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A new role for the A2b adenosine receptor in regulating platelet function
2010-01-01
Summary. Background: Activation of platelets is a critical component of atherothrombosis and plays a central role in the progression of unstable cardiovascular syndromes. Adenosine, acting through adenosine receptors, increases intracellular cAMP levels and inhibits platelet aggregation. The A2a adenosine receptor has already been recognized as a mediator of adenosine-dependent effects on platelet aggregation, and here we present a new role for the A2b adenosine receptor (A2bAR) in this process. Methods and Results: As compared with platelets from wild-type controls, platelets derived from A2bAR knockout mice have significantly greater ADP receptor activation-induced aggregation. Although mouse megakaryocytes and platelets express low levels of the A2bAR transcript, this gene is highly upr...
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation...Full Text Available
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Full Text Available.Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (A2BAR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9±3.4-fold, p<0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS-exposure. Studies utilizing A2BAR promoter constructs and RNA-protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR-bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.
2010-01-01
The synthesis and SAR of a series of N-(5,6-diarylpyridin-2-yl)amide derivatives as potent A2B adenosine receptor antagonists is described. Several compounds showed good selectivity versus other adenosine receptors. The potent and selective analogue 9 was shown to have good oral bioavailability in the rat.
2010-01-01
Several new potent and selective A2B adenosine receptor antagonists have been prepared in which the aryl-amide moiety of the lead series, exemplified by 1a, has been replaced by bioisosteric bicyclic moieties. Although the majority of compounds had generally improved microsomal stability as compared to 1a, this was not translated into overall improvements in the pharmacokinetic profiles of a representative set of compounds.
1995-01-20
Adenosine receptors have been implicated as important mediators of a diversity of physiological processes throughout the body. These receptors are members of the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Four different subtypes of adenosine receptor have been identified through molecular cloning and subsequent pharmacological and biochemical analyses. Of these subtypes, the Al and A2a receptors have been mapped to chromosome 22q11.2-q13.1 and 11q11-q13, respectively. The A3 receptor has been localized to chromosome 3 in the mouse, by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse human linkage homologies. In determining the chromosomal localization of the A2a receptor, a minor hybridization peak was detected on chromosome 10q25.3-q26.3, and the authors of this study concluded that this site was likely to correspond to an adenosine A2-like receptor. With the molecular identification of the adenosine A2b receptor, the hybridization site on chromosome 10 was assigned to the A2b receptor (MIM 102777). We have used fluorescence in situ hybridization (FISH) and PCR screening of a somatic cell hybrid panel to determine the true chromosomal localization of the A2b adenosine receptor subtype. 12 refs., 1 fig., 1 tab.
2010-01-01
Hypoxia is a common characteristic of many pathological and physiological conditions that can markedly change cellular metabolism and cause the accumulation of extracellular adenosine. Recent studies have shown that adenosine can modulate the function of certain immune cell types through binding with different adenosine receptors. Our previous studies have shown that hypoxia has an effect on the biological activity of dendritic cells (DCs) by inducing their differentiation towards a Th2 polarising phenotype. However, the mechanisms underlying this suppression remain unclear. In this study, we have demonstrated that hypoxic mDCs predominantly express adenosine receptor A2b. The A2b receptor antagonist MRS1754 was able to increase the production of IL-12p70 and TNF-α by hypoxic mDCs and...
Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the...Full Text Available
Full Text Available.Infections with intracellular bacteria such as chlamydiae affect the majority of the world population. Infected tissue inflammation and granuloma formation help contain the short-term expansion of the invading pathogen, leading also to local tissue damage and hypoxia. However, the effects of key aspects of damaged inflamed tissues and hypoxia on continued infection with intracellular bacteria remain unknown. We find that development of Chlamydia trachomatis is reversibly retarded by prolonged exposure of infected cells to extracellular adenosine, a hallmark of hypoxia and advanced inflammation. In epithelial cells, this effect was mediated by the A2b adenosine receptor, unique in the adenosine receptor family for having a hypoxia-inducible factor (HIF1-α) binding site at its promoter region, and was dependent on an increase in the intracellular cAMP levels, but was independent of cAMP-dependent protein kinase (PKA). Further study of adenosine receptor signaling during intracellular bacterial infection could lead to breakthroughs in our understanding of persistent infections with these ubiquitous pathogens.
2010-01-01
Ligand-based and receptor-based methods were used to investigate the binding modes of human adenosine A2B antagonists. At first, pharmacophore models were developed based on 140 diverse A2B antagonists from literature. Meanwhile, the structural model of A2B receptor was built up based on the crystal structure of human A2A receptor and validated by Induced Fit docking, Glide-XP and Glide-SP docking. Two models matched each other very well and some important implications were hence obtained. The residues of Phe173 and Glu174 in the second extracellular loop and Asn254 were crucial to the antagonists binding to form p-p stacking and hydrogen-bonding interactions. These findings would be very helpful for the discovery of novel and potent A2B antagonists.
http://hdl.handle.net/10072/5645
Adenosine is an important cardioprotective agent that works via several adenosine receptor (ADOR) subtypes to regulate cardiovascular activity. It is well established that functional responses to adenosine decline with age. What is unclear, though, is whether these changes occur at the receptor, second messenger or translational level. In this study we determined the effect of age on cardiac adenosine receptor expression using the housekeeping gene 18S rRNA versus the adenosine A2B receptor gene as internal controls. Absolute quantification showed that no age-related changes occurred in the expression of 18S rRNA or adenosine A2B receptor internal control genes. Subsequently, relative analysis of the adenosine receptor subtypes using 18S rRNA found a significant age-related reduction in the expression of the adenosine A1 receptor (5.5-fold), with no changes in the expression of the adenosine A2A, A2B and A3 receptors. When using the expression of the adenosine A2B receptor as the internal control gene, a significant down regulation of both the adenosine A1 (5.4-fold) and A2A (2.2-fold) receptors with no change in the expression of adenosine A3 receptor was found. Therefore, the high level of expression of the 18S rRNA housekeeping gene was found to mask a significant change in expression of the adenosine A2A receptor with age. Ultimately, these findings show an age-related reduction in adenosine A1 and A2A receptor expression in rat heart. Publisher: http://dx.doi.org/10.1016/j.mad.2003.11.016; Elsevier; Ireland; http://www.elsevier.com/wps/find/journaldescription.cws_home/506026/description#description Relation: Mechansims of Ageing and Development; 211; 217; 125 Other identifier: 0047-6374 Language: en_AU Rights: Copyright 2004 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links.
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060913.103656
Pyrazolo[3,4-d]pyrimidines were known as adenosine antagonists at the rat A1 and A2A adenosine receptors based on our previous studies. In this study, 245 pyrazolo[3,4-d]pyrimidines derivatives with various benzyl substitutents at N-1 and various hydrophobic side chains at C-4 and C-6 were synthesized and screened at the human A1, A2A and A3 adenosine receptors. 14 out of 245 compounds were resynthesized and purified to determine the Ki values of these compounds at the human A1 adenosine receptor. Chapter 1 of the thesis is a literature review of adenosine research. It describes the physiology of adenosine and the discovery and characterization of all adenosine receptors namely A1, A2A, A2B and A3. It also looks at the medical application of adenosine, adenosine analogs, adenosine agonists and adenosine antagonists. The final part of the chapter discusses the discovery and development of adenosine agonists and antagonists. Chapter 2 of the thesis describes the rational design of the pyrazolo[3,4-d]pyrimidines template using ligand-based molecular modelling technique and describes the synthesis of the template. Chapter 3 and chapter 4 describe the application of silicon chemistry and attempts to synthesise a series of pyrazolo[3,4-d]pyrimidines heterocycle by solid phase synthesis. Chapter 5 and chapter 6 describe the synthesis of a series of pyrazolo[3,4-d]pyrimidines heterocycle using the solution phase parallel synthesis and the binding studies of a library of 245 compounds and the resynthesis of 14 target compounds. Chapter 7 describes the cell culture and membrane preparation of the human A1, A2A, A2B and A3 adenosine receptors and radioligands binding assays. Publisher: Griffith University. School of Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Khoa Quang Do
2003-01-01
Elevated extracellular adenosine has been found to stimulate hematopoiesis in experimental mice exposed to radiotherapy (gamma-rays), chemotherapy (5-fluorouracil), or combined action of both these modalities (gamma-rays + carboplatin). These findings have been obtained after treatment of the animals with the combination of dipyridamole (DP), preventing the cellular uptake of adenosine, and adenosine monophosphate (AMP), acting as adenosine prodrug. Increased cycling of hematopoietic progenitor cells following the administration of DP + AMP has been shown to represent an important mechanism of acceleration of regeneration of suppressed hematopoiesis. In recent experiments, non-degradable synthetic adenosine receptor agonists, more or less specific for individual subtypes of adenosine receptors (A1, A2A, A2B, and A3 subtypes) have been studied. These ... >>
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20061011.163451
