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1.  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
2.  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
3.  Partial adenosine A1 receptor agonists for cardiovascular therapies 
Purinergic Signalling  2011;8(Suppl 1):91-99.
Adenosine, a purine nucleoside, is present in all cells in tightly regulated concentrations. It has many different physiological effects in the whole body and in the heart. Adenosine activates four G protein-coupled receptors A1, A2a, A2b, and A3. Activation of myocardial A1 receptors has been shown to inhibit a variety of myocardial pathologies associated with ischemia and reperfusion injury, including stunning, arrhythmogenesis, coronary and ventricular dysfunction, acute myocardial infarction, apoptosis, and chronic heart failure, implying several options for new cardiovascular therapies for diseases, like angina pectoris, control of cardiac rhythm, ischemic injury during an acute coronary syndrome, or heart failure. However, the main issue of using full A1 receptor agonists in such indications is the broad physiologic spectrum of cardiac and extracardiac effects. Desired A1 receptor-mediated protective and regenerative cardiovascular effects might be counter-regulated by unintended side effects when considering full A1 receptor agonists. These effects can be overcome by partial A1 agonists. Partial A1 agonists can be used to trigger only some of the physiological responses of receptor activation depending on endogenous adenosine levels and on receptor reserve in different tissues. CV-Therapeutics reported the identification of a partial A1 receptor agonist CVT-3619, and recently, another partial A1 receptor agonist VCP28 was published. Both compounds are adenosine derivatives. Adenosine-like A1 receptor agonists often have the drawback of a short half-life and low bioavailability, making them not suitable for chronic oral therapy. We identified the first non-adenosine-like partial A1 receptor agonist(s) with pharmacokinetics optimal for oral once daily treatment and characterized the qualities of the partial character of the A1 receptor agonist(s) in preclinical and clinical studies.
PMCID: PMC3265704  PMID: 22081230
Adenosine A1 receptor; Partial adenosine A1 receptor agonists; Cardiovascular diseases
4.  Protection against ischemic damage by adenosine amine congener, a potent and selective adenosine A1 receptor agonist 
European journal of pharmacology  1999;369(3):313-317.
Although the selectivity and potency of adenosine amine congener (ADAC) at adenosine A1receptors are similar to other highly selective agonists at this receptor type, the chemical structure of the N6 substituent is completely different. We now demonstrate that the characteristics of the therapeutic profile of ADAC are distinct from those observed during our previous studies of adenosine A1receptor agonist-mediated neuroprotection. Most significantly, chronic treatment with low microgram doses of ADAC (25–100 µg/kg) protects against both mortality and neuronal damage induced by 10 min bilateral carotid occlusion in gerbils. At higher chronic doses, the statistical significance of the protective effect is lost. Acute preischemic administration of the drug at 75–200 µg/kg also results in a statistically significant reduction of postischemic mortality and morbidity. These data indicate that, contrary to other adenosine A1 receptor agonists whose chronic administration enhances postocclusive brain damage, ADAC may be a promising agent in treatment of both acute (e.g., cerebral ischemia) and chronic (seizures) disorders of the central nervous system in which adenosine A1 receptors appear to be involved.
PMCID: PMC3438899  PMID: 10225368
Cerebral ischemia; Adenosine A1 receptor; Therapy; Gerbil
5.  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
6.  Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils 
European journal of pharmacology  1996;316(2-3):171-179.
Although adenosine receptor-based treatment of cerebral ischemia and other neurodegenerative disorders has been frequently advocated, cardiovascular side effects and an uncertain therapeutic time window of such treatment have constituted major obstacles to clinical implementation. Therefore, we have investigated the neuroprotective effects of the adenosine A1 receptor agonist adenosine amine congener (ADAC) injected after either 5 or 10 min ischemia at 100 μg/kg. When the drug was administered at either 6 or 12 h following 5 min forebrain ischemia, all animals were still alive on the 14th day after the occlusion. In both ADAC treated groups neuronal survival was approximately 85% vs. 50% in controls. Administration of a single dose of ADAC at times 15 min to 12 h after 10 min ischemia resulted in a significant improvement of survival in animals injected either at 15 or 30 min, or at 1, 2, or 3 h after the insult. In all 10 min ischemia groups, administration of ADAC resulted in a significant protection of neuronal morphology and preservation of microtubule associated protein 2 (MAP-2). However, postischemic Morris’ water maze tests revealed full preservation of spatial memory and learning ability in animals injected at 6 h. On the other hand, the performance of gerbils treated at 12 h postischemia was indistinguishable from that of the controls. Administration of ADAC at 100 μg/kg in non-ischemic animals did not result in bradycardia, hypotension, or hypothermia. The data indicate that when ADAC is used postischemically, the most optimal level of protection is obtained when drugs are given at 30 min to 6 h after the insult. Although the mechanisms involved in neuroprotective effects of adenosine A1 receptor agonists require further studies, the present results demonstrate the feasibility of their clinical applications.
