Adenosine‐induced apoptosis in chick embryonic sympathetic neurons: a new physiological role for adenosine.
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1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1‐100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13‐dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100‐300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase‐cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose‐dependently inhibited by 5'‐iodotubercidin (3‐100 nM). 5'‐Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2‐deoxycytidine, but not by nicotinamide or 2‐deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase‐mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.Keywords:
Adenosine kinase
Purinergic Signalling
Adenosine A1 receptor
Adenosine A3 receptor
Adenosine A2B receptor
Aminophylline
Inosine
Hypoxanthine
Adenosine Deaminase Inhibitor
Abstract Extracellular adenosine mediates diverse anti-inflammatory, angiogenic, and other signaling effects via binding to adenosine receptors, and it also regulates cell proliferation and death via activation of the intrinsic signaling pathways. Given the emerging role of adenosine and other purines in tumor growth and metastasis, this study evaluated the effects of adenosine on the invasion of metastatic prostate and breast cancer cells. Treatment with low micromolar concentrations of adenosine, but not other nucleosides or adenosine receptor agonists, inhibited subsequent cell invasion and migration through Matrigel- and laminin-coated inserts. These inhibitory effects occurred via intrinsic receptor-independent mechanisms, despite the abundant expression of A2B adenosine receptors (ADORA2B). Extracellular nucleotides and adenosine were shown to be rapidly metabolized on tumor cell surfaces via sequential ecto-5′-nucleotidase (CD73/NT5E) and adenosine deaminase reactions with subsequent cellular uptake of nucleoside metabolites and their intracellular interconversion into ADP/ATP. This was accompanied by concurrent inhibition of AMP-activated protein kinase and other signaling pathways. No differences in the proliferation rates, cytoskeleton assembly, expression of major adhesion molecules [integrin-1β (ITGB1), CD44, focal adhesion kinase], and secretion of matrix metalloproteinases were detected between the control and treated cells, thus excluding the contribution of these components of invasion cascade to the inhibitory effects of adenosine. These data provide a novel insight into the ability of adenosine to dampen immune responses and prevent tumor invasion via two different, adenosine receptor–dependent and –independent mechanisms. Implications: This study suggests that the combined targeting of adenosine receptors and modulation of intracellular purine levels can affect tumor growth and metastasis phenotypes. Mol Cancer Res; 12(12); 1863–74. ©2014 AACR.
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CD73 (ecto-5'-nucleotidase) on human gingival fibroblasts plays a role in the regulation of intracellular cAMP levels through the generation of adenosine, which subsequently activates adenosine receptors. In this study, we examined the involvement of ecto-adenosine deaminase, which can be anchored to CD26 on human gingival fibroblasts, in metabolizing adenosine generated by CD73, and thus attenuating adenosine receptor activation. Ecto-adenosine deaminase expression on fibroblasts could be increased by pre-treatment with a lysate of Jurkat cells, a cell line rich in cytoplasmic adenosine deaminase. Interestingly, the cAMP response to adenosine generated from 5'-AMP via CD73 and the ability of 5'-AMP to induce hyaluronan synthase 1 mRNA were significantly decreased by the pre-treatment of fibroblasts with Jurkat cell lysate. This inhibitory effect was reversed by the specific adenosine deaminase inhibitor. These results suggest that ecto-adenosine deaminase metabolizes CD73-generated adenosine and regulates adenosine receptor activation.