Ischaemic heart disease is a major contributor to premature death and heart failure in the Westernised world. Ischaemic injury within the heart may be beneficially modulated by the nucleoside adenosine. Derived from catabolism of ATP, adenosine was initially known as a potent bradycardic and hypotensive agent. However, more recently the protective function of adenosine has been investigated. The protective effects of adenosine are mediated via activation of adenosine receptors: A1, A2A, A2B, and A3 receptors. Activation of these potentially protective (or retaliatory) adenosine receptors hinges upon accumulation of adenosine during ischaemia-reperfusion. This Thesis examines the role and mechanisms of adenosine mediated cardioprotection in young and aged hearts, exploring endogenous and exogenous adenosine receptor activation, genetic manipulation of A1 receptors and adenosine deaminase and pharmacological manipulation of adenosine metabolism. The effects of age on ischaemic responses and adenosine handling and protection are also assessed. The core approach to assess each of the above issues involved the Langendorff isolated mouse heart preparation. Experiments within Chapter 3 focuses on the contractile effects of adenosine receptors in normoxic hearts. This study indicates A2A receptors have no direct effect on contractility, while adenosine exerts positive inotropy independently of coronary flow and perfusion pressure (i.e. Independent of the Gregg phenomenon). In addition, investigations in genetically modified hearts hint at positive inotropy in response to A1 receptors. Since the latter is only evidenced in modified lines, it is possible A1-mediated inotropy may be abnormal or supraphysiological. In Chapter 4 the impact of genetic 'deletion' of A1ARs and/or adenosine deaminase (ADA) on intrinsic tolerance to ischaemia were studied. Data demonstrate that genetic deletion of A1 receptors significantly limits the ability of the mouse myocardium to withstand injury during ischaemic insult. Thus, providing strong support for a role of A1ARs in determining intrinsic tolerance to ischaemia-reperfusion. ADA KO mice confirm protection afforded by endogenous adenosine and the notion of adenosine metabolism modification as a protective strategy. Interestingly, effects of A1AR KO differ from A1AR overexpression or A1AR agonism in that the latter decrease contractile diastolic dysfunction while A1AR KO selectively increase systolic dysfunction and increase oncosis without altering diastolic injury. This challenges current dogma regarding the action of A1 adenosine receptors on ischaemic injury. In Chapter 5 the effects of adenosine metabolism inhibition (via adenosine deaminase (ADA) and adenosine kinase (AK) inhibitors) were studied. Inhibition of adenosine deaminase with the drug EHNA, and adenosine phosphorylation with iodotubercidin significantly protected mouse hearts from ischaemia-reperfusion, reducing contractile dysfunction and cardiac enzyme efflux. However, inhibitors failed to improve the outcome of the aged myocardium. 8-SPT and 5-HD reduced the protective effects of EHNA and iodotubercidin demonstrating thus; cardioprotection via ADA and AK appears to rely on adenosine receptor activation and involves a mitoK ATP dependent mechanism. Since aging is of considerable importance with regard to outcomes of ischaemic heart disease, experiments in Chapter 6 focused on effects of aging (and gender) on cardiovascular function and injury during ischaemia-reperfusion. In assessing post ischaemic outcomes in hearts from young adult (2-4 months), mature adult (8 months), middle aged (12 months), aged (18 months) and senescent (24-28 months) C57/BL/6J mice, data reveal a substantial age-related decline in ischaemic tolerance (which appears selective for myocardial vs. vascular injury). The decline in ischaemic tolerance is expressed primarily within the initial 12 months in both males and females with relatively little further decline with continued aging. There is evidence of a modest improvement in tolerance in senescence vs. aged hearts possibly reflecting selection of a protected phenotype in senescent populations. In addition, mature and middle-aged female hearts showed improved tolerance to ischaemia-reperfusion compared to males, supporting a role for hormonal changes. Effects of aging and purine metabolism were studied in Chapter 7. Data suggest impaired tolerance to ischaemia-reperfusion in older hearts may stem in part from shifts in myocardial purine catabolism. Data reveal reduced accumulation of salvageable and cardioprotective adenosine and enhanced accumulation of poorly salvaged (and potentially injurious) hypoxanthine and xanthine. These changes may potentially predispose the aged myocardium to ischaemic injury and radical generation via the xanthine oxidase reaction. The final data Chapter of this Thesis describes preliminary data regarding aging, signalling and adenosine mediated protection. It was found that protein kinase C (PKC) and A1 receptors mediate protection in young hearts and also that A1 receptors appear to mediate protection via a PKC LindependentLi signalling cascade. In addition, experiments in aged hearts (attempting to elucidate mechanisms behind impaired adenosinergic protection with age) suggest a PKC-independent A1-mediated protection path may be preserved with aging, since A1 receptors continue to offer some protection while PKC activation does not. It is possible the failure of exogenous adenosine to offer protection in aged hearts may result from a lesion at or downstream of PKC. Publisher: Griffith University. School of Medical Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Laura E Willems
http://eprints.qut.edu.au/15127/
Herein we report the synthesis and biological evaluation of some potent and selective A1 adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N6-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (Ki = 50 nM) and good selectivity (A3/A1 400) for the A1 adenosine receptor. N6-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (Ki = 7 nM), but lower selectivity (A3/A1 = 3). All compounds bind weakly (Ki > 1 μM) to A2A and A2B receptors. The combination of A1 agonist activity and antioxidant activity has the potential to produce cardioprotective effects. Publisher: Elsevier Relation: DOI:10.1016/j.bmcl.2007.07.035; Gregg, Alison D. and Bottle, Steven E. and Devine, Shane M. and Figler, Heidi and Linden, Joel and White, Paul and Pouton, Colin W. and Urmaliya, Vijay and Scammells, Peter J. (2007) Dual acting antioxidant A1 adenosine receptor agonists. Bioorganic & Medicinal Chemistry Letters, 17(9). pp. 5437-5441. Format: application/pdf Rights: Copyright 2007 Elsevier; Reproduced in accordance with the copyright policy of the publisher.
Localization of the adenosine A1 receptor subtype gene (ADORA1) to chromosome 1q32.1
1995-03-20
Adenosine, acting through its receptors, exerts effects on almost all organ systems, influencing a diversity of physiological responses, including the inhibition of neurotransmitter release, the modulation of cardiac rhythmicity and contractility, and the potentiation of IgE-dependent mediator release. Adenosine receptors belong to the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Molecular cloning and subsequent pharmacological and biochemical analyses have led to the identification of four different subtypes of adenosine receptor. The A3 receptor has been localized to chromosome 3 in the mouse by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse-human linkage homologies. We have previously mapped the A2b adenosine receptor subtype to chromosome 17p11.2-p12 using fluorescence in situ hybridization (FISH) and PCR-based screening of somatic cell hybrid DNAs. A previous report has concluded that the Al and A2a receptor subtypes are localized on chromosome 22q11.2-q13.1 and 11q11-q13, respectively, but conflicts with that of MacCollin et al., who have mapped the A2a gene to chromosome 22. In this report, we show that the human A1 adenosine receptor subtype does not map to chromosome 22q11.2-q13.1, but is instead localized on chromosome 1q32. 13 refs., 1 fig.
http://eprints.jcu.edu.au/10022/1/aging_and_adenosine_transcription.pdf
The well-documented age-related change in ischemic tolerance may result from impaired adenosine-mediated cardioprotection. Additionally, ischemia itself may potentially modify adenosine signalling, contributing to the post-ischemic phenotype. This study investigates age- and ischemia-dependent changes in adenosine receptor transcript levels (Adora) for the A1, A2A, A2B, and A3 receptor subtypes in mouse myocardium. Hearts from young (2–4 months) and moderately aged (16–18 months) mice were subjected to 20-min ischemia and 45-min reperfusion. Ischemic tolerance was impaired in aged hearts, which recovered less than 30% ventricular pressure development (compared with ~70% in young hearts), and lost 2-fold higher levels of lactate dehydrogenase during reperfusion (reflecting cellular disruption). Real-time PCR analyses revealed an age-related decline in Adora3 levels and induction of Adora2B. Curiously, this effect was mimicked by ischemia, which acutely reduced Adora3 levels and induced Adora2B in young (but not old) hearts. In contrast, in aged hearts ischemia selectively reduced levels of Adora1 transcript (~2-fold) without altering transcript levels for the other receptors. These results demonstrate selective modulation of cardioprotective adenosine receptor transcription by both aging and ischemia. Reduced A3 adenosine receptor transcription may contribute to impaired ischemic tolerance in aged hearts, whereas changes in Adora transcription induced by ischemia may impact on the post-ischemic phenotype at later time points. Publisher: Elsevier Format: application/pdf Other identifier: Ashton, Kevin J., Nilsson, Ulrika, Willems, Laura, Holmgren, Kirsty, and Headrick, John P. (2003) Effects of aging and ischemia on adenosine receptor transcription in mouse myocardium. Biochemical and Biophysical Research Communications, 312 (2). pp. 367-372. ISSN 1090-2104
2010-01-01
Adenosine can show anti-inflammatory as well as pro-inflammatory activities. The contribution of the specific adenosine receptor subtypes in various cells, tissues and organs is complex. In this study, we examined the effect of the adenosine A2A receptor agonist CGS 21680 and the A2BR antagonist PSB-1115 on acute inflammation induced experimentally by 2,4,6-trinitrobenzenesulfonic acid (TNBS) on rat ileum/jejunum preparations. Pre-incubation of the ileum/jejunum segments with TNBS for 30 min resulted in a concentration-dependent inhibition of acetylcholine (ACh)-induced contractions. Pharmacological activation of the A2AR with CGS 21680 (0.1â10 µM) pre-incubated simultaneously with TNBS (10 mM) prevented concentration-dependently the TNBS-induced inhibition of the ACh contractions. S...