PMCID: PMC3449162  PMID: 8982684
Ischemia; treatment; Adenosine; Memory; MAP2 (microtubule-associated protein 2); (Gerbil)
7.  Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry. Implications for drug addiction, sleep and pain 
Progress in neurobiology  2007;83(5):332-347.
Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of L-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where high intensity stimuli are prevalent.
PMCID: PMC2141681  PMID: 17532111
Adenosine A2A receptor; striatum; hypothalamus; peripheral nervous system; drug addiction; sleep; pain
8.  Treating lung inflammation with agonists of the adenosine A2A receptor: promises, problems and potential solutions 
British Journal of Pharmacology  2008;155(4):463-474.
Adenosine A2A receptor agonists may be important regulators of inflammation. Such conclusions have come from studies demonstrating that, (i) adenosine A2A agonists exhibit anti-inflammatory properties in vitro and in vivo, (ii) selective A2A antagonists enhance inflammation in vivo and, (iii) knock outs of this receptor aggravate inflammation in a wide variety of in vivo models. Inflammation is a hallmark of asthma and COPD and adenosine has long been suggested to be involved in disease pathology. Two recent publications, however, suggested that an inhaled adenosine A2A receptor agonist (GW328267X) did not affect either the early and late asthmatic response or symptoms associated with allergic rhinitis suggesting that the rationale for treating inflammation with an adenosine A2A receptor agonist may be incorrect. A barrier to fully investigating the role of adenosine A2A receptor agonists as anti-inflammatory agents in the lung is the side effect profile due to systemic exposure, even with inhalation. Unless strategies can be evolved to limit the systemic exposure of inhaled adenosine A2A receptor agonists, the promise of treating lung inflammation with such agents may never be fully explored. Using strategies similar to that devised to improve the therapeutic index of inhaled corticosteroids, UK371,104 was identified as a selective agonist of the adenosine A2A receptor that has a lung focus of pharmacological activity following delivery to the lung in a pre clinical in vivo model of lung function. Lung-focussed agents such as UK371,104 may be suitable for assessing the anti-inflammatory potential of inhaled adenosine A2A receptor agonists.
PMCID: PMC2579671  PMID: 18846036
lung inflammation; adenosine receptors; adenosine A2A receptor agonists; inhalation UK371,104
9.  The Role of Muscarinic Receptors in the Beneficial Effects of Adenosine against Myocardial Reperfusion Injury in Rats 
PLoS ONE  2011;6(11):e25618.
Adenosine, a catabolite of ATP, displays a wide variety of effects in the heart including regulation of cardiac response to myocardial ischemia and reperfusion injury. Nonetheless, the precise mechanism of adenosine-induced cardioprotection is still elusive. Isolated Sprague-Dawley rat hearts underwent 30 min global ischemia and 120 min reperfusion using a Langendorff apparatus. Both adenosine and acetylcholine treatment recovered the post-reperfusion cardiac function associated with adenosine and muscarinic receptors activation. Simultaneous administration of adenosine and acetylcholine failed to exert any additive protective effect, suggesting a shared mechanism between the two. Our data further revealed a cross-talk between the adenosine and acetylcholine receptor signaling in reperfused rat hearts. Interestingly, the selective M2 muscarinic acetylcholine receptor antagonist methoctramine significantly attenuated the cardioprotective effect of adenosine. In addition, treatment with adenosine upregulated the expression and the maximal binding capacity of muscarinic acetylcholine receptor, which were inhibited by the selective A1 adenosine receptor antagonist 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) and the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME). These data suggested a possible functional coupling between the adenosine and muscarinic receptors behind the observed cardioprotection. Furthermore, nitric oxide was found involved in triggering the response to each of the two receptor agonist. In summary, there may be a cross-talk between the adenosine and muscarinic receptors in ischemic/reperfused myocardium with nitric oxide synthase might serve as the distal converging point. In addition, adenosine contributes to the invigorating effect of adenosine on muscarinic receptor thereby prompting to regulation of cardiac function. These findings argue for a potentially novel mechanism behind the adenosine-mediated cardioprotection.
