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1.  Adenosine signaling in normal and sickle erythrocytes and beyond 
Microbes and infection / Institut Pasteur  2012;14(10):10.1016/j.micinf.2012.05.005.
Sickle cell disease (SCD) is a debilitating hemolytic genetic disorder with high morbidity and mortality affecting millions of individuals worldwide. Although SCD was discovered more than a century ago, no effective mechanism-based prevention and treatment are available due to poorly understood molecular basis of sickling, the fundamental pathogenic process of the disease. SCD patients constantly face hypoxia. One of the best-known signaling molecules to be induced under hypoxic conditions is adenosine. Recent studies demonstrate that hypoxia-mediated elevated adenosine signaling plays an important role in normal erythrocyte physiology. In contrast, elevated adenosine signaling contributes to sickling and multiple life threatening complications including tissue damage, pulmonary dysfunction and priapism. Here, we summarize recent research on the role of adenosine signaling in normal and sickle erythrocytes, progression of the disease and therapeutic implications.
In normal erythrocytes, both genetic and pharmacological studies demonstrate that adenosine can enhance 2,3-bisphosphoglycerate (2,3-BPG) production via A2B receptor (ADORA2B) activation, suggesting that elevated adenosine has an unrecognized role in normal erythrocytes to promote O2 release and prevent acute ischemic tissue injury. However, in sickle erythrocytes, the beneficial role of excessive adenosine-mediated 2,3-BPG induction becomes detrimental by promoting deoxygenation, polymerization of sickle hemoglobin and subsequent sickling. Additionally, adenosine signaling via the A2A receptor (ADORA2A) on invariant natural killer T (iNKT) cells inhibits iNKT cell activation and attenuates pulmonary dysfunction in SCD mice. Finally, elevated adenosine coupled with ADORA2BR activation is responsible for priapism, a dangerous complication seen in SCD.
Overall, the research reviewed here reveals a differential role of elevated adenosine in normal erythrocytes, sickle erythrocytes, iNK cells and progression of disease. Thus, adenosine signaling represents a potentially important therapeutic target for the treatment and prevention of disease.
PMCID: PMC3842013  PMID: 22634345
sickle cell disease; malaria; adenosine; adenosine A2B receptor; 2,3-diphosphoglycerate; adenosine deaminase
2.  Adenosine and the Auditory System 
Current Neuropharmacology  2009;7(3):246-256.
Adenosine is a signalling molecule that modulates cellular activity in the central nervous system and peripheral organs via four G protein-coupled receptors designated A1, A2A, A2B, and A3. This review surveys the literature on the role of adenosine in auditory function, particularly cochlear function and its protection from oxidative stress. The specific tissue distribution of adenosine receptors in the mammalian cochlea implicates adenosine signalling in sensory transduction and auditory neurotransmission although functional studies have demonstrated that adenosine stimulates cochlear blood flow, but does not alter the resting and sound-evoked auditory potentials. An interest in a potential otoprotective role for adenosine has recently evolved, fuelled by the capacity of A1 adenosine receptors to prevent cochlear injury caused by acoustic trauma and ototoxic drugs. The balance between A1 and A2A receptors is conceived as critical for cochlear response to oxidative stress, which is an underlying mechanism of the most common inner ear pathologies (e.g. noise-induced and age-related hearing loss, drug ototoxicity). Enzymes involved in adenosine metabolism, adenosine kinase and adenosine deaminase, are also emerging as attractive targets for controlling oxidative stress in the cochlea. Other possible targets include ectonucleotidases that generate adenosine from extracellular ATP, and nucleoside transporters, which regulate adenosine concentrations on both sides of the plasma membrane. Developments of selective adenosine receptor agonists and antagonists that can cross the blood-cochlea barrier are bolstering efforts to develop therapeutic interventions aimed at ameliorating cochlear injury. Manipulations of the adenosine signalling system thus hold significant promise in the therapeutic management of oxidative stress in the cochlea.
PMCID: PMC2769008  PMID: 20190966
Adenosine; adenosine receptors; cochlea; hearing; deafness; oxidative stress; noise; ototoxicity.
3.  Adenosine A2A receptors in Parkinson’s disease treatment 
Purinergic Signalling  2008;4(4):305-312.
Latest results on the action of adenosine A2A receptor antagonists indicate their potential therapeutic usefulness in the treatment of Parkinson’s disease. Basal ganglia possess high levels of adenosine A2A receptors, mainly on the external surfaces of neurons located at the indirect tracts between the striatum, globus pallidus, and substantia nigra. Experiments with animal models of Parkinson’s disease indicate that adenosine A2A receptors are strongly involved in the regulation of the central nervous system. Co-localization of adenosine A2A and dopaminergic D2 receptors in striatum creates a milieu for antagonistic interaction between adenosine and dopamine. The experimental data prove that the best improvement of mobility in patients with Parkinson’s disease could be achieved with simultaneous activation of dopaminergic D2 receptors and inhibition of adenosine A2A receptors. In animal models of Parkinson’s disease, the use of selective antagonists of adenosine A2A receptors, such as istradefylline, led to the reversibility of movement dysfunction. These compounds might improve mobility during both monotherapy and co-administration with L-DOPA and dopamine receptor agonists. The use of adenosine A2A receptor antagonists in combination therapy enables the reduction of the L-DOPA doses, as well as a reduction of side effects. In combination therapy, the adenosine A2A receptor antagonists might be used in both moderate and advanced stages of Parkinson’s disease. The long-lasting administration of adenosine A2A receptor antagonists does not decrease the patient response and does not cause side effects typical of L-DOPA therapy. It was demonstrated in various animal models that inhibition of adenosine A2A receptors not only decreases the movement disturbance, but also reveals a neuroprotective activity, which might impede or stop the progression of the disease. Recently, clinical trials were completed on the use of istradefylline (KW-6002), an inhibitor of adenosine A2A receptors, as an anti-Parkinson drug.
PMCID: PMC2583202  PMID: 18438720
Parkinson’s disease; Adenosine; Adenosine receptors; Dopamine receptors; Neuroprotection
4.  Adenosine: An Old Drug Newly Discovered 
Anesthesiology  2009;111(4):904-915.