http://researchbank.rmit.edu.au/view/rmit:4110
The expression pattern of purinergic receptors was examined in subventricular zone-derived primary neurospheres. Primary neurospheres expressed mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of P2Y4, and the A1, A2a and A2b adenosine receptors. ATPgammaS, ADPbetaS and UTP evoked transient increases in cytoplasmic Ca2+ concentration in dissociated primary neurospheres, demonstrating the functional expression of P2Y1 and P2Y2 receptors. Ca2+ transients were not attenuated by the removal of extracellular Ca2+ and were reversibly inhibited by the P2Y1 selective antagonist, MRS 2179. P2Y and adenosine receptor agonists reduced the size and frequency of primary neurospheres. The effects of ADPbetaS and adenosine were reversed by subtype-selective receptor antagonists, demonstrating that P2Y1 and A2a receptors mediate inhibitory effects on primary neurosphere proliferation. The modulation of neural precursor cell proliferation by P2Y and adenosine receptors therefore represents a potential regulatory mechanism within the neurogenic microenvironment. Coverage: 2007-01-01T00:00:00Z
http://hdl.handle.net/10072/29336
Adenosine, a catabolite of ATP, exerts numerous effects in the heart, including modulation of the cardiac response to stress, such as occurs during myocardial ischemia and reperfusion. Over the past 20 years substantial evidence has accumulated that adenosine, administered either prior to ischemia or during reperfusion, reduces both reversible and irreversible myocardial injury. The latter effect results in reduction of both necrosis or myocardial infarction (MI) and apoptosis. These effects appear to be mediated via the activation of one or more G-protein coupled receptors (GPCRs), referred to as A1, A2A, A2B and A3 adenosine receptor (AR) subtypes. Experimental studies in different species and models suggest that activation of the A1 or A3ARs prior to ischemia is cardioprotective. Further experimental studies reveal that the administration of A2AAR agonists during reperfusion can also reduce MI, and recent reports suggest that A2BARs may also play an important role in modulating myocardial reperfusion injury. Despite convincing experimental evidence for AR-mediated cardioprotection, there have been only a limited number of clinical trials examining the beneficial effects of adenosine or adenosine-based therapeutics in humans, and the results of these studies have been equivocal. This review summarizes our current knowledge of AR-mediated cardioprotection, and the roles of the four known ARs in experimental models of ischemia-reperfusion. The chapter concludes with an examination of the clinical trials to date assessing the safety and efficacy of adenosine as a cardioprotective agent during coronary thrombolysis in humans. Publisher: Springer-Verlag; Berlin Contributor: CN Wilson, J Mustafa Relation: Handbook of Experimental Pharmacology, Adenosine Receptors in Health and Disease; 7; 19; 189; 214; N Other identifier: 978-3-540-89614-2 Language: en_AU Rights: Y
http://espace.library.uq.edu.au/view/UQ:129712
The expression pattern of purinergic receptors was examined in subventricular zone-derived primary neurospheres. Primary neurospheres expressed mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of P2Y4, and the A1, A2a and A2b adenosine receptors. ATPyS, ADP beta S and UTP evoked transient increases in cytoplasmic Ca2+ concentration in dissociated primary neurospheres, demonstrating the functional expression of P2Y1 and P2Y2 receptors. Ca2+ transients were not attenuated by the removal of extracellular Ca2+ and were reversibly inhibited by the P2Y1 selective antagonist, MRS 2179. P2Y and adenosine receptor agonists reduced the size and frequency of primary neurospheres. The effects of ADP beta S and adenosine were reversed by subtype-selective receptor antagonists, demonstrating that P2Y1 and A2a receptors mediate inhibitory effects on primary neurosphere proliferation. The modulation of neural precursor cell proliferation by P2Y and adenosine receptors therefore represents a potential regulatory mechanism within the neurogenic microenvironment. (c) 2007 Elsevier Inc. All rights reserved. Publisher: Elsevier Inc. Coverage: 2007-08-01T00:00:00Z
Adenosine A2 Receptor Presence and Synergy with Cholinergic Stimulation in Rabbit Lacrimal Gland
2010-01-01
Purpose: Secretion from the lacrimal gland is an important part of the well-being of the eye, and a central part in the search for treatment of dry eye syndrome. Adenosine has stimulatory effects on the lacrimal gland, and can potentiate the effect of the cholinergic agonist carbachol (Cch). The aim of the present study is to investigate the presence of the adenosine A2 receptor subtypes A2A and A2B in the rabbit lacrimal gland, and to characterize their role in regulated acinar cell secretion. Methods: Expression of the receptors was investigated using reverse transcriptase-PCR (RT-PCR) and immunofluorescence, and secretion effects were studied using a secretion assay in isolated lacrimal gland acinar cells. Results: Presence of both receptors was detected by RT-PCR and immunofluorescence...
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20060309.084525
Adenosine is a multi-functional physiological molecule found abundantly in the body. It is one of the important components of ATP cellular energy metabolism. Adenosine has diverse actions as a ligand on many different types of cells and tissues acting via specific receptors. Currently, four subtypes of adenosine receptors are described, namely, the A1, A2A, A2B and A3 receptors. Neuroblastoma, mostly found in young children, is a malignant tumor derived from peripheral neurons in the body. Several different types of neuroblastoma cell lines of human origin have been established and contributed to the studies of neuroblastoma itself, neuronal differentiation, neurotransmitters, alcoholism, Alzheimer's disease and other neuronal diseases and disorders. In 1987, it was shown by Abbracchio et al. that a human neuroblastoma cell line, IMR32, could be induced to differentiate into cells that have a more neuronal morphology, with long neurites, by an adenosine receptor agonist 5'-N-ethylcarboxamideadenosine (NECA) 2. 'Neuronal differentiation' is expected to be a new alternative to the conventional clinical therapies, such as surgery, chemotherapy and radiotherapy. Unlike IMR32, PC12 cells, a rat adrenal pheochromocytoma cell line, resembling human neuroblastoma cell lines and also expressing the A2 subtype of adenosine receptors, was shown not to differentiate under stimulation of the A2A subtype of adenosine receptors 3. Moreover, adenosine inhibited neuronal differentiation in mouse dorsal root ganglion cells presumably via the A1 subtype 4. The mechanism(s) of these confusing effects of adenosine on neuronal differentiation require examination. First, a detection method for each of the adenosine receptor subtypes was developed using reverse transcriptase polymerase chain reaction (RT-PCR). This provided a sensitive, non-radioactive, analytical tool. Subtype-specific, four pairs of PCR primers, corresponding to the A1, A2A, A2B and A3 receptors, were designed and synthesized. The RT-PCR study revealed the presence of adenosine A1, A2A and A2B receptor mRNAs in untreated SH-SY5Y cells. These PCR primers were also designed so that they would allow multiplex PCR. Optimization of conditions for multiplex PCR was conducted, allowing it to detect several adenosine receptor subtypes simultaneously, and it was proven to be partially successful. In the study of differentiation, the use of the designed PCR primers was not quantitative to measure the levels of adenosine receptors due to variations of the expressions levels of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, a house-keeping gene commonly used as the internal control in PCR or northern blot analysis. An adequate neuronal differentiation model system was established in order to study the possible role(s) of adenosine in neuronal differentiation. Nerve growth factor (NGF), a well-known inducer of differentiation of rat PC12 cells, did not show any apparent differentiation effects on human neuroblastoma SH-SY5Y cells. All-trans retinoic acid (50 µM) induced distinct neuronal differentiation in SH-SY5Y cells, however ethanol, used as a vehicle for retinoic acid, was also shown to have effects on this cell line causing morphological changes. Adenosine (100 µM) alone also did not induce marked differentiation in this cell line probably due to the presence of adenosine in serum. Adenosine deaminase-resistant, synthetic adenosine analogues were used and demonstrated enhancement of differentiation. A serum deprivation-induced differentiation in SH-SY5Y was found to be a consistent and useful model to evaluate the effects of other factors on differentiation in this cell line. This serum deprivation-induced differentiation was also found to accompany a substantial rise in the expression of neurofilament-H (NF-H), one of the marker proteins for neuronal differentiation, at the protein level. Using this model, the possible involvement of adenosine signaling via its receptors was investigated. Treatment of cells with selective adenosine analogues for the A1 and A2A subtypes, 2-chloro-N6-cyclopentyladenosine (CCPA, 100 nM) and 2-[4-(2-carboxylethyl)phenylamino]-5'-N-ethylcarboxamido (CGS21680, 30 and 100 nM), respectively, enhanced the differentiation induced by serum deprivation at day 7 by approximately 60% and 70%, respectively. These enhancing effects of agonists were blocked by selective antagonists, 8-cyclophenyl-1,3-dipropylxanthine (DPCPX) and 9-chloro-2-(2-furyl)[1,2,4]triazolo[1,5-c]quinalzolin-5-amine (CGS15943), respectively. Simultaneous co-stimulation of the A1 and A2A subtypes with these agonists gave no further effects compared to the enhancing effects exerted by CCPA or CGS21680 alone. Signal transduction pathways were examined using various protein kinase inhibitors. A selective protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89, 100 nM) alone greatly enhanced the differentiation induced by serum deprivation in this cell line. No additive or synergistic effects of 10 nM H-89 with either the A1 or A2A receptor agonist were seen. A selective mitogen-activated protein kinase kinase (MAPKK) inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098,059) showed a similar pattern to H-89: 100 nM PD098,059 alone caused enhanced differentiation in serum deprivation-induced SH-SY5Y cells. The combination of PD098,059 and adenosine agonists did not show any further enhancement of differentiation. On the contrary, a selective protein kinase C (PKC) inhibitor, chelerythrine, suppressed the differentiation (by 51%) by serum deprivation at 1 uM, and at 100 nM, chelerythrine suppressed the enhancement of differentiation caused by CCPA and CGS21680 with no effect on the basic level of differentiation, indicating the possible involvement of PKC both in the differentiation induced by serum deprivation and the adenosine receptor-induced potentiation. Surprisingly, contrary to the assumption that the stimulation of PKA induces or assists neuronal differentiation, H-89 (20 uM) alone exerted a prompt differentiation (44% at day 2) in SH-SY5Y cells in the presence of the normal serum concentration (10%). This data suggests that the previously assumed role of PKA in differentiation must be re-evaluated. This H-89-induced differentiation model was shown to have a different differentiation mechanism to the previous serum deprivation-induced differentiation. Establishment of these new differentiation study models will add further options to explore neuronal differentiation, especially, of human type. Publisher: Griffith University. School of Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Rocky Hiroki Nishimura
2010-01-01
A number of novel xanthines bearing a variety of substituents at positions 1, 3, 7 and 8 were prepared and evaluated for their binding affinity to the human adenosine receptor A1, A2A, A2B and A3 subtypes. Several of the 1,3,8- and 1,3,7,8-substituted xanthines showed moderate-to-high affinity at human A2B and A1 receptors, with the most active compound (14q) having a pKi of 7.57 nM for hA2B receptors and a selectivity over hA2A receptors of 8.1-fold and hA1 receptors of 3.7-fold.