PMCID: PMC3208538  PMID: 22073139
10.  Oral antiplatelet therapy for atherothrombotic disease: overview of current and emerging treatment options 
Clinical presentations of atherothrombotic vascular disease, such as acute coronary syndromes, ischemic stroke or transient ischemic attack, and symptomatic peripheral arterial disease, are major causes of morbidity and mortality worldwide. Platelet activation and aggregation play a seminal role in the arterial thrombus formation that precipitates acute manifestations of atherothrombotic disease. As a result, antiplatelet therapy has become the cornerstone of therapy for the prevention and treatment of atherothrombotic disease. Dual antiplatelet therapy with aspirin and a P2Y12 adenosine diphosphate (ADP) receptor inhibitor, such as clopidogrel or prasugrel, is the current standard-of-care antiplatelet therapy in patients with acute coronary syndromes managed with an early invasive strategy. However, these agents are associated with several important clinical limitations, including significant residual risk for ischemic events, bleeding risk, and variability in the degree of platelet inhibition. The residual risk can be attributed to the fact that aspirin and P2Y12 inhibitors block only the thromboxane A2 and ADP platelet activation pathways but do not affect the other pathways that lead to thrombosis, such as the protease-activated receptor-1 pathway stimulated by thrombin, the most potent platelet agonist. Bleeding risk associated with aspirin and P2Y12 inhibitors can be explained by their inhibitory effects on the thromboxane A2 and ADP pathways, which are critical for protective hemostasis. Interpatient variability in the degree of platelet inhibition in response to antiplatelet therapy may have a genetic component and contribute to poor clinical outcomes. These considerations underscore the clinical need for therapies with a novel mechanism of action that may reduce ischemic events without increasing the bleeding risk.
PMCID: PMC3292409  PMID: 22393298
acute coronary syndromes; antiplatelet therapy; ADP; thromboxane A2; PAR-1; bleeding
11.  Adenosine and Stroke: Maximizing the Therapeutic Potential of Adenosine as a Prophylactic and Acute Neuroprotectant 
Current Neuropharmacology  2009;7(3):217-227.
Stroke is a leading cause of morbidity and mortality in the United States. Despite intensive research into the development of treatments that lessen the severity of cerebrovascular injury, no major therapies exist. Though the potential use of adenosine as a neuroprotective agent in the context of stroke has long been realized, there are currently no adenosine-based therapies for the treatment of cerebral ischemia and reperfusion. One of the major obstacles to developing adenosine-based therapies for the treatment of stroke is the prevalence of functional adenosine receptors outside the central nervous system. The activities of peripheral immune and vascular endothelial cells are particularly vulnerable to modulation via adenosine receptors. Many of the pathophysiological processes in stroke are a direct result of peripheral immune infiltration into the brain. Ischemic preconditioning, which can be induced by a number of stimuli, has emerged as a promising area of focus in the development of stroke therapeutics. Reprogramming of the brain and immune responses to adenosine signaling may be an underlying principle of tolerance to cerebral ischemia. Insight into the role of adenosine in various preconditioning paradigms may lead to new uses for adenosine as both an acute and prophylactic neuroprotectant.
PMCID: PMC2769005  PMID: 20190963
Adenosine; adenosine receptors; cerebral ischemia; neuroprotection; preconditioning; stroke; treatment.
Trends in pharmacological sciences  2009;30(10):528-535.
Asthma and chronic obstructive pulmonary disease (COPD) are pulmonary disorders characterized by various degrees of inflammation and tissue remodeling. Adenosine is a signaling molecule that is elevated in the lungs of patients with asthma and COPD. Adenosine elicits its actions by engaging cell surface adenosine receptors, and substantial preclinical evidence suggests that targeting these receptors will provide novel approaches for the treatment of asthma and COPD. Studies in animal models of airway disease suggest that there may be clinical benefit to the use of A1, A3 and A2B adenosine receptor antagonists in the treatment of features of asthma and/or COPD, while A2A agonists may also prove effective. Several adenosine receptor based pharmacologic agents have entered clinical development for the treatment of asthma and COPD; however, the studies have been limited and the efficacy of such approaches is not yet clear.