Over decades, anesthesiologists have used intravenous adenosine as mainstay therapy for diagnosing or treating supraventricular tachycardia in the perioperative setting. More recently, specific adenosine receptor therapeutics or gene-targeted mice deficient in extracellular adenosine production or individual adenosine receptors became available. These models enabled physicians and scientists to learn more about the biological functions of extracellular nucleotide metabolism and adenosine signaling. Such functions include specific signaling effects through adenosine receptors expressed by many mammalian tissues, for example vascular endothelia, myocytes, heptocytes, intestinal epithelia or immune cells. At present, pharmacological approaches to modulate extracellular adenosine signaling are evaluated for their potential use in perioperative medicine, including attenuation of acute lung injury, renal, intestinal, hepatic and myocardial ischemia, or vascular leakage. If these laboratory studies can be translated into clinical practice, adenosine receptor based therapeutics may become an integral pharmacological component of daily anesthesiology practice.
PMCID: PMC2797575  PMID: 19741501
5.  Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells1 
Pharmacological studies suggest that A2B adenosine receptors mediate proinflammatory effects of adenosine in human mast cells in part by up-regulating production of Th2 cytokines and angiogenic factors. This concept has been recently challenged by the finding that mast cells cultured from bone marrow-derived mast cells (BMMCs) of A2B knockout mice display an enhanced degranulation in response to FcεRI stimulation. This finding was interpreted as evidence of anti-inflammatory functions of A2B receptors and it was suggested that antagonists with inverse agonist activity could promote activation of mast cells. In this report, we demonstrate that genetic ablation of the A2B receptor protein has two distinct effects on BMMCs, one is the previously reported enhancement of Ag-induced degranulation, which is unrelated to adenosine signaling; the other is the loss of adenosine signaling via this receptor subtype that up-regulates IL-13 and vascular endothelial growth factor secretion. Genetic ablation of A2B receptors had no effect on A3 adenosine receptor-dependent potentiation of Ag-induced degranulation in mouse BMMCs, but abrogated A2B adenosine receptor-dependent stimulation of IL-13 and vascular endothelial growth factor secretion. Adenosine receptor antagonists MRS1706 and DPCPX with known inverse agonist activity at the A2B subtype inhibited IL-13 secretion induced by the adenosine analog NECA, but did not mimic the enhanced Ag-induced degranulation observed in A2B knockout BMMCs. Thus, our study confirmed the proinflammatory role of adenosine signaling via A2B receptors and the anti-inflammatory actions of A2B antagonists in mouse BMMCs.
PMCID: PMC3628765  PMID: 18490720
6.  The Role of the Adenosinergic Pathway in Immunosuppression Mediated by Human Regulatory T Cells (Treg) 
Current medicinal chemistry  2011;18(34):5217-5223.
Tumor-induced dysfunction of immune cells is a common problem in cancer. Tumors induce immune suppression by many different mechanisms, including accumulation of regulatory T cells (Treg). Adaptive Treg (Tr1) generated in the tumor microenvironment express CD39 and CD73 ectonucleotidases, produce adenosine and are COX2+PGE2+. Adenosine and PGE2 produced by Tr1 or tumor cells bind to their respective receptors on the surface of T effector cells (Teff) and cooperate in up-regulating cytosolic 3′5′-cAMP levels utilizing adenylyl cyclase isoform 7 (AC-7). In Teff, increased cAMP mediates suppression of anti-tumor functions. Treg, in contrast to Teff, seem to require high cAMP levels for mediating suppression. This differential requirement of Treg and Teff for cAMP offers an opportunity for pharmacologic interventions using selected inhibitors of the adenosine/PGE2 pathways. Blocking of adenosine/PGE2 production by Tr1 or blocking binding of these factors to their receptors on T cells or inhibition of cAMP synthesis in Teff all represent novel therapeutic strategies that used in combination with conventional therapies could restore anti-tumor functions of Teff. At the same time, these inhibitors could disarm Tr1 cells by depriving them of the factors promoting their generation and activity or by down-regulating 3′5′-cAMP levels. Thus, the pharmacologic control of Treg-Teff interactions offers a novel strategy for restoration of anti-tumor Teff functions and silencing of Treg. Used in conjunction with anti-cancer drugs or with immune therapies, this strategy has a potential to improve therapeutic effects by preventing or reversing tumor-induced immune suppression.
PMCID: PMC3721332  PMID: 22087822
Adenosine; anti-tumor immunity; effector T cells (Teff); pharmacologic inhibitors; prostaglandin E2 (PGE2); regulatory T cells (Treg)
7.  Evaluation of neuronal phosphoproteins as effectors of caffeine and mediators of striatal adenosine A2A receptor signaling 
Brain research  2006;1129(1):1-14.
Adenosine A2A receptors are predominantly expressed in the dendrites of enkephalin-positive γ-aminobutyric acidergic medium spiny neurons in the striatum. Evidence indicates that these receptors modulate striatal dopaminergic neurotransmission and regulate motor control, vigilance, alertness, and arousal. Although the physiological and behavioral correlates of adenosine A2A receptor signaling have been extensively studied using a combination of pharmacological and genetic tools, relatively little is known about the signal transduction pathways that mediate the diverse biological functions attributed to this adenosine receptor subtype. Using a candidate approach based on the coupling of these receptors to adenylate cyclase-activating G proteins, a number of membranal, cytosolic, and nuclear phosphoproteins regulated by PKA were evaluated as potential mediators of adenosine A2A receptor signaling in the striatum. Specifically, the adenosine A2A receptor agonist, CGS 21680, was used to determine whether the phosphorylation state of each of the following PKA targets is responsive to adenosine A2A receptor stimulation in this tissue: Ser40 of tyrosine hydroxylase, Ser9 of synapsin, Ser897 of the NR1 subunit of the N-methyl-D-aspartate-type glutamate receptor, Ser845 of the GluR1 subunit of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid-type glutamate receptor, Ser94 of spinophilin, Thr34 of the dopamine- and cAMP-regulated phosphoprotein, Mr32,000, Ser133 of the cAMP-response element-binding protein, Thr286 of Ca2+/calmodulin-dependent protein kinase II, and Thr202/Tyr204 and Thr183/Tyr185 of the p44 and p42 isoforms, respectively, of mitogen-activated protein kinase. Although the substrates studied differed considerably in their responsiveness to selective adenosine A2A receptor activation, the phosphorylation state of all postsynaptic PKA targets was up-regulated in a time- and dose-dependent manner by treatment with CGS 21680, whereas presynaptic PKA substrates were unresponsive to this agent, consistent with the postsynaptic localization of adenosine A2A receptors. Finally, the phosphorylation state of these proteins was further assessed in vivo by systemic administration of caffeine.