2010-01-01
G protein-coupled receptors (GPCRs) are therapeutic targets for many diseases, but progress in developing active and selective therapeutics has been severely hampered by the difficulty in obtaining accurate structures. We have been developing methods for predicting the structures for GPCR ligand complexes, but validation has been hampered by a lack of experimental structures with which to compare our predictions. We report here the predicted structures of the human adenosine GPCR subtypes (A1, A2A, A2B, and A3) and the binding sites for adenosine agonist and eight antagonists to this predicted structure, making no use of structural data, and compare with recent experimental crystal structure for ZM241385 bound human A2A receptor. The predicted structure correctly identifies 9 of the 12 cry...
http://hdl.handle.net/10072/29784
Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 μM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10-7M) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA; 10-7M) reduced the proportion of nonviable cells to 30.87 ± 2.49% and 35.18 ± 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 ± 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 ± 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 ± 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection. Publisher: http://dx.doi.org/10.1097/FJC.0b013e3181a443e2; Lippincott Williams & Wilkins; United States; http://journals.lww.com/cardiovascularpharm/pages/default.aspx Contributor: Michael R Rosen Relation: 5; Journal of Cardiovascular Pharmacology; 424; 433; Y; 53 Other identifier: 0160-2446 Language: en_AU Rights: Y
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20051130.124230
While previous research into the pathogenesis of ischaemic and reperfusion injuries has focussed on the cardiac myocyte, there is increasing evidence for a crucial role for coronary vascular injury in the genesis of the post-ischaemic phenotype [1-3]. Post-ischaemic vascular injury may be manifest as a transient or sustained loss of competent vessels, impairment of vascular regulatory mechanisms, and ultimately as the 'no-reflow' phenomenon (inability to sufficiently reperfuse previously ischaemic tissue despite the removal of the initial obstruction or occlusion). It is now appreciated that the earliest distinguishing feature of various forms of vascular injury (including atherosclerosis and infarction) is 'endothelial dysfunction', which is the marked reduction in endothelial-dependent relaxation due to reduced release or action of endothelial nitric oxide (NO). Importantly, vascular injury may worsen myocardial damage in vivo [4,5], significantly limiting tissue salvage and recovery. The pathogenesis of post-ischaemic vascular injury and endothelial dysfunction is incompletely understood, but has generally been considered to reflect a cardiovascular inflammatory response, neutrophils playing a key role. However, while blood-borne cells and inflammatory elements are undoubtedly involved in the 'progression' of vascular injury [6,7], accumulating evidence indicates that they are not the primary 'instigators' [8]. It should be noted that a wealth of controversial findings exists in the vascular injury literature and mechanisms involved remain unclear. Indeed, multiple mechanisms are likely to contribute to post-ischaemic vascular injury. Adenosine receptors are unique in playing a role in physical regulation of coronary function, and also in attenuating injury during and following ischaemia. While the adenosine receptor system has been extensively investigated in terms of effects on myocardial injury [9,10], little is known regarding potential effects of this receptor system on post-ischaemic coronary vascular injury. This thesis initially attempts to further our understanding of the role of adenosine in normal coronary vascular function, subsequent chapters assess the effect of ischaemia-reperfusion on vascular function, and adenosine receptor modification of vascular dysfunction in the isolated asanguinous mouse heart. Specifically, in Chapter 3 the receptor subtype and mechanisms involved in adenosine-receptor mediated coronary vasodilation were assessed in Langendorff perfused mouse and rat hearts. The study revealed that A2A adenosine receptors (A2AARs) mediate coronary dilation in the mouse vs. A2B adenosine receptors (A2BARs) in rat. Furthermore, responses in mouse involve a sensitive endothelial-dependent (NO-dependent) response and NO-independent (KATP-mediated) dilation. Interestingly, the ATP-sensitive potassium channel component predominates over NO-dependent dilation at moderate to high agonist levels. However, the high-sensitivity NO-dependent response may play an important role under physiological conditions when adenosine concentrations and the level of A2AAR activation are low. In Chapter 4 the mechanisms regulating coronary tone under basal conditions and during reactive hyperaemic responses were assessed in Langendorff perfused mouse hearts. The data support a primary role for KATP channels and NO in mediating sustained elevations in flow, irrespective of occlusion duration (5-40 s). However, KATP channels are of primary importance in mediating initial flow adjustments after brief (5-10 s) occlusions, while KATP (and NO) independent processes are increasingly important with longer (20-40 s) occlusion. Evidence is also presented for compensatory changes in KATP and/or NO mediated dilation when one pathway is blocked, and for a modest role for A2AARs in reactive hyperaemia. In Chapter 5 the impact of ischaemia-reperfusion on coronary function was examined, and the role of A1 adenosine receptor (A1AR) activation by endogenous adenosine in modifying post-ischaemic vascular function was assessed in isolated buffer perfused mouse hearts. The results demonstrate that ischaemia does modify vascular control and signficantly impairs coronary endothelial dilation in a model devoid of blood cells. Additionally, the data indicate that post-ischaemic reflow is significantly determined by A2AAR activation by endogenous adenosine, and that A1AR activation by endogenous adenosine protects against this model of vascular injury. Following from Chapter 5, the potential of A1, A2A and A3AR activation by exogenous and endogenous agonists to modulate post-ischaemic vascular dysfunction was examined in Chapter 6. Furthermore, potential mechanisms involved injury and protection were assessed by comparing effects of adenosine receptors to other 'vasoprotective' interventions, including anti-oxidant treatment, Na+/H+ exchange (NHE) inhibition, endothelin (ET) antagonism, and 2,3-butanedione monoxime (BDM). The data acquired confirm that post-ischaemic endothelial dysfunction is reduced by intrinsic A1AR activation, and also that exogenous A3AR activation potently reduces vascular injury. Protection appears unrelated to inhibition of ET or oxidant stress. However, preliminary data suggest A3AR vasoprotection may share signalling with NHE inhibition. Finally, the data reveal that coronary reflow in isolated buffer perfused hearts is not a critical determinant of cardiac injury, suggesting independent injury processes in post-ischaemic myocardium vs. vasculature. Collectively, these studies show that adenosine has a significant role in regulating coronary vascular tone and reactive hyperaemic responses via NO and KATP dependent mechanisms. Ischaemia-reperfusion modifies vascular control and induces significant endothelial dysfunction in the absence of blood, implicating neutrophil independent injury processes. Endogenous adenosine affords intrinsic vasoprotection via A1AR activation, while adenosinergic therapy via exogenous A3AR activation represents a new strategy for directly protecting against post-ischaemic vascular injury. Publisher: Griffith University. School of Health Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Amanda J Zatta
http://espace.library.uq.edu.au/view/UQ:158524
Purinergic receptors mediate a wide variety of biological effects in response to extracellular nucleotides and nucleosides, such as ATP, ADP, UTP, UDP and adenosine. To date, nineteen purinergic receptor subtypes have been identified and classified based on their sequence identity, pharmacological properties and signal transduction mechanisms and include the ionotropic P2X receptors (P2X1 - 7) and the G-protein coupled P2Y (P2Y1, 2, 4, 6, 12 - 14) and adenosine receptors (A1, A2a, A2b and A3). Purinergic receptor activation leads to changes in cytoplasmic [Ca2+] and/or [cAMP] and the modulation of mitogen activated protein kinase (MAP kinase) signalling cascades, which can result in mitogenic and/or inhibitory effects on cellular proliferation, migration and differentiation. In the central nervous system (CNS), extracellular ATP is released by neurons, astrocytes and vascular endothelial cells and plays a role in neurotransmission and neuromodulation. Furthermore, extracellular ATP acts as a critical signalling molecule for neuron-glial communication and is involved in the propagation of intercellular Ca2+ waves between astrocytes. The observation that purinergic signalling is an important factor in intercellular signalling, and has been shown to regulate proliferation, differentiation and survival in many cell types, has raised the possibility that purinergic receptors may also play a role in neurogenesis and development. Results presented in Chapter 3 demonstrate that undifferentiated neural progenitor cells isolated from the adult rat hippocampus express mRNA for all P2X receptor subtypes. The analysis of intracellular [Ca2+] transients in response to purinergic receptor agonists and other neurotransmitters show that hippocampal NPCs respond predominantly to ATP, ADP and glutamate via P2X7/P2X5 receptors, P2Y1 receptors and metabotropic glutamate receptors, respectively. Other neurotransmitters, such as GABA, glycine