PMCID: PMC2778268  PMID: 19762093
respiratory diseases; COPD; asthma; fibrosis; G-Protein coupled receptors; inflammation
13.  Amelioration of Cerebral Ischemic Injury by a Synthetic Seco-nucleoside LMT497 
Experimental Neurobiology  2015;24(1):31-40.
Recently, we reported that the A3 adenosine receptor (A3AR) agonist LJ529 (2-chloro-N6-(3-iodobnzyl)-5'-N-methylcarbamoyl-4'-thioadenosine) reduces cerebral ischemic injury via inhibition of recruitment of peripheral inflammatory cells into ischemic brain lesion. A3AR agonists, however, are known to possess anti-platelet activity, which may deter the combination therapy with tissue plasminogen activator for the therapy of cerebral ischemic stroke. Thus, the present study investigates the neuroprotective/anti-ischemic effect of a synthetic seco-nucleoside, LMT497 ((S)-2-((R)-1-(2-chloro-6-(3-iodobenzylamino)-9H-purin-9-yl)-2-hydroxyethoxy)-3-hydroxy-N-methylpropanamide) with little anti-platelet activity. LMT497 neither showed A3AR binding activity nor anti-platelet activity. In our present study LMT497 significantly attenuated the injury/death of cortical neurons exposed to oxygen-glucose deprivation (OGD) followed by re-oxygenation (R). LMT497 significantly reduced the ascending cellular level of reactive oxygen species under ischemic conditions by increasing the superoxide dismutase (SOD) levels. LMT497 also inhibited the migration of microglia which mediates inflammatory responses in ischemia. In rats subjected to middle cerebral artery occlusion (MCAO, 1.5 h) followed by reperfusion, LMT497 largely reduced brain infarction volume, and edema, and improved neurological score. Therapeutic efficacy of LMT497 was obtained by twice treatments even at 10 h and 18 h after the onset of ischemia. Collectively, LMT497 could be a therapeutic drug candidate with a wide therapeutic time window for the treatment of cerebral ischemic stroke.
PMCID: PMC4363332  PMID: 25792868
cerebral ischemia; MCAO; oxidative stress; inflammation; LMT497; Cl-IB-MECA
14.  A Novel Pharmacological Approach to Treating Cardiac Ischemia 
The Journal of biological chemistry  2000;275(39):30272-30279.
Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A1 or A3 receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerted a cardioprotective effect that was significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A1 and A3 receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A1 and A3 receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A1 or A3 receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A1 and A3 receptors (Ki values of 0.7–3.5 nm) versus A2A receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A1 and A3 receptors, or engineered atrial cells, in which either human A3 receptors alone or both human A1 and A3 receptors were expressed. The binary agonist MRS 1741 coactivated A1 and A3 receptors simultaneously, with full cardioprotection (EC50 ~0.1 nm) dependent on expression of both receptors. Thus, co-activation of both adenosine A1 and A3 receptors by the binary A1/A3 agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.
PMCID: PMC3561767  PMID: 10887176
15.  Novel allosteric agonists of M1 muscarinic acetylcholine receptors induce brain region-specific responses that correspond with behavioral effects in animal models 
The Journal of Neuroscience  2012;32(25):8532-8544.
M1 muscarinic acetylcholine receptors (mAChRs) represent a viable target for treatment of multiple disorders of the central nervous system (CNS) including Alzheimer’s disease and schizophrenia. The recent discovery of highly selective allosteric agonists of M1 receptors has provided a major breakthrough in developing a viable approach for discovery of novel therapeutic agents that target these receptors. Here, we describe the characterization of two novel M1 allosteric agonists VU0357017 and VU0364572 that display profound differences in their efficacy in activating M1 coupling to different signaling pathways including Ca++ and β-arrestin responses. Interestingly, the ability of these agents to differentially activate coupling of M1 to specific signaling pathways leads to selective actions on some but not all M1-mediated responses in brain circuits. These novel M1 allosteric agonists induced robust electrophysiological effects in rat hippocampal slices but showed lower efficacy in striatum and no measureable effects on M1-mediated responses in medial prefrontal cortical pyramidal cells in mice. Consistent with these actions, both M1 agonists enhanced acquisition of hippocampal-dependent cognitive function but did not reverse amphetamine-induced hyperlocomotion in rats. Together, these data reveal that M1 allosteric agonists can differentially regulate coupling of M1 to different signaling pathways and this can dramatically alter the actions of these compounds on specific brain circuits important for learning and memory and psychosis.