Section: Cellular and Molecular Biology of Nervous Systems
PMCID: PMC1847645  PMID: 17157277
Caffeine; Striatum; Adenosine A2A receptor PKA; MAPK; Signal transduction
8.  Involvement of Peripheral Adenosine A2 Receptors in Adenosine A1 Receptor–Mediated Recovery of Respiratory Motor Function After Upper Cervical Spinal Cord Hemisection 
In an animal model of spinal cord injury, a latent respiratory motor pathway can be pharmacologically activated through central adenosine A1 receptor antagonism to restore respiratory function after cervical (C2) spinal cord hemisection that paralyzes the hemidiaphragm ipsilateral to injury. Although respiration is modulated by central and peripheral mechanisms, putative involvement of peripheral adenosine A2 receptors in functional recovery in our model is untested. The objective of this study was to assess the effects of peripherally located adenosine A2 receptors on recovery of respiratory function after cervical (C2) spinal cord hemisection.
Respiratory activity was electrophysiologically assessed (under standardized recording conditions) in C2-hemisected adult rats with the carotid bodies intact (H-CBI; n =12) or excised (H-CBE; n =12). Animals were administered the adenosine A2 receptor agonist, CGS-21680, followed by the A1 receptor antagonist, 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX), or administered DPCPX alone. Recovered respiratory activity, characterized as drug-induced activity in the previously quiescent left phrenic nerve of C2-hemisected animals in H-CBI and H-CBE rats, was compared. Recovered respiratory activity was calculated by dividing drug-induced activity in the left phrenic nerve by activity in the right phrenic nerve.
Administration of CGS-21680 before DPCPX (n = 6) in H-CBI rats induced a significantly greater recovery (58.5 ± 3.6%) than when DPCPX (42.6 ± 4.6%) was administered (n = 6) alone. In H-CBE rats, prior administration of CGS-21680 (n = 6) did not enhance recovery over that induced by DPCPX (n = 6) alone. Recovery in H-CBE rats amounted to 39.7 ± 3.7% and 38.4 + 4.2%, respectively.
Our results suggest that adenosine A2 receptors located in the carotid bodies can enhance the magnitude of adenosine A1 receptor–mediated recovery of respiratory function after C2 hemisection. We conclude that a novel approach of targeting peripheral and central adenosine receptors can be therapeutically beneficial in alleviating compromised respiratory function after cervical spinal cord injury.
PMCID: PMC1864794  PMID: 16572566
Cervical spinal cord hemisection; Carotid bodies; Adenosine A1 and A2 receptors; Respiratory function
9.  2012 Philip S. Portoghese Medicinal Chemistry Lectureship: Structure-Based Approaches to Ligands for G Protein-Coupled Adenosine and P2Y Receptors, From Small Molecules to Nanoconjugates +  
Journal of medicinal chemistry  2013;56(10):3749-3767.
Adenosine receptor (ARs) and P2Y receptors (P2YRs) that respond to extracellular nucleosides/tides are associated with new directions for therapeutics. The X-ray structures of the A 2A AR complexes with agonists and antagonists are examined in relationship to the G protein-coupled receptor (GPCR) superfamily and applied to drug discovery. Much of the data on AR ligand structure from early SAR studies, now is explainable from the A 2A AR X-ray crystallography. The ligand-receptor interactions in related GPCR complexes can be identified by means of modeling approaches, e.g. molecular docking. Thus, molecular recognition in binding and activation processes has been studied effectively using homology modeling and applied to ligand design. Virtual screening has yielded new nonnucleoside AR antagonists, and existing ligands have been improved with knowledge of the receptor interactions. New agonists are being explored for CNS and peripheral therapeutics based on in vivo activity, such as chronic neuropathic pain. Ligands for receptors more distantly related to the X-ray template, i.e. P2YRs, have been introduced and are mainly used as pharmacological tools for elucidating the physiological role of extracellular nucleotides. Other ligand tools for drug discovery include fluorescent probes, radioactive probes, multivalent probes, and functionalized nanoparticles.
PMCID: PMC3701956  PMID: 23597047
G protein-coupled receptor ;  purines ;  molecular modeling ;  adenosine receptor ;  P2Y receptor
10.  Homeostatic Control of Synaptic Activity by Endogenous Adenosine is Mediated by Adenosine Kinase 
Cerebral Cortex (New York, NY)  2012;24(1):67-80.
Extracellular adenosine, a key regulator of neuronal excitability, is metabolized by astrocyte-based enzyme adenosine kinase (ADK). We hypothesized that ADK might be an upstream regulator of adenosine-based homeostatic brain functions by simultaneously affecting several downstream pathways. We therefore studied the relationship between ADK expression, levels of extracellular adenosine, synaptic transmission, intrinsic excitability, and brain-derived neurotrophic factor (BDNF)-dependent synaptic actions in transgenic mice underexpressing or overexpressing ADK. We demonstrate that ADK: 1) Critically influences the basal tone of adenosine, evaluated by microelectrode adenosine biosensors, and its release following stimulation; 2) determines the degree of tonic adenosine-dependent synaptic inhibition, which correlates with differential plasticity at hippocampal synapses with low release probability; 3) modulates the age-dependent effects of BDNF on hippocampal synaptic transmission, an action dependent upon co-activation of adenosine A2A receptors; and 4) influences GABAA receptor-mediated currents in CA3 pyramidal neurons. We conclude that ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. These mechanistic studies support previous therapeutic studies and implicate ADK as a promising therapeutic target for upstream control of multiple neuronal signaling pathways crucial for a variety of neurological disorders.
PMCID: PMC3862265  PMID: 22997174
adenosine; brain-derived neurotrophic factor; GABA; homeostasis; transgenic mice
11.  The Reno-Vascular A2B Adenosine Receptor Protects the Kidney from Ischemia 
PLoS Medicine  2008;5(6):e137.
