-induced p-ERK increase, indicating that activation of CB(2) receptors reduced effects of LPS on ERK-1/2 phosphorylation through NO. JWH-015 increased LPS-induced NO release at 30 min, while at 4 h CB(2) receptor stimulation had an inhibitory effect. All the effects of JWH-015 were significantly blocked by the CB(2) receptor antagonist AM 630 and, as the inhibition of CB(2) receptor expression by siRNA abolished the effects of JWH-015, were shown to be mediated specifically by activation of CB(2) receptors., Conclusions and Implications: Our results demonstrate that CB(2) receptor stimulation activated the MAPK pathway, but the presence of a second stimulus blocked MAPK signal transduction, inhibiting pro-inflammatory LPS-induced production of NO. Therefore, CB(2) receptor agonists may promote anti-inflammatory therapeutic responses in activated microglia., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

A(3) adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A(2A) and A(3)AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A(2A) and/or A(3)ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A(3)ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A(2A) and A(3)ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A(2A) and A(3)ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A(3)ARs are involved in the increase of p53 expression. A(3)AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A(3)AR activation. The effect of the A(3)AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A(3)ARs.

Introduction: The adenosine receptors A(1), A(2A), A(2B) and A(3) are important and ubiquitous mediators of cellular signaling that play vital roles in protecting tissues and organs from damage. In particular, adenosine triggers tissue protection and repair by different receptor-mediated mechanisms, including increasing the oxygen supply:demand ratio, pre-conditioning, anti-inflammatory effects and the stimulation of angiogenesis., Areas Covered: The state of the art of the role of adenosine receptors which have been proposed as targets for drug design and discovery, in health and disease, and an overview of the ligands for these receptors in clinical development., Expert Opinion: Selective ligands of A(1), A(2A), A(2B) and A(3) adenosine receptors are likely to find applications in the treatment of pain, ischemic conditions, glaucoma, asthma, arthritis, cancer and other disorders in which inflammation is a feature. The aim of this review is to provide an overview of the present knowledge regarding the role of these adenosine receptors in health and disease.

Adenosine is a ubiquitous signaling molecule whose physiological functions are mediated by its interaction with four G-protein-coupled receptor subtypes, termed A(1), A(2A), A(2B) and A(3). As a result of increased metabolic rates, this nucleoside is released from a variety of cells throughout the body in concentrations that can have a profound impact on vasculature and immunoescape. However, as high concentrations of adenosine have been reported in cancer tissues, it also appears to be implicated in the growth of tumors. Thus, full characterisation of the role of adenosine in tumor development, by addressing the question of whether adenosine receptors are present in cancer tissues, and, if so, which receptor subtype mediates its effects in cancer growth, is a vital research goal. To this end, this review focuses on the most relevant aspects of adenosine receptor subtype activation in tumors reported so far. Although all adenosine receptors now have an increasing number of recognised biological roles in tumors, it seems that the A(2A) and A(3) subtypes are the most promising as regards drug development. In particular, activation of A(2A) receptors leads to immunosuppressive effects, which decreases anti-tumoral immunity and thereby encourages tumor growth. Due to this behavior, the addition of A(2A) antagonists to cancer immunotherapeutic protocols has been suggested as a way of enhancing tumor immunotherapy. Interestingly, the safety of such compounds has already been demonstrated in trials employing A(2A) antagonists in the treatment of Parkinson's disease. As for A(3) receptors, the effectiveness of their agonists in several animal tumor models has led to the introduction of these molecules into a programme of pre-clinical and clinical trials. Paradoxically, A(3) receptor antagonists also appear to be promising candidates in human cancer treatment of regimes. Clearly, research in this still field is still in its infancy, with several important and challenging issues remaining to be addressed, although purine scientists do seem to be getting closer to their goal: the incorporation of adenosine ligands into drugs with the ability to save lives and improve human health., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The adenosine receptors A(1), A(2A), A(2B), and A(3) are important and ubiquitous mediators of cellular signaling, which play vital roles in protecting tissues and organs from damage. In particular, adenosine triggers tissue protection and repair by different receptor-mediated mechanisms, including an increase of oxygen supply/demand ratio, preconditioning, anti-inflammatory effects, and stimulation of angiogenesis. Considerable advances have been recently achieved in the pharmacological and molecular characterization of adenosine receptors, which have been proposed as targets for drug design and discovery. At the present time, it can be speculated that adenosine A(1), A(2A), A(2B), and A(3) receptor-selective ligands may show utility in the treatment of pain, ischemic conditions, glaucoma, asthma, arthritis, cancer, and other disorders in which inflammation is a feature. This chapter documents the present state of knowledge of adenosine receptors' role in health and disease., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In this work, we investigated the biological functions of adenosine (ado) in metalloproteinase-9 (MMP-9) regulation in U87MG human glioblastoma cells. The nucleoside was able to increase both MMP-9 mRNA and protein levels through A3 receptors activation. We revealed that A3 receptor stimulation induced an increase of MMP-9 protein levels in cellular extracts of U87MG cells by phosphorylation of extracellular signal-regulated protein kinases (ERK1/2), c-Jun N-terminal kinase/stress-activated protein kinase (pJNK/SAPK), protein kinase B (Akt/PKB) and finally activator protein 1 (AP-1). A3 receptor activation stimulated also an increase of extracellular MMP-9 in the supernatants from U87MG glioblastoma cells. Finally, the Matrigel invasion assay demonstrated that A3 receptors, by inducing an increase in MMP-9 levels, was responsible for an increase of glioblastoma cells invasion. Collectively, these results suggest that ado, through A3 receptors activation, modulates MMP-9 protein levels and plays a role in increasing invasion of U87MG cells., (2010 Elsevier Inc. All rights reserved.)