PMCID: PMC3398407  PMID: 22723693
16.  Activation of adenosine1 (A1) receptors suppresses head shakes induced by a serotonergic hallucinogen in rats 
Neuropharmacology  2009;56(8):1082-1087.
Modulation of glutamatergic neurotransmission by metabotropic glutamate2/3 (mGlu2/3) receptor agonists effectively treats seemingly diverse neuropsychiatric illness such as generalized anxiety disorder and schizophrenia. Activation of adenosine A1 heteroceptors, like mGlu2 autoreceptors, decreases glutamate release in the medial prefrontal cortex (mPFC) and other limbic brain regions. Previously, we have reported electrophysiological, neurochemical and behavioral evidence for interactions between the 5-hydroxytryptamine2A (5-HT2A) and mGlu2/3 receptors in the mPFC. The present studies were designed to investigate the effects in rats of adenosine A1 receptor activation/blockade on a behavior modulated by 5-HT2A receptor activation/blockade in the mPFC: head shakes induced in the rat by phenethylamine hallucinogens. An adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA) suppressed head shakes induced by activation of 5-HT2A receptors with the phenethylamine hallucinogen (±)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI). An adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), enhanced DOI-induced head shakes and blocked the suppressant action of an adenosine A1 receptor agonist on DOI-induced head shakes. Thus, the pattern of activity for an agonist and antagonist at the adenosine A1 receptor with respect to modulating DOI-induced head shakes is similar to the pattern observed with mGlu2/3 receptor agonists and antagonists. These novel observations with an adenosine A1 receptor agonist suggests that this pharmacological action could contribute to antipsychotic effects in addition to thymoleptic effects.
PMCID: PMC2706691  PMID: 19324062
head shakes; phenethylamine hallucinogens; DOI; adenosine; glutamate; medial prefrontal cortex
17.  Adenosine receptors and asthma 
British Journal of Pharmacology  2008;153(Suppl 1):S446-S456.
The accumulation of evidence implicating a role for adenosine in the pathogenesis of asthma has led to investigations into all adenosine receptor subtypes as potential therapeutic targets for the treatment of asthma. Selective A1 receptor antagonists are currently in preclinical development since adenosine has been shown experimentally to mediate various features of asthma through this receptor such as bronchoconstriction, mucus secretion and inflammation. The A2A receptor is expressed on most inflammatory cells implicated in asthma, and as A2A stimulation activates adenylate cyclase and consequently elevates cAMP, selective A2A receptor agonists have now reached clinical development. However, initial reports concerning their efficacy are inconclusive. A2B receptor antagonists are also under investigation based on the rationale that inhibiting the effects of adenosine on mast cells would be beneficial, in addition to other reported pro-inflammatory effects mediated by the A2B receptor on cells such as airway smooth muscle, epithelial cells and fibroblasts. Whilst the effects in pre-clinical models are promising, their efficacy in the clinical setting has also yet to be reported. Finally, adenosine A3 receptor stimulation has been demonstrated to mediate inhibitory effects on eosinophils since it also elevates cAMP. However, some experimental reports suggest that A3 antagonists mediate anti-inflammatory effects, thus the rationale for A3 receptor ligands as therapeutic agents remains to be determined. In conclusion, establishing the precise role of adenosine in the pathogenesis of asthma and developing appropriate subtype selective agonists/antagonists represents an exciting opportunity for the development of novel therapeutics for the treatment of asthma.
PMCID: PMC2268070  PMID: 18311158
adenosine receptor; inflammation; asthma; mast cells; airway smooth muscle
18.  Interactions between responses mediated by activation of adenosine A2 receptors and alpha 1-adrenoceptors in the rabbit isolated aorta. 
British Journal of Pharmacology  1993;109(2):394-404.