Acute renal failure from ischemia significantly contributes to morbidity and mortality in clinical settings, and strategies to improve renal resistance to ischemia are urgently needed. Here, we identified a novel pathway of renal protection from ischemia using ischemic preconditioning (IP).
Methods and Findings
For this purpose, we utilized a recently developed model of renal ischemia and IP via a hanging weight system that allows repeated and atraumatic occlusion of the renal artery in mice, followed by measurements of specific parameters or renal functions. Studies in gene-targeted mice for each individual adenosine receptor (AR) confirmed renal protection by IP in A1−/−, A2A−/−, or A3AR−/− mice. In contrast, protection from ischemia was abolished in A2BAR−/− mice. This protection was associated with corresponding changes in tissue inflammation and nitric oxide production. In accordance, the A2BAR-antagonist PSB1115 blocked renal protection by IP, while treatment with the selective A2BAR-agonist BAY 60–6583 dramatically improved renal function and histology following ischemia alone. Using an A2BAR-reporter model, we found exclusive expression of A2BARs within the reno-vasculature. Studies using A2BAR bone-marrow chimera conferred kidney protection selectively to renal A2BARs.
These results identify the A2BAR as a novel therapeutic target for providing potent protection from renal ischemia.
Using gene-targeted mice, Holger Eltzschig and colleagues identify the A2B adenosine receptor as a novel therapeutic target for providing protection from renal ischemia.
Editors' Summary
Throughout life, the kidneys perform the essential task of filtering waste products and excess water from the blood to make urine. Each kidney contains about a million small structures called nephrons, each of which contains a filtration unit consisting of a glomerulus (a small blood vessel) intertwined with a urine-collecting tube called a tubule. If the nephrons stop working for any reason, the rate at which the blood is filtered (the glomerular filtration rate or GFR) decreases and dangerous amounts of waste products such as creatinine build up in the blood. Most kidney diseases destroy the nephrons slowly over years, producing an irreversible condition called chronic renal failure. But the kidneys can also stop working suddenly because of injury or poisoning. One common cause of “acute” renal failure in hospital patients is ischemia—an inadequate blood supply to an organ that results in the death of part of that organ. Heart surgery and other types of surgery in which the blood supply to the kidneys is temporarily disrupted are associated with high rates of acute renal failure.
Why Was This Study Done?
Although the kidneys usually recover from acute failure within a few weeks if the appropriate intensive treatment (for example, dialysis) is provided, acute renal failure after surgery can be fatal. Thus, new strategies to protect the kidneys from ischemia are badly needed. Like other organs, the kidneys can be protected from lethal ischemia by pre-exposure to several short, nonlethal episodes of ischemia. It is not clear how this “ischemic preconditioning” increases renal resistance to ischemia but some data suggest that the protection of tissues from ischemia might involve a signaling molecule called extracellular adenosine. This molecule binds to proteins called receptors on the surface of cells and sends signals into them that change their behavior. There are four different adenosine receptor—A1AR, A2AAR, A2BAR, and A3AR—and in this study, the researchers use ischemic preconditioning as an experimental strategy to investigate which of these receptors protects the kidneys from ischemia in mice, information that might provide clues about how to protect the kidneys from ischemia.
What Did the Researchers Do and Find?
The researchers first asked whether ischemic preconditioning protects the kidneys of mice strains that lack the genes for individual adenosine receptors (A1AR−/−, A2AAR−/−, A2BAR−/−, and A3AR−/− mice) from subsequent ischemia. Using a hanging-weight system, they intermittently blocked the renal artery of these mice before exposing them to a longer period of renal ischemia. Twenty-four hours later, they assessed the renal function of the mice by measuring their blood creatinine levels, GFRs, and urine production. Ischemic preconditioning protected all the mice from ischemia-induced loss of kidney function except the A2BAR−/− mice. It also prevented ischemia-induced structural damage and inflammation in the kidneys of wild-type but not A2BAR−/− mice. These results suggest that A2BAR may help to protect the kidneys from ischemia. Consistent with this idea, ischemic preconditioning did not prevent ischemia-induced renal damage in wild-type mice treated with a compound that specifically blocks the activity of A2BAR. However, wild-type mice (but not A2BAR−/− mice) treated with an A2BAR agonist (which activates the receptor) retained their kidney function after renal ischemia without ischemic preconditioning. Finally, the researchers report that A2BAR has to be present on the blood vessels in the kidney to prevent ischemia-induced acute renal failure.
What Do These Findings Mean?
These findings suggest that the protection of the kidneys from ischemia and the renal resistance to ischemia that is provided by ischemic preconditioning involve adenosine signaling through A2BAR. They also suggest that adenosine might provide protection against ischemia-induced damage by blocking inflammation in the kidney although other possible mechanisms of action need to be investigated. Importantly, these findings suggest that A2BAR might be a therapeutic target for the prevention of renal ischemia. However, results obtained in animals do not always reflect the situation in people, so before A2BAR agonists can be used to reduce the chances of patients developing acute renal failure after surgery, these results need confirming in people and the safety of A2BAR agonists need to be thoroughly investigated.
Additional Information.
Please access these Web sites via the online version of this summary at
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on how the kidneys work and what can go wrong with them, including a list of links to further information about kidney disease
The MedlinePlus encyclopedia has a page on acute kidney failure (in English and Spanish)
Wikipedia has pages on acute renal failure, ischemia, ischemic preconditioning, and adenosine (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2504049  PMID: 18578565
12.  Adenosine and Ischemic Preconditioning 
Current pharmaceutical design  1999;5(12):1029-1041.
Adenosine is released in large amounts during myocardial ischemia and is capable of exerting potent cardioprotective effects in the heart. Although these observations on adenosine have been known for a long time, how adenosine acts to achieve its anti-ischemic effect remains incompletely understood. However, recent advances on the chemistry and pharmacology of adenosine receptor ligands have provided important and novel information on the function of adenosine receptor subtypes in the cardiovascular system. The development of model systems for the cardiac actions of adenosine has yielded important insights into its mechanism of action and have begun to elucidate the sequence of signalling events from receptor activation to the actual exertion of its cardioprotective effect. The present review will focus on the adenosine receptors that mediate the potent anti-ischemic effect of adenosine, new ligands at the receptors, potential molecular signalling mechanisms downstream of the receptor, mediators for cardioprotection, and possible clinical applications in cardiovascular disorders.