and acetylcholine were without effect. Electrophysiological measurements support these data and show that undifferentiated hippocampal NPCs share similar passive electrical properties to astrocytes, including electrical inexcitability and a high resting membrane potential (Q - 90 mV), providing a substantial electrochemical gradient for Ca2+ influx from the extracellular space via open P2X receptor ion channels. A more detailed analysis of purinergic receptor expression and function was conducted in primary neurosphere-derived NPCs generated in vitro from the subventricular zone (SVZ) of adult mice and in neural stem cells purified using flow cytometry (FACS) from acutely dissociated SVZ tissue. Results presented in Chapter 4 demonstrate that primary neurosphere-derived NPCs express similar purinergic receptors to FACS-purified neural stem cells. Both populations express mRNA for P2X4 and P2X7 receptors, all P2Y receptors, with the exception of the P2Y4 and the A1, A2a and A2b adenosine receptors. However, the neural stem cell population also expressed P2X1 and A3 receptor mRNA. ATP and the P2X7 receptor agonist, BzATP, evoked transient increases in cytoplasmic [Ca2+] in FACSpurified neural stem cells acutely dissociated from the SVZ, suggesting the functional expression of purinergic receptors. Furthermore, in similarity to NPCs isolated from the adult rat hippocampus, SVZ-derived NPCs exhibited transient increases in cytoplasmic [Ca2+] in response to purinergic and glutamatergic agonists but were insensitive to other neurotransmitters. Pharmacological profiling demonstrated that SVZ-derived NPCs express functional P2Y1 and P2Y2 receptors and a proportion of cells (Q 30%) showed transient increases in cytoplasmic [Ca2+] in response to the P2X7 receptor agonist BzATP, suggesting the functional expression of P2X7 and/or P2X4 receptors. Adenosine failed to elicit a Ca2+ response and did not significantly modulate basal levels of cAMP, however, A1 and A2a adenosine receptor protein was detected in primary neurospheres by western blot analysis. Having demonstrated that NPCs and neural stem cells derived from the SVZ of adult mice express functional purinergic receptors, we then investigated the effects of purinergic receptor activation on primary neurosphere frequency and proliferation in vitro. Experiments presented in Chapter 5 demonstrate that ATPyS, ADPbS and adenosine reduce the size and frequency of primary neurospheres in the presence of mitogens; epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), suggesting that crosstalk may occur between purinergic receptor and growth factor receptor signalling cascades associated with neural progenitor cell mitogenesis and survival. The inhibitory effect of ADPbS was antagonized by the P2Y1 receptor antagonist MRS 2179 and fully reversed by the P2Y12 receptor antagonist MRS 2395, which also antagonized adenosine receptors. The inhibitory effect of adenosine was attenuated by the A2a antagonist SCH 58261, and the A3 antagonist MRS 1523. Taken together, P2Y1, A2a and/or A3 receptors mediate inhibitory effects on growth factor-stimulated proliferation in primary neurospheres derived from the adult mouse SVZ. In passaged neurospheres, however, P2Y1 receptor activation potentiated NPC proliferation, whereas adenosine was inhibitory suggesting a change in ADP-sensitive P2Y receptor signalling occurs in NPCs selected for, and enriched over successive passages in the presence of mitogens EGF and bFGF. Neural progenitor cells have been reported to reside in a niche environment in close proximity to neurons astrocytes and the brain microvasculature. The functional expression of P2Y1, P2Y2 and P2X7 receptors in NPCs may enable these cells to participate in and/or respond to localized intercellular Ca2+ signalling within the neurogenic niche. Moreover, the modulation of neural stem cell and NPC proliferation by P2Y and adenosine receptor signalling pathways may represent a potential modulatory mechanism within the stem cell microenvironment (Stafford et al., in press).
2010-01-01
Adenosine receptors (ARs) belong to the G-protein-coupled receptor (GPCR) superfamily and consist of four subtypes referred to as A1, A2A, A2B, and A3. It is important to develop potent and selective modulators of ARs for therapeutic applications. In order to develop reliable in silico models that can effectively classify antagonists of each AR, we carried out three machine learning methods: Laplacian-modified naive Bayesian, recursive partitioning, and support vector machine. The results for each classification model showed values high in accuracy, sensitivity, specificity, area under the receiver operating characteristic curve and Matthews correlation coefficient. By highlighting representative antagonists, the models demonstrated their power and usefulness, and these models could be uti...
2010-01-01
The synthesis of oligo(ethylene glycol)-alkene substituted theophyllines in positions 7 and/or 8 is described. The binding activity at adenosine receptors of selected derivatives was studied. Compound 2 showed high affinity for human A2B receptor (Ki=4.16nM) with a selectivity KiA2A/KiA2B of 24.1, and a solubility in water of 1mM. The alkenyl substituent in some of the theophylline derivatives allows for covalent attachment of them onto hydrogen-terminated silicon substrate surfaces via hydrosilylation. Alternatively, an azido group was incorporated to an oligo(ethylene glycol)theophylline derivative as an anchor for tethering the molecules on ethynyl presenting surfaces via click reaction.
2010-01-01
Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A1 and A2A adenosine receptors (ARs), but not of A2B and A3ARs. Activation of the heterologously expressed human A3AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H2O2 (750mM) but not in untransfected cells. The A3 agonist IB-MECA (EC50 3.8mM) and the non-selective agonist NECA (EC50 3.9mM) protected A3 AR-transfected cells against H2O2 in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendri...
2010-01-01
A new series of 9-deazaxanthine derivatives with various substituents at the heterocyclic system were synthesized and evaluated for their binding affinities for the four human recombinant adenosine receptors, A1–A3 subtypes. A number of the 9-deazaxanthines derivatives 3a–m showed moderate-to-high affinity for hA2B receptors, with compound 3f showing a 32-fold selectivity for A2B over A1 and a 2750-fold selectivity for A2B over A2A.
http://hdl.handle.net/10072/6190
In our laboratory we have developed a quantitative-polymerase chain reaction (Q-PCR) strategy to examine the differential expression of adenosine receptor (ADOR), A1, A2A, A2B and A3, and estrogen receptors (ER) α and β. Brain and uterine mRNA were first used to optimise specific amplification conditions prior to SYBR Green I real time analysis of receptor subtype expression. SYBR Green I provided a convenient and sensitive means of examining specific PCR amplification product in real time, and allowed the generation of standard curves from which relative receptor abundance could be determined. Real time Q-PCR analysis was then performed, to examine changes in receptor expression levels in brains of adult female Wistar rats 3-month post ovariectomy. Comparison with sham-operated age-matched control rats demonstrated both comparative and absolute-copy number changes in receptor levels. Evaluation of both analytical methods investigated 18S rRNA as an internal reference for comparative gene expression analysis in the brain. The results of this study revealed preferential repression of ADORA2A (>4-fold down) and consistent (>2-fold) down-regulation of ADORA1, ADORA3, and ER-β, following ovariectomy. No change was found in ADORA2B or ER-α. Analysis of absolute copy number in this study revealed a correlation between receptor expression in response to ovariectomy, and relative receptor subtype abundance in the brain. Publisher: http://dx.doi.org/10.1016/S1385-299X(02)00219-2; Elsevier BV; Netherlands; http://www.elsevier.com/wps/find/journaldescription.cws_home/622287/description#description Contributor: F G Wouterlood Relation: Brain Research Protocols; 9; 18; 11 Other identifier: 1385-299X Language: en_AU Rights: Copyright 2003 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links
http://hdl.handle.net/10072/29430
We tested the impact of A1 adenosine receptor (AR) deletion on injury and oxidant damage in mouse hearts subjected to 25 min ischemia/45 min reperfusion (I/R). Wild-type hearts recovered ~50% of contractile function, and released 8.2±0.7 IU/g of lactate dehydrogenase (LDH). A1AR deletion worsened dysfunction and LDH efflux (15.2±2.6 IU/g). Tissue cholesterol and native cholesteryl esters were unchanged, while cholesteryl ester-derived lipid hydroperoxides and hydroxides (CE-O(O)H; a marker of lipid oxidation) increased 3-fold, and α-tocopherylquinone (α-TQ; oxidation product of α-tocopherol (α-TOH)) increased 6-fold. Elevations in α-TQ were augmented 2- to 3-fold by A1AR deletion, whereas CE-O(O)H was unaltered. A1AR deletion also decreased glutathione redox status ([GSH]/[GSSG+GSH]) and enhanced expression of the antioxidant response element heme oxygenase-1 (HO-1) during I/R: 4-fold elevations in HO-1 mRNA and activity were doubled by A1AR deletion. Broad-spectrum AR agonism (10 µM 2-chloroadenosine; 2-CAD) countered effects of A1AR deletion on oxidant damage, HO-1, and tissue injury, indicating additional ARs (A2A, A2B and/or A3) can mediate similar actions. These data reveal local adenosine engages A1ARs during I/R to selectively limit oxidant damage and enhance outcome. Control of α-TOH/α-TQ levels may contribute to A1AR-dependent cardioprotection. Publisher: http://dx.doi.org/10.1089/ars.2009.2644; Mary Ann Liebert, Inc.; United States Relation: 11; Antioxidants & Redox Signaling; 2641; 2650; N; 11 Other identifier: 1523-0864 Language: en_AU Rights: Copyright 2009 Mary Ann Liebert, Inc., publishers. Self-archiving of the author-manuscript version is not yet supported by this publisher. Please refer to the journal link for access to the definitive, published version or contact the authors for more information.; Y