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This review focuses on the potential role of site- and event-selective adenosinergic drugs in the treatment of cardiovascular diseases. Adenosine is released from the myocardium and vessels in response to various forms of stress and acts on four receptor subtypes (A1, A2A, A2B and A3). Adenosine is an important endogenous substance with important homeostatic activity in the regulation of cardiac function and circulation. Adenosine receptors are also involved in the modulation of various cellular events playing crucial role in physiological and pathological processes of the cardiovascular system. These actions are associated to activation of distinct adenosine receptor subtypes, therefore drugs targeting specific adenosine receptors might be promising therapeutic tools in treatment of several disorders including various forms of cardiac arrhythmia, myocardial ischemia-reperfusion injury, angina pectoris, chronic heart failure, etc. Recently, in addition to subtype-specific adenosine receptor agonists and antagonists, a number of substances that enhance adenosine receptor activation locally at the site where the release of endogenous adenosine is the most intensive have been developed. Thus global actions of adenosine receptor agonists and antagonists, as well as desensitization or down-regulation following chronic administration of these orthosteric compounds can possibly be avoided. We discuss the chemical, pharmacological and clinical features of these compounds: (1) inhibitors of membrane adenosine transporters (NBTI, dipyridamole), (2) inhibitors of adenosine deaminase (coformycin, EHNA), (3) inhibitors of adenosine kinase (tubercidin, aristeromycin), (4) inhibitors of AMP deaminase (GP3269), (5) activators of 5'- nucleotidase (methotrexate), (6) adenosine regulators (acadesine) and (7) allosteric adenosine receptor modulators (PD81723, LUF6000). The development of this type of substances might offer a novel therapeutic approach for treating cardiovascular diseases in the near future. Keywords: Site and event specific action, adenosinergic drugs, membrane adenosine transport, adenosine deaminase, adenosine kinase, AMP deaminase, 5'-nucleotidase, adenosine regulators, allosteric receptor modulators, Cardiovascular Disorders
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Adenosine, acting both through G-protein coupled adenosine receptors and intracellularly, plays a complex role in multiple physiological and pathophysiological processes by modulating neuronal plasticity, astrocytic activity, learning and memory, motor function, feeding, control of sleep and aging. Adenosine is involved in stroke, epilepsy and neurodegenerative pathologies. Extracellular concentration of adenosine in the brain is tightly regulated. Adenosine may be generated intracellularly in the central nervous system from degradation of AMP or from the hydrolysis of S-adenosyl homocysteine, and then exit via bi-directional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. Inactivation of extracellular adenosine occurs by transport into neurons or neighboring cells, followed by either phosphorylation to AMP by adenosine kinase or deamination to inosine by adenosine deaminase. Modulation of the nucleoside transporters or of the enzymatic activities involved in the metabolism of adenosine, by affecting the levels of this nucleoside and the activity of adenosine receptors, could have a role in the onset or the development of central nervous system disorders, and can also be target of drugs for their treatment. In this review, we focus on the contribution of 5′-nucleotidases, adenosine kinase, adenosine deaminase, AMP deaminase, AMP-activated protein kinase and nucleoside transporters in epilepsy, cognition, and neurodegenerative diseases with a particular attention on amyotrophic lateral sclerosis and Huntington’s disease. We include several examples of the involvement of components of the adenosine metabolism in learning and of the possible use of modulators of enzymes involved in adenosine metabolism or nucleoside transporters in the amelioration of cognition deficits.
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1. A newly found action of adenosine in neurons, which may have an important physiological function in the growth and development of the sympathetic nervous system, is described. Adenosine (1‐100 microM) inhibited neurite outgrowth within the first 24 h and killed about 80% of sympathetic neurons supported by nerve growth factor over the next 2 days in culture. Neurons supported by excess KCl, forskolin or phorbol 12,13‐dibutyrate were equally susceptible to the toxic actions of adenosine. Inosine, guanosine or hypoxanthine (all 100‐300 microM) were without effect on neuronal growth and survival. 2. Specific agonists of adenosine A1 and A2 receptors were not neurotoxic, and toxic effects of adenosine were not antagonized by aminophylline. These results rule out involvement of adenosine receptors and the adenylyl cyclase‐cAMP signalling system in neurotoxic actions of adenosine. 3. Adenosine toxicity was prevented by inhibitors of the adenosine membrane transporter, suggesting an intracellular site of action of adenosine. 4. Inhibitors of adenosine deaminase dramatically facilitated the toxic action so that physiologically relevant concentrations of adenosine were neurotoxic. 5. Adenosine kinase activity of sympathetic neurons was dose‐dependently inhibited by 5'‐iodotubercidin (3‐100 nM). 5'‐Iodotubercidin (100 nM) completely protected neurons against toxicity of adenosine plus adenosine deaminase inhibitors. These results provide convincing evidence that phosphorylation of the nucleoside is an essential requirement for initiation of adenosine toxicity. 6. Sympathetic neurons were successfully rescued from the lethal effects of adenosine deaminase inhibitor plus adenosine by uridine or 2‐deoxycytidine, but not by nicotinamide or 2‐deoxyguanosine, suggesting that depletion of pyrimidine nucleotides by phosphorylated adenosine compounds and consequent inhibition of DNA synthesis produces neuronal death. 7. DNA fragmentation, assessed by the fluorescent dye bisbenzimide and by the TUNEL (terminal deoxynucleotidyl transferase‐mediated nick end labelling) method, indicated that neuronal death induced by adenosine was apoptotic. 8. We conclude that adenosine deaminase and adenosine kinase play an important role in the metabolism of intracellular concentrations of adenosine and thereby regulate the growth and development of sympathetic neurons. Our study highlights, for the first time, the importance of adenosine as a mediator of programmed cell death of neurons supported by nerve growth factor.
Adenosine kinase
Purinergic Signalling
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Adenosine A3 receptor
Adenosine A2B receptor
Aminophylline
Inosine
Hypoxanthine
Adenosine Deaminase Inhibitor
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