PMCID: PMC3561763  PMID: 10607860
13.  Anti-inflammatory effects of adenosine N1-oxide 
Adenosine is a potent endogenous anti-inflammatory and immunoregulatory molecule. Despite its promise, adenosine’s extremely short half-life in blood limits its clinical application. Here, we examined adenosine N1-oxide (ANO), which is found in royal jelly. ANO is an oxidized product of adenosine at the N1 position of the adenine base moiety. We found that it is refractory to adenosine deaminase-mediated conversion to inosine. We further examined the anti-inflammatory activities of ANO in vitro and in vivo.
The effect of ANO on pro-inflammatory cytokine secretion was examined in mouse peritoneal macrophages and the human monocytic cell line THP-1, and compared with that of adenosine, synthetic adenosine receptor (AR)-selective agonists and dipotassium glycyrrhizate (GK2). The anti-inflammatory activity of ANO in vivo was examined in an LPS-induced endotoxin shock model in mice.
ANO inhibited secretion of inflammatory mediators at much lower concentrations than adenosine and GK2 when used with peritoneal macrophages and THP-1 cells that were stimulated by LPS plus IFN-γ. The potent anti-inflammatory activity of ANO could not be solely accounted for by its refractoriness to adenosine deaminase. ANO was superior to the synthetic A1 AR-selective agonist, 2-chloro-N6-cyclopentyladenosine (CCPA), A2A AR-selective agonist, 2-[p-(2-carboxyethyl)phenethylamino]-5’-N-ethylcarboxamideadenosine hydrochloride (CGS21680), and A3 AR-selective agonist, N6-(3-iodobenzyl)adenosine-5’-N-methyluronamide (IB-MECA), in suppressing the secretion of a broad spectrum of pro-inflammatory cytokines by peritoneal macrophages. The capacities of ANO to inhibit pro-inflammatory cytokine production by THP-1 cells were comparable with those of CCPA and IB-MECA. Reflecting its potent anti-inflammatory effects in vitro, intravenous administration of ANO significantly reduced lethality of LPS-induced endotoxin shock. A significant increase in survival rate was also observed by oral administration of ANO. Mechanistic analysis suggested that the up-regulation of the anti-inflammatory transcription factor c-Fos was, at least in part, involved in the ANO-induced suppression of pro-inflammatory cytokine secretion.
Our data suggest that ANO, a naturally occurring molecule that is structurally close to adenosine but is functionally more potent, presents potential strategies for the treatment of inflammatory disorders.
PMCID: PMC4308844  PMID: 25632271
Adenosine; Anti-inflammatory effect; Pro-inflammatory cytokines; Adenosine receptor agonists; Endotoxin shock
14.  Allosteric Modulation of A3 Adenosine Receptors by a Series of 3-(2-Pyridinyl)isoquinoline Derivatives 
Molecular pharmacology  2001;60(5):1057-1063.
Allosteric modulators of A1 and A2A adenosine receptors have been described; however, for the A3 adenosine receptor, neither an allosteric site nor a compound with allosteric effects has been described. In this study, the allosteric modulation of human A3 adenosine receptors by a series of 3-(2-pyridinyl)isoquinoline derivatives was investigated by examining their effects on the dissociation of the agonist radioligand, [125I]N6-(4-amino-3-iodobenzyl)-5′ -N-methylcarboxamidoadenosine (I-AB-MECA), from the receptor. Several 3-(2-pyridinyl)isoquinoline derivatives, including VUF5455, VUF8502, VUF8504, and VUF8507, slowed the dissociation of the agonist radioligand [125I]I-AB-MECA in a concentration-dependent manner, suggesting an allosteric interaction. These compounds had no effect on the dissociation of the radiolabeled antagonist [3H]PSB-11 from the A3 adenosine receptor, suggesting a selective enhancement of agonist binding. By comparison, compounds of similar structure (VUF8501, VUF8503, VUF8505), the classical adenosine receptor antagonist CGS15943 and the A1 receptor allosteric enhancer PD81723 did not significantly influence the dissociation rate of [125I]I-AB-MECA. The effect of agonist on forskolin-induced cAMP production was significantly enhanced by VUF5455. When the subtype-selectivity of the allosteric enhancement was tested the compounds had no effect on the dissociation of either [3H]N6-[(R)-phenylisopropyl]adenosine from the A1 adenosine receptor or [3H]CGS21680 from the A2A adenosine receptor. Probing of structure-activity relationships suggested that a carbonyl group is essential for allosterism but preferred only for competitive antagonism. The presence of a 7-methyl group decreased the competitive binding affinity without a major loss of the allosteric enhancing activity, suggesting that the structural requirements for allosteric enhancement might be distinct from those for competitive antagonism.
PMCID: PMC3953614  PMID: 11641434
15.  Kappa opioid antagonists: Past successes and future prospects 
The AAPS Journal  2005;7(3):E704-E722.
Antagonists of the kappa opioid receptor were initially investigated as pharmacological tools that would reverse the effects of kappa opioid receptor agonists. In the years following the discovery of the first selective kappa opioid antagonists, much information about their chemistry and pharmacology has been elicited and their potential therapeutic uses have been investigated. The review presents the current chemistry, ligand-based structure activity relationships, and pharmacology of the known nonpeptidic selective kappa opioid receptor antagonists. This manuscript endeavors to provide the reader with a useful reference of the investigations made to define the structure-activity relationships and pharmacology of selective kappa opioid receptor antagonists and their potential uses as pharmacological tools and as therapeutic agents in the treatment of disease states.
PMCID: PMC2751273  PMID: 16353947
Opioid; Opiate; Receptor; Kappa; Antagonist
16.  A role of p38 mitogen-activated protein kinase in adenosine A1 receptor-mediated synaptic depotentiation in area CA1 of the rat hippocampus 
Molecular Brain  2008;1:13.