http://hdl.handle.net/10536/DRO/DU:30023463
N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor. Publisher: Deakin University, Faculty of Science and Technology, School of Biological and Chemical Sciences Relation: isMemberOf: Higher degree theses (full text) collection Coverage: 1999-01-01 Language: eng
http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20050915.160941
N-Ethylcarboxamidoadenosine (12) was synthesised from adenosine (1) and the 6-chloro-2’,3’-O-isopropylidene-AT-ethylcarboxamidoadenosine (25) was synthesised from inosine (19). Employing molecular modelling techniques and the results from previous structure activity relationships it was possible to design and synthesise a N6-substituted N-ethylcarboxamidoadenosines which possessed an oxygen in the N6-substituent either in the form of an epoxide (which was obtained by cpoxidising an alkene with m-CPBA or dimethyldioxirane) or in the form of a cyclic ether as was the case for N6-((tetrahydro-2H--pyran--2-yl)methyl-N-ethylcarboxamidoadenosine (78). These compounds were tested for their biological activity at the A1 adenosine receptor by their ability to inhibit cAMP accumulation in DDT, MF2 cells. The EC50 values obtained indicated that the N6-(norborn-5-en-2-yl)-N-ethylcarboxamidoadenosines were the most potent. Of theseN6-(S-endo-norbrn-5-en-2-yI)-N-ethylcarboxaniidoadenosine (56) was the most potent (0.2 nM). N6-(exo-norborn-5-en-2-yl)-2-iodo-N-ethylcarboxamidoadenosine (79) was synthesised from guanosine (22) and was also evaluated for its potency at the A, receptor (24.8 ± 1.5 nM). At present 79 is being evaluated for its selectivity for the A1 receptor compared to the other three receptor subtypes (A2a, A2b, A3). A series of N6-(benzyl)-N-ethylcarboxamidoadenosines were synthesised with substitutions at the 4-position of the phenyl ring. Another series of compounds were synthesised which replaced the methylene spacer between the N6H and the N6-aromatic or lipophilic substituent The replacement groups -were carbonyl and trans-2- cyclopropyl moieties. The N6-acyl compounds were obtained by reacting 2’,3’-O- di(tert-butyldimethylsilyl)-AT-ethylcarboxamidoadenosinc (59) with the appropriate acid chloride and then deprotecting with lelrabutylammonium fluoride in tetrahydrofuran. The compound N6-(4-(1,2-dihydroxy)ethyl)benzyl-N- ethylcarboxamidoadenosine (125) was synthesised by the reaction of 4-(1,2-0- isopropylidene-ethyl)benzyl aminc (123) with 6-chloro-2,3-0-isopropylidene-N- ethylcarboxamidoadenosine (25). Compound 123 was synthesised from an epoxidation of vinylbenzyl phthalimide (118) followed by an acidic ring opening to yield the diol which was isopropylidenated to yield 4-(l,2-O-isopropylidene- elhyl)benzyl phlhalimide (122), It was hoped that the presence of the diol functionality in 125 would increase water solubility whilst maintaining potency at the A3 receptor. Publisher: Deakin University. School of Biological and Chemical Sciences Language: en Rights: http://www.deakin.edu.au/disclaimer.html); Copyright Denis Matthew John Wright
http://hdl.handle.net/10072/14270
This study evaluated the ability of A1 and A3 adenosine receptor (AR) agonism, and A1, A2A, A2B and A3AR antagonism (revealing "intrinsic" responses), to modify post-ischemic coronary dysfunction in mouse heart. Vascular function was assessed before and after 20 min global ischemia and 30-45 min reperfusion in Langendorff perfused C57/Bl6 mouse hearts. Ischemic insult impaired coronary sensitivity to the endothelial-dependent dilators ADP (pEC50=6.8+/-0.1 vs. 7.6+/-0.1, non-ischemic) and acetylcholine (pEC50=6.1+/-0.1 vs. 7.3+/-0.1 in non-ischemic), and for the mixed endothelial-dependent/independent dilator 2-chloroadenosine (pEC50=7.5+/-0.1 vs. 8.4+/-0.1, non-ischemic). Endothelium-independent dilation in response to nitroprusside was unaltered (pEC50=7.0+/-0.1 vs. 7.1+/-0.1 in non-ischemic). Pre-treatment with a selective A1AR agonist (50 nM CHA) failed to modify coronary dysfunction, whereas A1AR antagonism (200 nM DPCPX) worsened the effects of I/R (2-chloroadenosine pEC50=6.9+/-0.1). Conversely, A3AR agonism (100 nM Cl-IB-MECA) did reduce effects of I/R (pEC50s=8.0+/-0.1 and 7.3+/-0.1 for 2-chloroadenosine and ADP, respectively), whereas antagonism (100 nM MRS1220) was without effect. While A2AAR agonism could not be assessed (due to pronounced vasodilatation), A2AAR antagonism (100 nM SCH58261) was found to exert no effect, and antagonism of A2BARs (50 nM MRS1754) was also ineffective. The protective actions of A3AR agonism were also manifest as improved reactive hyperemic responses. Interestingly, post-ischemic coronary dysfunction was also limited by: Na+-H+ exchange (NHE) inhibition with 10 or 50 microM BIIB-513 (2-chloroadenosine pEC50s=7.8+/-0.1, either dose), an effect not additive with A3AR agonism; Ca2+ antagonism with 0.3 microM verapamil (2-chloroadenosine pEC50=7.9+/-0.1); and Ca2+ desensitization with 5 mM BDM (2-chloroadenosine pEC50=7.8+/-0.1). In contrast, endothelin antagonism (200 nM PD142893) and anti-oxidant therapy (300 microM MPG+150 U/ml SOD+600 U/ml catalase) were ineffective. Our data collectively confirm that ischemia selectively impairs endothelial function and reactive hyperemia independently of blood cells. Vascular injury is intrinsically limited by endogenous (but not exogenous) activation of A1ARs, whereas exogenous A3AR activation further limits dysfunction (improving post-ischemic vasoregulation). Finally, findings suggest this form of post-ischemic coronary injury is unrelated to endothelin or oxidant stress, but may involve modulation of Ca2+ overload and/or related ionic perturbations. Publisher: Elsevier Inc; New York, NY Relation: 2006; 21; Life sciences; 2426; 2437; N; 78 Other identifier: 0024-3205 Language: en_AU Rights: N
Molecular mechanisms involved in adenosine-induced endothelial cell barrier enhancement
2010-01-01
Extracellular adenosine is a physiologically relevant agonist released by various sources, including endothelial cells (EC) and activated platelets, with complex effects mediated via activation of P1 purinergic receptors. Adenosine-induced EC production of glutathione peroxidase1 and nitric oxide is recognized, and an anti-inflammatory mechanism has been described. Effects of extracellular adenosine on the pulmonary EC barrier function and vascular permeability, however, remain poorly characterized. In this study, we demonstrated the adenosine-induced rapid dose-dependent barrier enhancement in human pulmonary artery EC (HPAEC) as measured by an increase in transendothelial electrical resistance (TER). We have shown that HPAEC express only A2A and A2B adenosine receptors. Pharmacological a...
http://hdl.handle.net/2440/7298
None Available Publisher: Academic Press Other identifier: Genomics. 25:605-607; 0888-7543; 0019951025 Language: en_US
Levocetirizine, the R-enantiomer of cetirizine dihydrochloride has pharmacodynamically and pharmacokinetically favourable characteristics, including rapid onset of action, high bioavailability, high...Full Text Available
Full Text Available.Levocetirizine, the R-enantiomer of cetirizine dihydrochloride has pharmacodynamically and pharmacokinetically favourable characteristics, including rapid onset of action, high bioavailability, high affinity for and occupancy of the H1-receptor, limited distribution, minimal hepatic metabolism together with minimal untoward effects. Several well conducted randomised clinical trials have demonstrated the effectiveness of levocetirizine for the treatment of allergic rhinitis and chronic idiopathic urticaria in adults and children. In addition to the treatment for the immediate short-term manifestations of allergic disease, there appears to be a growing trend for the use of levocetirizine as long-term therapy. In addition to its being a potent antihistamine, levocetirizine has several documented anti-inflammatory effects that are observed at clinically relevant concentrations that may enhance its therapeutic benefit. This review will consider the potential or otherwise of the reported anti-inflammatory effects of levocetirizine to enhance its effectiveness in the treatment of allergic disease.
TNF-α and neuropathic pain - a review
Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous...Full Text Available
TNF-α and neuropathic pain - a review
Full Text Available.Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous system. Neuropathic pain is a recognized type of pathological pain where nociceptive responses persist beyond the resolution of damage to the nerve or its surrounding tissue. Very often, neuropathic pain is disproportionately enhanced in intensity (hyperalgesia) or altered in modality (hyperpathia or allodynia) in relation to the stimuli. At time of this writing, there is as yet no common consensus about the etiology of neuropathic pain - possible mechanisms can be categorized into peripheral sensitization and central sensitization of the nervous system in response to the nociceptive stimuli. Animal models of neuropathic pain based on various types of nerve injuries (peripheral versus spinal nerve, ligation versus chronic constrictive injury) have persistently implicated a pivotal role for TNF-α at both peripheral and central levels of sensitization. Despite a lack of success in clinical trials of anti-TNF-α therapy in alleviating the sciatic type of neuropathic pain, the intricate link of TNF-α with other neuro-inflammatory signaling systems (e.g., chemokines and p38 MAPK) has indeed inspired a systems approach perspective for future drug development in treating neuropathic pain.