The antagonistic interaction between adenosine and dopamine receptors could have important pathophysiological and therapeutic implications in Parkinson's disease (PD). The primary aim of this study was to investigate the expression, affinity, and density of A(1), A(2A), A(2B), and A(3) adenosine receptors (ARs) and D(2) dopamine receptors (D(2)Rs) in PD. An increase in A(2A)AR density in putamen was found. The presence and functionality of ARs in human lymphocyte and neutrophil membranes from patients with PD revealed a specific A(2A)AR alteration compared with healthy subjects. A statistically significant linear correlation among the A(2A)AR density, functionality, or tumor necrosis factor-alpha (TNF-alpha) levels and Unified Parkinson's Disease Rating Scale (UPDRS) motor score was reported. Adenosine concentration and TNF-alpha levels were increased in plasma of patients with PD. In rat adrenal pheochromocytoma (PC12) cells, a widely useful model, adenosine antagonists decreased dopamine uptake, and an opposite effect was mediated by A(2A) agonists. This is the first report showing the presence of an A(2A)AR alteration in putamen in PD that mirrors a similar up-regulation in human peripheral blood cells. Moreover, the correlation found between A(2A)AR density or A(2A) agonist potency and UPDRS motor score highlights the central role of A(2A)ARs in the pharmacological treatment of PD.

The thermodynamic parameters DeltaG degrees , DeltaH degrees and DeltaS degrees of the binding equilibrium of agonists and antagonists at cannabinoid CB(1) and CB(2) receptors were determined by means of affinity measurements at different temperatures and van't Hoff plots were constructed. Affinity constants were measured on CHO cells transfected with the human CB(1) and CB(2) receptors by inhibition assays of the binding of the cannabinoid receptor agonist [(3)H]-CP-55,940. van't Hoff plots were linear for agonists and antagonists in the temperature range 0-30 degrees C. The thermodynamic parameters for CB(1) receptors fall in the ranges 17< or =DeltaH degrees < or =59 kJ/mol and 213< or =DeltaS degrees < or =361 kJ/mol for agonists and -52< or =DeltaH degrees < or =-26 kJ/mol and -12< or =DeltaS degrees < or =38 kJ/mol for antagonists. The thermodynamic parameters for CB(2) receptors fall in the ranges 27< or =DeltaH degrees < or =48 kJ/mol and 234< or =DeltaS degrees < or =300 kJ/mol for agonists and -19< or =DeltaH degrees < or =-17 kJ/mol and 43< or =DeltaS degrees < or =74 kJ/mol for antagonists. Collectively, these data show that agonist binding is always totally entropy-driven while antagonist binding is enthalpy and entropy-driven, indicating that CB(1) and CB(2) receptors are thermodynamically discriminated. These data could give new details on the nature of the forces driving the CB(1) and CB(2) binding at a molecular level. Enthalpy, entropy, free energy and binding affinity for each ligand to its receptor can all be assessed and therefore the optimal binding profile discovered. Carrying out these binding investigations as early as possible in the discovery process increases the probability that a lead compound will become a successful pharmaceutical compound.