Although long-term potentiation (LTP) of synaptic strength is very persistent, current studies have provided evidence that various manipulations or pharmacological treatment when applied shortly after LTP induction can reverse it. This kind of reversal of synaptic strength is termed as depotentiation and may have a function to increase the flexibility and storage capacity of neuronal networks. Our previous studies have demonstrated that an increase in extracellular levels of adenosine and subsequent activation of adenosine A1 receptors are important for the induction of depotentiation; however, the signaling downstream of adenosine A1 receptors to mediate depotentiation induction remains elusive.
We confirm that depotentiation induced by low-frequency stimulation (LFS) (2 Hz, 10 min, 1200 pulses) was dependent on adenosine A1 receptor activation, because it was mimicked by bath-applied adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and was inhibited by the selective adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Pretreatment of the hippocampal slices with the selective p38 mitogen-activated protein kinase (MAPK) inhibitors, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl]-5-(4-pyrudyl)-1H-imidazole (SB203580) or trans-1-(4-hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxypyrimidin-4-yl)imidazole (SB239063), prevented the induction of depotentiation by LFS and CPA. In agreement with electrophysiological observation, both LFS- and CPA-induced depotentiation are associated with an increase in p38 MAPK activation, which are blocked by DPCPX or SB203580 application.
These results suggest that activation of adenosine A1 receptor and in turn triggering p38 MAPK signaling may contribute to the LFS-induced depotentiation at hippocampal CA1 synapses.
PMCID: PMC2579284  PMID: 18947392
17.  Adenosine Receptors and the Heart: Role in Regulation of Coronary Blood Flow and Cardiac Electrophysiology 
Adenosine is an autacoid that plays a critical role in regulating cardiac function, including heart rate, contractility, and coronary flow. In this chapter, current knowledge of the functions and mechanisms of action of coronary flow regulation and electrophysiology will be discussed. Currently, there are four known adenosine receptor (AR) subtypes, namely A1, A2A, A2B, and A3. All four subtypes are known to regulate coronary flow. In general, A2AAR is the predominant receptor subtype responsible for coronary blood flow regulation, which dilates coronary arteries in both an endothelial-dependent and -independent manner. The roles of other ARs and their mechanisms of action will also be discussed. The increasing popularity of gene-modified models with targeted deletion or overexpression of a single AR subtype has helped to elucidate the roles of each receptor subtype. Combining pharmacologic tools with targeted gene deletion of individual AR subtypes has proven invaluable for discriminating the vascular effects unique to the activation of each AR subtype.
Adenosine exerts its cardiac electrophysiologic effects mainly through the activation of A1AR. This receptor mediates direct as well as indirect effects of adenosine (i.e., anti-β-adrenergic effects). In supraventricular tissues (atrial myocytes, sinua-trial node and atriovetricular node), adenosine exerts both direct and indirect effects, while it exerts only indirect effects in the ventricle. Adenosine exerts a negative chronotropic effect by suppressing the automaticity of cardiac pacemakers, and a negative dromotropic effect through inhibition of AV-nodal conduction. These effects of adenosine constitute the rationale for its use as a diagnostic and therapeutic agent. In recent years, efforts have been made to develop A1R-selective agonists as drug candidates that do not induce vasodilation, which is considered an undesirable effect in the clinical setting.
PMCID: PMC2913612  PMID: 19639282
A1 adenosine receptor; A2A adenosine receptor; A2B adenosine receptor; A3 adenosine receptor; Endothelium; Coronary artery; Smooth muscle; Adenosine receptor knockout; Phospholipase C; MAPK; Adenosine receptor agonist; Adenosine receptor antagonist; Sinus node; AV node; Cardiac electrophysiology; PSVT; Anti-beta adrenergic action
18.  Partial Agonists for A3 Adenosine Receptors 
Selective agonists for A3 adenosine receptors (ARs) could potentially be therapeutic agents for a variety of disorders, including brain and heart ischemic conditions, while partial agonists may have advantages over full agonists as a result of an increased selectivity of action. A number of structural determinants for A3AR activation have recently been identified, including the N6-benzyl group, methanocarba substitution of ribose, 2-chloro and 2-fluoro substituents, various 2’- and 3’-substitutions and 4’-thio substitution of oxygen. The 2-chloro substitution of CPA and R-PIA led to A3 antagonism (CCPA) and partial agonism (Cl-R-PIA). 2-Chloroadenosine was a full agonist, while 2-fluoroadenosine was a partial agonist. Both 2’- and 3’- substitutions have a pronounced effect on its efficacy, although the effect of 2’-substitution was more dramatic. The 4-thio substitution of oxygen may also diminish efficacy, depending on other substitutions. Both N6-methyl and N6-benzyl groups may contribute to the A3 affinity and selectivity; however, an N6-benzyl group but not an N6-methyl group diminishes A3AR efficacy. N6-benzyl substituted adenosine derivatives have similar potency for human and rat A3ARS while N6-methyl substitution was preferable for the human A3AR. The combination of 2-chloro and N6-benzyl substitutions appeared to reduce efficacy further than either modification alone. The A2AAR agonist DPMA was shown to be an antagonist for the human A3AR. Thus, the efficacy of adenosine derivatives at the A3AR appears to be more sensitive to small structural changes than at other subtypes. Potent and selective partial agonists for the A3AR could be identified by screening known adenosine derivatives and by modifying adenosine and the adenosine derivatives.
PMCID: PMC3425644  PMID: 15078216
19.  Positive allosteric modulation of the adenosine A2a receptor attenuates inflammation 
Adenosine is produced at high levels at inflamed sites as a by-product of cellular activation and breakdown. Adenosine mediates its anti-inflammatory activity primarily through the adenosine A2a receptor (A2aR), a member of the G-protein coupled receptors. A2aR agonists have demonstrated anti-inflammatory efficacy, however, their therapeutic utility is hindered by a lack of adenosine receptor subtype selectivity upon systemic exposure. We sought to harness the anti-inflammatory effects of adenosine by enhancing the responsiveness of A2aR to endogenously produced adenosine through allosteric modulation. We have identified a family of positive allosteric modulators (PAMs) of the A2aR. Using one member of this PAM family, AEA061, we demonstrate that A2aRs are amenable to allosteric enhancement and such enhancement produces increased A2aR signaling and diminished inflammation in vivo.