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
The recent determination of X-ray structures of pharmacologically relevant...Full Text Available
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
Full Text Available.The recent determination of X-ray structures of pharmacologically relevant GPCRs has made these targets accessible to structure-based ligand discovery. Here we explore whether novel chemotypes may be discovered for the A2A adenosine receptor, based on complementarity to its recently determined structure. The A2A adenosine receptor signals in the periphery and the CNS, with agonists explored as anti-inflammatory drugs and antagonists explored for neurodegenerative diseases. We used molecular docking to screen a 1.4 million compound database against the X-ray structure computationally and tested 20 high-ranking, previously unknown molecules experimentally. Of these 35% showed substantial activity with affinities between 200 nM and 9 μM. For the most potent of these new inhibitors, over 50-fold specificity was observed for the A2A versus the related A1 and A3 subtypes. These high hit rates and affinities at least partly reflect the bias of commercial libraries toward GPCR-like chemotypes, an issue that we attempt to investigate quantitatively. Despite this bias, many of the most potent new ligands were novel, dissimilar from known ligands, providing new lead structures for modulation of this medically important target.
Signaling from Axon Guidance Receptors
2010-05-01
Full Text Available.Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues—netrins, semaphorins, ephrins, and slits—have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.
Signaling from Axon Guidance Receptors
2010-05-01
Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote...Full Text Available
Role of adenosine A2 receptors in regulation of cerebral blood flow during induced hypotension
2010-01-01
The mechanisms responsible for vascular autoregulation in the brain during changes in mean arterial blood pressure are ambiguous. Potentially, adenosine, a purine nucleoside and potent vasodilator, may be involved as earlier studies have documented an increase in brain adenosine concentrations with cerebral ischemia and hypotension. Consequently, we tested the hypothesis that adenosine is involved in vasodilatation during hypotension within the autoregulatory range (>50 mm Hg) by exposing adenosine 2a receptor (A2aR) knockout and wild type (WT) mice to short (2 to 5 mins) periods of hypotension. We found that autoregulation was significantly (PA2b:10–85>1), ...
Full Text Available.BackgroundChronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions of people worldwide, but their pathogenesis is still not well understood.It is also not well known if distinct changes in gene expression characterize these diseases and if these patterns can discriminate between diseased and control patients and/or stratify the disease. The main focus of our work was the identification of novel markers that overlap among the 3 diseases or discriminate them from each other.MethodsDiseased (n = 13, n = 15 and n = 12 in IBD, psoriasis and RA respectively) and healthy patients (n = 18) were recruited based on strict inclusion and exclusion criteria; peripheral blood samples were collected by clinicians (30 ml) in Venous Blood Vacuum Collection Tubes containing EDTA and peripheral blood mononuclear cells were separated by Ficoll gradient centrifugation. RNA was extracted using Trizol reagent. Gene expression data was obtained using TaqMan Low Density Array (TLDA) containing 96 genes that were selected by an algorithm and the statistical analyses were performed in Prism by using non-parametric Mann-Whitney U test (P-values < 0.05).ResultsHere we show that using a panel of 96 disease associated genes and measuring mRNA expression levels in peripheral blood derived mononuclear cells; we could identify disease-specific gene panels that separate each disease from healthy controls. In addition, a panel of five genes such as ADM, AQP9, CXCL2, IL10 and NAMPT discriminates between all samples from patients with chronic inflammation and healthy controls. We also found genes that stratify the diseases and separate different subtypes or different states of prognosis in each condition.ConclusionsThese findings and the identification of five universal markers of chronic inflammation suggest that these diseases have a common background in pathomechanism, but still can be separated by peripheral blood gene expression. Importantly, the identified genes can be associated with overlapping biological processes including changed inflammatory response. Gene panels based on such markers can play a major role in the development of personalized medicine, in monitoring disease progression and can lead to the identification of new potential drug targets in chronic inflammation.
BackgroundChronic inflammatory diseases including inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis), psoriasis and rheumatoid arthritis (RA) afflict millions...Full Text Available
Full Text Available.BackgroundMany studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes with cocaine abstinence. Persistent changes in gene expression, as well as alterations induced during abstinence may underlie long-lasting drug craving and relapse liability.ResultsWhole-genome expression analysis was conducted on a rat cocaine binge-abstinence model that has previously been demonstrated to engender increased drug seeking and taking with abstinence. Gene expression changes in two mesolimbic terminal fields (mPFC and NAc) were identified in a comparison of cocaine-naïve rats with rats after 10 days of cocaine self-administration followed by 1, 10, or 100 days of enforced abstinence (n = 6-11 per group). A total of 1,461 genes in the mPFC and 414 genes in the NAc were altered between at least two time points (ANOVA, p < 0.05; ± 1.4 fold-change). These genes can be subdivided into: 1) changes with cocaine self-administration that do not persist into periods of abstinence, 2) changes with cocaine self-administration that persist with abstinence, 3) and those not changed with cocaine self-administration, but changed during enforced abstinence. qPCR analysis was conducted to confirm gene expression changes observed in the microarray analysis.ConclusionsTogether, these changes help to illuminate processes and networks involved in abstinence-induced behaviors, including synaptic plasticity, MAPK signaling, and TNF signaling.
BackgroundMany studies of cocaine-responsive gene expression have focused on changes occurring during cocaine exposure, but few studies have examined the persistence of these changes...Full Text Available
2010-01-01
Proteins of the G-protein coupled receptor (GPCR) family present numerous attractive targets for rational drug design, but also a formidable challenge for identification and conformational modeling...Full Text Available
2010-01-01
Full Text Available.Proteins of the G-protein coupled receptor (GPCR) family present numerous attractive targets for rational drug design, but also a formidable challenge for identification and conformational modeling of their 3D structure. A recently performed assessment of blind predictions of adenosine A2a receptor (AA2AR) structure in complex with ZM241385 (ZMA) antagonist provided a first example of unbiased evaluation of the current modeling algorithms on a GPCR target with ~30% sequence identity to the closest structural template. Several of the 29 groups participating in this assessment exercise (Michino et al., doi:10.1038/nrd2877) successfully predicted the overall position of the ligand ZMA in the AA2AR ligand binding pocket, however models from only three groups captured more than 40% of the ligand-receptor contacts.Here we describe two of these top performing approaches, in which all-atom models of the AA2AR were generated by homology modeling followed by ligand guided backbone ensemble receptor optimization (LiBERO). The resulting AA2AR-ZMA models, along with the best models from other groups are assessed here for their virtual ligand screening (VLS) performance on a large set of GPCR ligands.We show that ligand guided optimization was critical for improvement of both ligand-receptor contacts and VLS performance as compared to the initial raw homology models. The best blindly predicted models performed on par with the crystal structure of AA2AR in selecting known antagonists from decoys, as well as from antagonists for other adenosine subtypes and AA2AR agonists. These results suggest that despite certain inaccuracies, the optimized homology models can be useful in the drug discovery process.
2009-11-04
Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A2AR) decrease pro-inflammatory cytokine release and increase release of the...Full Text Available
2009-11-04
Full Text Available.Previous studies of peripheral immune cells have documented that activation of adenosine 2A receptors (A2AR) decrease pro-inflammatory cytokine release and increase release of the potent anti-inflammatory cytokine, interleukin-10 (IL-10). Given the growing literature supporting that glial proinflammatory cytokines importantly contribute to neuropathic pain, and that IL-10 can suppress such pain, we evaluated the effects of intrathecally (i.t.) administered A2AR agonists on neuropathic pain using the chronic constriction injury (CCI) model. A single i.t. injection of the A2AR agonists ATL313 or CGS21680, 10-14 d after CCI versus sham surgery, produced a long-duration reversal of mechanical allodynia and thermal hyperalgesia for at least 4 wk. Neither drug altered the nociceptive responses of sham-operated controls. An A2AR antagonist (ZM241385) co-administered i.t. with ATL313 abolished the action of ATL313 in rats with neuropathy-induced allodynia, but had no effect on allodynia in the absence of the A2AR agonist. ATL313 attenuated CCI-induced upregulation of spinal cord activation markers for microglia and astrocytes in the L4-L6 spinal cord segments both 1 wk and 4 wk after a single i.t. ATL313 administration. Neutralizing IL-10 antibodies administered i.t. transiently abolished the effect of ATL313 on neuropathic pain. In addition, IL-10 mRNA was significantly elevated in the CSF cells collected from the lumbar region. Activation of A2ARs following i.t. administration may be a novel, therapeutic approach for the treatment of neuropathic pain by increasing IL-10 in the immunocompetent cells of the CNS.
CD73 represses pro-inflammatory responses in human endothelial cells
Full Text Available.BackgroundCD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been reported to regulate expression of pro-inflammatory molecules in mouse endothelium. Our aim is to determine the function of CD73 in human endothelial cells.MethodsWe used RNAi to deplete CD73 levels in human umbilical cord endothelial cells (HUVECs).ResultsCD73 depletion resulted in a strong reduction in adenosine production, indicating that CD73 is the major source of extracellular adenosine in HUVECs. We find that CD73 depletion induces a similar response to pro-inflammatory stimuli such as the cytokine TNF-α. In CD73-depleted cells, surface levels of the leukocyte adhesion molecules ICAM-1, VCAM-1 and E-selectin increase. This correlates with increased translocation of the transcription factor NF-kB to the nucleus, which is known to regulate ICAM-1, VCAM-1 and E-selectin expression in response to TNF-α. Adhesion of monocytic cells to endothelial cells is enhanced. In addition, CD73-depleted cells become elongated, have higher levels of stress fibres and increased endothelial permeability, resembling known responses to TNF-α.ConclusionsThese results indicate that CD73 normally suppresses pro-inflammatory responses in human endothelial cells.