Glucocorticoids (GCs) constitute the most important and widely prescribed class of anti-inflammatory and immunosuppressive drugs. Since their first clinical use more than half a century ago, they have been employed for therapeutic treatment in a variety of diseases such as atopic disorders, autoimmune diseases and cancer. Their efficacy is related to their capability to reduce the expression and activity of many pro-inflammatory agents, that is named "transrepression". Unfortunately, they also induce serious adverse effects, in particular upon high dosage and prolonged usage, many of which are due to the transcription of genes involved in metabolic processes, that is called "transactivation". This discrepancy is the driving force for discovery of novel safer GC receptor ligands, such as selective GCs receptor agonists (SEGRAs) able to induce transrepression, whilst avoiding transactivation. The aim of this review is to give an overview of the above-mentioned mechanisms at the basis of GCs action and to summarize the results obtained with SEGRAs in terms of efficacy versus side effects and discuss the opportunities and challenges that this class of ligands might offer for clinical arena.

Objective: Foam cell (FC) formation by oxidized low-density lipoprotein (oxLDL) accumulation in macrophages is crucial for development of atherosclerosis. Hypoxia has been demonstrated in atherosclerosis and hypoxia-inducible factor-1 (HIF-1) has been shown to promote intraplaque angiogenesis and FC development. As hypoxia induces HIF-1alpha stabilization and adenosine (ado) accumulation, we investigated whether this nucleoside regulates HIF-1alpha in FCs., Methods and Results: Ado, under hypoxia, stimulates HIF-1alpha accumulation by activating all adenosine receptors (ARs). HIF-1alpha modulation involved extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38 MAPK), and protein kinase B (Akt) phosphorylation in the case of A(1), A(2A), A(2B), and ERK 1/2 phosphorylation in the case of A(3) receptors. Ado, through the activation of A(3) and A(2B) receptors, stimulates vascular endothelial growth factor (VEGF) secretion in a HIF-1alpha-dependent way. Furthermore, ado, through the A(2B) subtype, induces an increase of Interleukin-8 (IL-8) secretion in a ERK 1/2, p38, and Akt kinase-dependent but not HIF-1alpha-mediated way. Finally, ado stimulates FC formation, and this effect is strongly reduced by A(3) and A(2B) blockers and by HIF-1alpha silencing., Conclusions: This study provides the first evidence that A(3,) A(2B), or mixed A(3)/A(2B) antagonists may be useful to block important steps in the atherosclerotic plaque development ado-induced.

Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator hypoxia-inducible factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF, and HIF-1 expressions in human melanoma cancer cells. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, namely, A(1), A(2A), A(2B), and A(3). We have demonstrated that A(2B) receptor blockade can impair IL-8 production, whereas blocking A(3) receptors, it is possible to further decrease VEGF secretion in melanoma cells treated with VP-16 and doxorubicin. This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.

Cyst growth and expansion in autosomal dominant polycystic kidney disease (ADPKD) has been attributed to numerous factors, including ATP, cAMP and adenosine signalling. Although the role of ATP and cAMP has been widely investigated in PKD1-deficient cells, no information is currently available on adenosine-mediated signalling. Here we investigate for the first time the impact of abnormalities of polycystin-1 (PC1) on the expression and functional activity of adenosine receptors, members of the G-protein-coupled receptor superfamily. Pharmacological, molecular and biochemical findings show that a siRNA-dependent PC1-depletion in HEK293 cells and a PKD1-nonsense mutation in cyst-derived cell lines result in increased expression of the A(3) adenosine receptor via an NFkB-dependent mechanism. Interestingly, A(3) adenosine receptor levels result higher in ADPKD than in normal renal tissues. Furthermore, the stimulation of this receptor subtype with the selective agonist Cl-IB-MECA causes a reduction in both cytosolic cAMP and cell proliferation in both PC1-deficient HEK293 cells and cystic cells. This reduction is associated with increased expression of p21(waf) and reduced activation not only of ERK1/2, but also of S6 kinase, the main target of mTOR signalling. In the light of these findings, the ability of Cl-IB-MECA to reduce disease progression in ADPKD should be further investigated. Moreover, our results suggest that NFkB, which is markedly activated in PC1-deficient and cystic cells, plays an important role in modulating A(3)AR expression in cystic cells.