A2aR activity was evaluated using a cell-based cAMP assay. Binding affinity of A2aR was determined using [3H]CGS 21680. A2aR-mediated G-protein activation was quantified using [35S]GTP-γS. The effect of AEA061 on cytokine production was evaluated using primary monocytes and splenocytes. The anti-inflammatory effect of AEA061 was evaluated in the LPS-induced mouse model of inflammation.
AEA061 had no detectable intrinsic agonist activity towards either rat or human A2aRs. AEA061 enhanced the efficacy of adenosine to rat and human A2aRs by 11.5 and 2.8 fold respectively. AEA061 also enhanced the maximal response by 4.2 and 2.1 fold for the rat and the human A2aR respectively. AEA061 potentiated agonist-mediated Gα activation by 3.7 fold. Additionally, AEA061 enhanced both the affinity as well as the Bmax at the human A2aR by 1.8 and 3 fold respectively. Consistent with the anti-inflammatory role of the A2aR, allosteric enhancement with AEA061 inhibited the production of TNF-α, MIP-1α, MIP-1β, MIP-2, IL-1α, KC and RANTES by LPS-stimulated macrophages and/or splenocytes. Moreover, AEA061 reduced circulating plasma TNF-α and MCP-1 levels and increased plasma IL-10 in endotoxemic A2aR intact, but not in A2aR deficient, mice.
AEA061 increases affinity and Bmax of A2aR to adenosine, thereby increasing adenosine potency and efficacy, which translates to enhanced A2aR responsiveness. Since the A2aR negatively regulates inflammation, PAMs of the receptor offer a novel means of modulating inflammatory processes.
PMCID: PMC4253011  PMID: 25473378
Adenosine A2a receptor; G protein-couple receptor; Allosteric enhancement; Inflammation; Positive allosteric modulator; Inhibition of TNF-α production
20.  Internalization and desensitization of adenosine receptors 
Purinergic Signalling  2007;4(1):21-37.
Until now, more than 800 distinct G protein-coupled receptors (GPCRs) have been identified in the human genome. The four subtypes of the adenosine receptor (A1, A2A, A2B and A3 receptor) belong to this large family of GPCRs that represent the most widely targeted pharmacological protein class. Since adenosine receptors are widespread throughout the body and involved in a variety of physiological processes and diseases, there is great interest in understanding how the different subtypes are regulated, as a basis for designing therapeutic drugs that either avoid or make use of this regulation. The major GPCR regulatory pathway involves phosphorylation of activated receptors by G protein-coupled receptor kinases (GRKs), a process that is followed by binding of arrestin proteins. This prevents receptors from activating downstream heterotrimeric G protein pathways, but at the same time allows activation of arrestin-dependent signalling pathways. Upon agonist treatment, adenosine receptor subtypes are differently regulated. For instance, the A1Rs are not (readily) phosphorylated and internalize slowly, showing a typical half-life of several hours, whereas the A2AR and A2BR undergo much faster downregulation, usually shorter than 1 h. The A3R is subject to even faster downregulation, often a matter of minutes. The fast desensitization of the A3R after agonist exposure may be therapeutically equivalent to antagonist occupancy of the receptor. This review describes the process of desensitization and internalization of the different adenosine subtypes in cell systems, tissues and in vivo studies. In addition, molecular mechanisms involved in adenosine receptor desensitization are discussed.
PMCID: PMC2245999  PMID: 18368531
Adenosine receptors; β-arrestins; Caveolae; Desensitization; G protein-coupled receptor kinase; Lipid rafts; Internalization; Palmitoylation; Phosphorylation
21.  Introduction to Adenosine Receptors as Therapeutic Targets 
Adenosine acts as a cytoprotective modulator in response to stress to an organ or tissue. Although short-lived in the circulation, it can activate four sub-types of G protein-coupled adenosine receptors (ARs): A1, A2A, A2B, and A3. The alkylxanthines caffeine and theophylline are the prototypical antagonists of ARs, and their stimulant actions occur primarily through this mechanism. For each of the four AR subtypes, selective agonists and antagonists have been introduced and used to develop new therapeutic drug concepts. ARs are notable among the GPCR family in the number and variety of agonist therapeutic candidates that have been proposed. The selective and potent synthetic AR agonists, which are typically much longer lasting in the body than adenosine, have potential therapeutic applications based on their anti-inflammatory (A2A and A3), cardioprotective (preconditioning by A1 and A3 and postconditioning by A2B), cerebroprotective (A1 and A3), and antinociceptive (A1) properties. Potent and selective AR antagonists display therapeutic potential as kidney protective (A1), antifibrotic (A2A), neuroprotective (A2A), and antiglaucoma (A3) agents. AR agonists for cardiac imaging and positron-emitting AR antagonists are in development for diagnostic applications. Allosteric modulators of A1 and A3 ARs have been described. In addition to the use of selective agonists/antagonists as pharmacological tools, mouse strains in which an AR has been genetically deleted have aided in developing novel drug concepts based on the modulation of ARs.
PMCID: PMC3415694  PMID: 19639277
Adenosine receptors; G protein-coupled receptors; Purines; Nucleosides; Imaging; Allosteric modulation; Agonists; Antagonists
22.  Investigational A3 adenosine receptor targeting agents 
Adenosine is an endogenous nucleoside that accumulates in the extracellular space in response to metabolic stress and cell damage. Extracellular adenosine is a signaling molecule and it signals by activating four G-protein coupled receptors: the A1, A2A, A2B and A3 receptors. Since the discovery of A3 adenosine receptors accumulating evidence has identified these receptors as potential targets for therapeutic intervention.
Areas covered
A3 adenosine receptors are expressed on the surface of most immune cell types, including neutrophils, macrophages, dendritic cells, lymphocytes and mast cells. A3 adenosine receptor activation on immune cells governs a broad array of immune cell functions, which include cytokine production, degranulation, chemotaxis, cytotoxicity, apoptosis, and proliferation. In accordance with their multitudinous immunoregulatory actions, targeting A3 adenosine receptors has been shown to impact the course of a wide spectrum of immune-related diseases, such as asthma, rheumatoid arthritis, cancer, ischaemia, and inflammatory disorders.