1. This paper describes aspects of the functional antagonism between the responses mediated by activated alpha 1-adrenoceptors and adenosine A2 receptors in the adventitia- and endothelium-denuded aorta of the rabbit. 2. Adenosine A2 receptor agonists relaxed aortic rings pre-contracted with phenylephrine. The relaxation response was agonist concentration-dependent and saturable. The respective contractile and relaxation responses were stable, reproducible, and reversible. 3. Increasing the phenylephrine concentration caused a progressive attenuation of the action of adenosine A2 receptor agonists, consisting of a decreased maximal response and a dextral shift of the adenosine agonist concentration-response curve. This functional antagonism could be completely reversed upon removal of adenosine by either the addition of adenosine deaminase or by wash-out of the adenosine agonist from the tissue. The relaxation response to the adenosine A2 receptor partial agonists, N6-cyclohexyladenosine and R-(-)-N6-(2-phenylisopropyl)adenosine, was abolished at higher phenylephrine concentrations (e.g. 30 EC50). 4. A 1000 fold increase in the adenosine concentration was required to shift the value of the EC50 of phenylephrine six fold, while a similar increase in the value of the EC50 of adenosine could be elicited by only a 32 fold increase in the phenylephrine concentration. A 30 fold increase in the phenylephrine concentration shifted the value of the EC50 of 5'-N-ethylcarboxamidoadenosine four fold. 5. Analysis of the functional antagonism between the responses mediated by these receptors using the Black & Leff (1983) operational model of agonism allowed for the estimation of the agonist dissociation constant, KA, and the apparent efficacy, tau, for both phenylephrine and adenosine A2 receptor agonists. Increasing the concentration of phenylephrine reduced the value of tau for adenosine agonists in a concentration-dependent and saturable manner. Similarly, increasing the concentration of adenosine reduced the value of tau for phenylephrine in a concentration-dependent and saturable manner. The phenylephrine KA value obtained by the method of functional antagonism (1.9 microM) was similar to that obtained by the receptor inactivation method (2.1 microM). 6. Partial occlusion of the alpha 1-adrenoceptor by the alkylating agent, dibenamine, demonstrated that the magnitude of the adenosine A2 receptor-mediated relaxation was inversely proportional to the number of functional alpha 1-adrenoceptors. 7. It is concluded that the magnitude of functional antagonism is proportional to the stimulus elicited through either receptor. We propose that this tissue preparation and pair of receptors is a good model to study quantitative aspects of functional antagonism between activated receptors.
PMCID: PMC2175693  PMID: 8395286
19.  Protective effects of traditional Chinese medicine formula NaoShuanTong capsule on haemorheology and cerebral energy metabolism disorders in rats with blood stasis 
NaoShuanTong capsule (NSTC), an oral traditional Chinese medicine formula, is composed of Pollen Typhae, Radix Paeoniae Rubra, Rhizoma Gastrodiae, Radix Rhapontici and Radix Curcumae. It has been widely used to treat ischemic stroke in clinic for many years in China. In addition to neuronal apoptosis, haemorheology and cerebral energy metabolism disorders also play an important role in the pathogenesis and development of ischemic stroke. The present study was designed to evaluate the in vivo protective effects of NSTC on haemorheology and cerebral energy metabolism disorders in rats with blood stasis. Sixty specific pathogen-free sprague-dawley rats, male only, were randomly divided into six groups (control group, model group, aspirin (100 mg/kg/d) group, NSTC low-dose (400 mg/kg/d) group, NSTC intermediate-dose (800 mg/kg/d) group, NSTC high-dose (1600 mg/kg/d) group) with 10 animals in each. The rats except those in the control group were placed in ice-cold water (0–4 °C) for 5 min during the time interval (4 h) of two adrenaline hydrochloride injections (0.8 mg/kg) to induce blood stasis. After treatment, whole blood viscosity at three shear rates, plasma viscosity and erythrocyte sedimentation rate significantly decreased in NSTC intermediate- and high-dose groups; erythrocyte aggregation index and red corpuscle electrophoresis index significantly decreased in all the three dose NSTC groups. Moreover, treatment with high-dose NSTC could significantly improve Na+–K+ adenosine triphosphatase (ATPase) and Ca2+ ATPase activity, as well as lower lactic acid level in brain tissues. These results demonstrated the protective effects of NSTC on haemorheology and cerebral energy metabolism disorders, which may provide scientific information for the further understanding of mechanism(s) of NSTC as a clinical treatment for ischemic stroke. Furthermore, the protective effects of activating blood circulation as observed in this study might create valuable insight for the utilisation of NSTC to be a feasible alternative therapeutic agent for patients with blood stasis.