Expert opinion
Given the existence of both pre-clinical and early clinical data supporting the utility of A3 adenosine receptor ligands in treating immune-related diseases, further development of A3 adenosine receptor ligands is anticipated.
PMCID: PMC3613226  PMID: 21457061
23.  Enhanced Actions of Adenosine in Medial Entorhinal Cortex Layer II Stellate Neurons in Temporal Lobe Epilepsy are Mediated via A1 Receptor Activation 
Epilepsia  2011;53(1):168-176.
The adenosinergic system is known to exert an inhibitory affect in the brain and as such adenosine has been considered an endogenous anticonvulsant. Entorhinal cortex (EC) layer II neurons, which serve as the primary input to the hippocampus, are spared in temporal lobe epilepsy (TLE) and become hyperexcitable. Since these neurons also express adenosine receptors, the activity of these neurons may be controlled by adenosine, specifically during seizure activity when adenosine levels are thought to rise. In light of this, we determined if the actions of adenosine on medial EC (mEC) layer II stellate neurons are augmented in TLE and by which receptor subtype.
Horizontal brain slices were prepared from rats exhibiting spontaneous seizures (TLE) induced by electrical stimulation and compared with age matched control rats. mEC layer II stellate neurons were visually identified and action potentials (AP) evoked by either a series of depolarizing current injection steps or via presynaptic stimulation of mEC deep layers. The effects of adenosine were compared with actions of adenosine A1 and A2A receptor-specific agonists (CPA and CGS 21680) and antagonists (DPCPX and ZM241385) respectively. Immunohistochemical and qPCR techniques were also employed to assess relative adenosine A1 receptor message and expression.
Key Findings
mEC layer II stellate neurons were hyper-excitable in TLE, evoking a higher frequency of AP's when depolarized and generating bursts of AP's when synaptically stimulated. Adenosine reduced AP frequency and synaptically evoked AP's in a dose dependent manner (500 nM – 100 μM); however, in TLE, the inhibitory actions of adenosine occurred at concentrations that were without affect in control neurons. In both cases, the inhibitory actions of adenosine were mediated via activation of the A1 and not the A2A receptor subtype. qPCR and immunohistochemical experiments revealed an up-regulation of the adenosine A1 mRNA and an increase in A1 receptor staining in TLE neurons compared to control.
Our data indicates the actions of adenosine on mEC layer II stellate neurons is accentuated in TLE due to an up-regulation of adenosine A1 receptors. Since adenosine levels are thought to rise during seizure activity, activation of adenosine A1 receptors could provide a possible endogenous mechanism to suppress seizure activity and spread within the temporal lobe.
PMCID: PMC3253213  PMID: 22126400
Adenosine; Temporal Lobe Epilepsy; Entorhinal Cortex; Action Potentials; A1 receptor
24.  Adenosine and blood platelets 
Purinergic Signalling  2011;7(3):357-365.
Adenosine is an important regulatory metabolite and an inhibitor of platelet activation. Adenosine released from different cells or generated through the activity of cell-surface ectoenzymes exerts its effects through the binding of four different G-protein-coupled adenosine receptors. In platelets, binding of A2 subtypes (A2A or A2B) leads to consequent elevation of intracellular cyclic adenosine monophosphate, an inhibitor of platelet activation. The significance of this ligand and its receptors for platelet activation is addressed in this review, including how adenosine metabolism and its A2 subtype receptors impact the expression and activity of adenosine diphosphate receptors. The expression of A2 adenosine receptors is induced by conditions such as oxidative stress, a hallmark of aging. The effect of adenosine receptors on platelet activation during aging is also discussed, as well as potential therapeutic applications.
PMCID: PMC3166992  PMID: 21484090
Adenosine; A2B adenosine receptor; A2A adenosine receptor; ADP-mediated platelet activation and aggregation; Cyclic adenosine monophosphate (cAMP)
25.  Structure–Activity Relationships and Molecular Modeling of 3,5-Diacyl-2,4-dialkylpyridine Derivatives as Selective A3 Adenosine Receptor Antagonists 
Journal of medicinal chemistry  1998;41(17):3186-3201.
The structure-activity relationships of 6-phenyl-1,4-dihydropyridine derivatives as selective antagonists at human A3 adenosine receptors have been explored (Jiang et al. J. Med. Chem. 1997, 39, 4667-4675). In the present study, related pyridine derivatives have been synthesized and tested for affinity at adenosine receptors in radioligand binding assays. Ki values in the nanomolar range were observed for certain 3,5-diacyl-2,4-dialkyl-6-phenylpyridine derivatives in displacement of [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5′-N-methylcarbamoyladenosine) at recombinant human A3 adenosine receptors. Selectivity for A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure–activity relationships at various positions of the pyridine ring (the 3- and 5-acyl substituents and the 2- and 4-alkyl substituents) were probed. A 4-phenylethynyl group did not enhance A3 selectivity of pyridine derivatives, as it did for the 4-substituted dihydropyridines. At the 2-and 4-positions ethyl was favored over methyl. Also, unlike the dihydropyridines, a thioester group at the 3-position was favored over an ester for affinity at A3 adenosine receptors, and a 5-position benzyl ester decreased affinity. Small cycloalkyl groups at the 6-position of 4-phenylethynyl-1,4-dihydropyridines were favorable for high affinity at human A3 adenosine receptors, while in the pyridine series a 6-cyclopentyl group decreased affinity. 5-Ethyl 2,4-diethyl-3-(ethylsulfanylcarbonyl)-6-phenylpyridine-5-carboxylate, 38, was highly potent at human A3 receptors, with a Ki value of 20 nM. A 4-propyl derivative, 39b, was selective and highly potent at both human and rat A3 receptors, with Ki values of 18.9 and 113 nM, respectively. A 6-(3-chlorophenyl) derivative, 44, displayed a Ki value of 7.94 nM at human A3 receptors and selectivity of 5200-fold. Molecular modeling, based on the steric and electrostatic alignment (SEAL) method, defined common pharmacophore elements for pyridine and dihydropyridine structures, e.g., the two ester groups and the 6-phenyl group. Moreover, a relationship between affinity and hydrophobicity was found for the pyridines.
PMCID: PMC3474377  PMID: 9703464

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