PMCID: PMC4433958  PMID: 26019500
NaoShuanTong capsule; blood stasis; haemorheology; cerebral energy metabolism; Chinese medicine formula
20.  The effects of saponin on the binding and functional properties of the human adenosine A1 receptor. 
British Journal of Pharmacology  1996;117(7):1521-1529.
1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.
PMCID: PMC1909466  PMID: 8730749
21.  The ischemic preconditioning effect of adenosine in patients with ischemic heart disease 
In vivo and in vitro evidence suggests that adenosine and its agonists play key roles in the process of ischemic preconditioning. The effects of low-dose adenosine infusion on ischemic preconditioning have not been thoroughly studied in humans.
We hypothesised that a low-dose adenosine infusion could reduce the ischemic burden evoked by physical exercise and improve the regional left ventricular (LV) systolic function.
Materials and methods
We studied nine severely symptomatic male patients with severe coronary artery disease. Myocardial ischemia was induced by exercise on two separate occasions and quantified by Tissue Doppler Echocardiography. Prior to the exercise test, intravenous low-dose adenosine or placebo was infused over ten minutes according to a randomized, double blind, cross-over protocol. The LV walls were defined as ischemic if a reduction, no increment, or an increment of < 15% in peak systolic velocity (PSV) was observed during maximal exercise compared to the baseline values observed prior to placebo-infusion. Otherwise, the LV walls were defined as non-ischemic.
PSV increased from baseline to maximal exercise in non-ischemic walls both during placebo (P = 0.0001) and low-dose adenosine infusion (P = 0.0009). However, in the ischemic walls, PSV increased only during low-dose adenosine infusion (P = 0.001), while no changes in PSV occurred during placebo infusion (P = NS).
Low-dose adenosine infusion reduced the ischemic burden and improved LV regional systolic function in the ischemic walls of patients with exercise-induced myocardial ischemia, confirming that adenosine is a potential preconditioning agent in humans.
PMCID: PMC2779183  PMID: 19891770
22.  Effect of adenosine receptor modulation on pentylenetetrazole-induced seizures in rats 
British Journal of Pharmacology  1997;120(2):282-288.
The effects of adenosine, the adenosine analogue, 2-chloroadenosine (2-CADO), the specific adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA) and A2 receptor agonist 5′-(N-cyclopropyl) carboxamidoadenosine (CPCA), were examined against seizures induced by acute administration of pentylenetetrazole (PTZ), 60 mg kg−1, and PTZ kindled seizures, in rats.Adenosine 1000 mg kg−1, i.p., 5 min pretreatment and CPA 10 mg kg−1 i.p., 60 min pretreatment, showed significant protection against acute PTZ-induced seizures while, CPCA up to 10 mg kg−1 was ineffective. The adenosine analogue 2-CADO in a dose of 5 mg kg−1 was only partially protective and on increasing the dose to 10 mg kg−1, this protection was lost.Theophylline, a non specific adenosine receptor antagonist at 50 mg kg−1 and the specific adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), at 1 mg kg−1, if administered before the maximally protective doses of adenosine and CPA, completely reversed the protection afforded by them against PTZ seizures. While, pretreatment with the adenosine A2 receptor antagonist, 3,7-dimethyl-1-propargylxanthine (DMPX), failed to reverse the protection.Adenosine and the adenosine A1 receptor agonist in doses that protected against seizures after acute PTZ administration, offered only incomplete protection when tested against PTZ kindled seizures.The effects of adenosine and adenosine receptor agonists on mean arterial pressure, heart rate and rectal temperature were studied, to rule out the possibility of their systemic effects mediating the protection of PTZ seizures. All these agents produced a fall in mean arterial pressure, heart rate and hypothermia in the doses exhibiting an anticonvulsant response. While the effect on blood pressure and heart rate was immediate i.e. seen within 5 min and, maintained throughout the observation period, the development of hypothermia lagged behind the onset of hypotension and bradycardia. However, there was no correlation between haemodynamic and hypothermic response and the anticonvulsant effect.The results indicate that the adenosine mediated anticonvulsant effect is via stimulation of A1 receptors. Hypotension and hypothermia do not appear to contribute to the protection observed with adenosine and the adenosine A1 receptor agonists.
PMCID: PMC1564359  PMID: 9117121
Adenosine; 2-chloroadenosine (2-CADO); N6-cyclopentyladenosine (CPA); 5′-(N-cyclopropyl) carboxamidoadenosine (CPCA); pentylenetetrazole (PTZ); PTZ kindled seizures; 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); theophylline; 3,7-dimethyl-1-propargylxanthine (DMPX)
23.  Delayed Adaptation of the Heart to Stress 
Stroke; a journal of cerebral circulation  2004;35(11 Suppl 1):2676-2679.
The early phase of preconditioning (PC) lasts 2 to 3 hours and protects against myocardial infarction, but not against stunning. In contrast, the late phase of PC lasts for 3 to 4 days and protects against both myocardial stunning and infarction, making this phenomenon more clinically relevant. Late PC is a genetic reprogramming of the heart that involves the activation of several stress-responsive genes, which ultimately results in the development of a cardioprotective phenotype. Sublethal ischemic insults release chemical signals (nitric oxide [NO], adenosine, and reactive oxygen species) that trigger a series of signaling events (eg, activation of protein kinase C, Src protein tyrosine kinases, Janus kinases 1/2, and nuclear factor-κB) and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, heme oxygenase-1, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. In addition to ischemia, heat stress, exercise, and cytokines can also induce a similar series of events. Perhaps most importantly, many pharmacologic agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists) can mimic the effects of ischemia in inducing the late phase of PC, suggesting that this phenomenon might be exploited therapeutically. The purpose of this review is to summarize the mechanisms that underlie the late phase of ischemic PC.
PMCID: PMC3242368  PMID: 15459441
ischemic preconditioning; myocardial ischemia; myocardial reperfusion; nitric oxide
24.  Periodic 17β-Estradiol Pretreatment Protects Rat Brain from Cerebral Ischemic Damage via Estrogen Receptor-β 
PLoS ONE  2013;8(4):e60716.
Although chronic 17β-estradiol (E2) has been shown to be a cognition-preserving and neuroprotective agent in animal brain injury models, concern regarding its safety was raised by the failed translation of this phenomenon to the clinic. Previously, we demonstrated that a single bolus of E2 48 hr prior to ischemia protected the hippocampus from damage in ovariectomized rats via phosphorylation of cyclic-AMP response element binding protein, which requires activation of estrogen receptor subtype beta (ER-β). The current study tests the hypothesis that long-term periodic E2-treatment improves cognition and reduces post-ischemic hippocampal injury by means of ER-β activation. Ovariectomized rats were given ten injections of E2 at 48 hr intervals for 21 days. Hippocampal-dependent learning, memory and ischemic neuronal loss were monitored. Results demonstrated that periodic E2 treatments improved spatial learning, memory and ischemic neuronal survival in ovariectomized rats. Additionally, periodic ER-β agonist treatments every 48 hr improved post-ischemic cognition. Silencing of hippocampal ER-β attenuated E2-mediated ischemic protection suggesting that ER-β plays a key role in mediating the beneficial effects of periodic E2 treatments. This study emphasizes the need to investigate a periodic estrogen replacement regimen to reduce cognitive decline and cerebral ischemia incidents/impact in post-menopausal women.
PMCID: PMC3625208  PMID: 23593292
25.  Reduction of postischemic brain damage and memory deficits following treatment with the selective adenosine A1 receptor agonist 
European journal of pharmacology  1996;302(1-3):43-48.
Agonists of adenosine A1 receptors have been frequently proposed as candidates for clinical development in treatment of cerebral ischemia and stroke. Numerous experimental studies have shown that pre- and postischemic administration of these drugs results in a very significant reduction of postischemic brain damage. However, only a few studies determined the impact of cerebral ischemia and drug treatment on postischemic recovery of spatial memory. The present paper demonstrates that preischemic i.p. administration of adenosine amine congener (ADAC) at 100 μg/kg in gerbils results in a significant (P < 0.05) reduction of postischemic mortality and hippocampal, cortical and striatal morbidity. Postischemic Morris’ water maze tests show that preischemic treatment with ADAC also leads to a very significant (P < 0.001) reduction of postischemic spatial memory loss. Our results indicate feasibility of further consideration of adenosine A1 receptor agonists as a clinically applicable acute treatment of brain ischemia. Recent development of neuroprotective adenosine A1 receptor agonists that are free of cardiovascular side effects supports such development.
PMCID: PMC3449166  PMID: 8790990
Cerebral ischemia; Adenosine receptor; Spatial memory; Water maze; (Gerbil)

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