CD73 represses pro-inflammatory responses in human endothelial cells
BackgroundCD73 is a 5'-ectonucleotidase that produces extracellular adenosine, which then acts on G protein-coupled purigenic receptors to induce cellular responses. CD73 has been...Full Text Available
2009-12-01
Full Text Available.Brucella spp. establish an intracellular replicative niche in macrophages, while macrophages attempt to eliminate the bacteria by innate defense mechanisms. Brucella spp. possess similar genomes yet exhibit different macrophage infections. Few B. melitensis and B. neotomae enter macrophages with intracellular adaptation occurring over 4–8 hr. Conversely, B. ovis are readily ingested by macrophages and exhibit a persistent plateau of infection. Evaluating early macrophage interaction with Brucella spp. allows discovery of host entry and intracellular translocation mechanisms. Microarray analysis of macrophage transcriptional response following a 4 hr infection by different Brucella spp. revealed common macrophage genes altered in expression compared to uninfected macrophages. Macrophage infection with three different Brucella spp. provokes a common innate immune theme with increased transcript levels of chemokines and defense response genes and decreased transcript levels of GTPase signaling and cytoskeletal function that may affect trafficking of Brucella containing vesicles. For example, transcript levels of genes associated with chemotaxis (IL-1β, MIP-1α), cytokine regulation (Socs3) and defense (Fas, Tnf) were increased, while transcript levels of genes associated with vesicular trafficking (Rab3d) and lysosomal associated enzymes (prosaposin) were decreased. Genes with altered macrophage transcript levels among Brucella spp. infections may correlate with species specific host defenses and intracellular survival strategies. Depending on the infecting Brucella species, gene ontology categorization identified genes differentially involved in cell growth and maintenance, endopeptidase inhibitor activity and G-protein mediated signaling. Examples of decreased gene expression in B. melitensis infection but not other Brucella spp. were growth arrest (Gas2), immunoglobulin receptor (FcγrI) and chemokine receptor (Cxcr4) genes, suggesting opposing effects on intracellular functions.
2009-12-01
Brucella spp. establish an intracellular replicative niche in macrophages, while macrophages attempt to eliminate the bacteria by innate defense mechanisms. Brucella...Full Text Available
2010-01-01
Abstract: The aim of the present study was to assess the purinoceptor functional responses of the urinary bladder by using isolated rat urinary bladder strip preparations. ATP elicited a transient bladder contraction followed by a sustained relaxation and ADP, UDP and UTP generated predominantly potent relaxations (relaxatory potencies: ADP = ATP > UDP = UTP). The ATP contractions were desensitized with the P2X1/3 purinoceptor agonist/desensitizer a,b-meATP and reduced by the P2 purinoceptor antagonist PPADS but unaffected by the P2 purinoceptor antagonist suramin. Electrical field stimulation (1-60 Hz) evoked frequency-dependent bladder contractions that were decreased by incubation with a,b-meATP but not further decreased by PPADS. Suramin antagonized relaxations generated by UDP but not...
Full Text Available.BackgroundEctonucleotidase dependent adenosine generation has been implicated in preconditioning related cardioprotection against ischemia-reperfusion injury, and treatment with a soluble ectonucleotidase has been shown to reduce myocardial infarct size (IS) when applied prior to induction of ischemia. However, ectonucleotidase treatment according to a clinically applicable protocol, with administration only after induction of ischemia, has not previously been evaluated. We therefore investigated if treatment with the ectonucleotidase apyrase, according to a clinically applicable protocol, would reduce IS and microvascular obstruction (MO) in a large animal model.MethodsA percutaneous coronary intervention balloon was inflated in the left anterior descending artery for 40 min, in 16 anesthetized pigs (40-50 kg). The pigs were randomized to 40 min of 1 ml/min intracoronary infusion of apyrase (10 U/ml, n = 8) or saline (0.9 mg/ml, n = 8), twenty minutes after balloon inflation. Area at risk (AAR) was evaluated by ex vivo SPECT. IS and MO were evaluated by ex vivo MRI.ResultsNo differences were observed between the apyrase group and saline group with respect to IS/AAR (75.7 ± 4.2% vs 69.4 ± 5.0%, p = NS) or MO (10.7 ± 4.8% vs 11.4 ± 4.8%, p = NS), but apyrase prolonged the post-ischemic reactive hyperemia.ConclusionApyrase treatment according to a clinically applicable protocol, with administration of apyrase after induction of ischemia, does not reduce myocardial infarct size or microvascular obstruction.
BackgroundEctonucleotidase dependent adenosine generation has been implicated in preconditioning related cardioprotection against ischemia-reperfusion injury, and treatment with...Full Text Available
Full Text Available.BackgroundEpendymal cells form a protective monolayer between the brain parenchyma and cerebrospinal fluid (CSF). They possess motile cilia important for directing the flow of CSF through the ventricular system. While ciliary beat frequency in airway epithelia has been extensively studied, fewer reports have looked at the mechanisms involved in regulating ciliary beat frequency in ependyma. Prior studies have demonstrated that ependymal cells express at least one purinergic receptor (P2X7). An understanding of the full range of purinergic receptors expressed by ependymal cells, however, is not yet complete. The objective of this study was to identify purinergic receptors which may be involved in regulating ciliary beat frequency in lateral ventricle ependymal cells.MethodsHigh-speed video analysis of ciliary movement in the presence and absence of purinergic agents was performed using differential interference contrast microscopy in slices of mouse brain (total number of animals = 67). Receptor identification by this pharmacological approach was corroborated by immunocytochemistry, calcium imaging experiments, and the use of two separate lines of knockout mice.ResultsCiliary beat frequency was enhanced by application of a commonly used P2X7 agonist. Subsequent experiments, however, demonstrated that this enhancement was observed in both P2X7+/+ and P2X7-/- mice and was reduced by pre-incubation with an ecto-5'-nucleotidase inhibitor. This suggested that enhancement was primarily due to a metabolic breakdown product acting on another purinergic receptor subtype. Further studies revealed that ciliary beat frequency enhancement was also induced by adenosine receptor agonists, and pharmacological studies revealed that ciliary beat frequency enhancement was primarily due to A2B receptor activation. A2B expression by ependymal cells was subsequently confirmed using A2B-/-/β-galactosidase reporter gene knock-in mice.ConclusionThis study demonstrates that A2B receptor activation enhances ciliary beat frequency in lateral ventricle ependymal cells. Ependymal cell ciliary beat frequency regulation may play an important role in cerebral fluid balance and cerebrospinal fluid dynamics.
BackgroundEpendymal cells form a protective monolayer between the brain parenchyma and cerebrospinal fluid (CSF). They possess motile cilia important for directing the flow of CSF...Full Text Available
Objective:The cardioprotective effects of activation of the A2A adenosine receptor (A2AAR) on ischemia/reperfusion injury in the heart remain controversial....Full Text Available
Full Text Available.Objective:The cardioprotective effects of activation of the A2A adenosine receptor (A2AAR) on ischemia/reperfusion injury in the heart remain controversial. We investigated whether ATL 313, a new selective A2AAR agonist, could reduce myocardial infarct size in a rat ischemia/reperfusion model.Methods:Sprague-Dawley rats were subjected to a 40 minute occlusion of the left coronary artery followed by 3 hours reperfusion. Hemodynamics were monitored during the procedure. The rats were divided into 3 groups: Group 1 received continuous intravenous infusion of saline given 10 min prior to ischemia and throughout reperfusion (n=8); Group 2 received continuous intravenous infusion of 10 ng/kg/min of ATL 313 given 10 min prior to ischemia, and throughout reperfusion (n=8); and group 3 received an intravenous bolus of ATL 313 (900 ng/Kg body weight) given 10 min prior to ischemia, and continuous intravenous infusion of 10 ng/kg/min of ATL 313 started at 20 min after ischemia and throughout reperfusion (n=8). After euthanasia of the rats, the hearts were harvested for the assessment of risk zone and zone of necrosis of the left ventricle.Results:The percentage of risk zone in the left ventricle was similar among group 1 (47 ± 3.7 %), group 2 (41.5 ± 4.2 %) and group 3 (42.4 ± 3.8 %). However, the infarct size, expressed as a percentage of the risk zone, was significantly decreased in group 3 (30.6 ± 5 %, P=0.01) compared with group 1 (53.8 ± 6.2 %) and group 2 (52.1 ± 4.8 %). In group 3, the bolus injection of ATL 313 caused a reduction in blood pressure during the procedure, and decreased heart rate and LV ±dp/dt before coronary artery occlusion; but increased LV +dp/dt at the end of reperfusion compared to the other 2 groups.Conclusion:A2AAR agonist ATL313 significantly reduced infarct size and improved LV contractility at the end of reperfusion assessed by LV dp/dt at a dose of 900 ng/Kg. The mechanisms for the observed cardioprotection effect of ATL313 remain to be determined.
2010-03-01
Full Text Available.
Adenosine: Tipping the balance towards hepatic steatosis and fibrosis
2010-01-01
COMMENTARY ON: Adenosine signaling contributes to ethanol induced fatty liver in mice. Zhongsheng Peng et al. J Clin Invest 9 2009; 119:582-94. doi: 10.1172/7CI37409. Copyright 2009 by AMERICAN SOCIETY FOR CLINICAL INVESTIGATION. Reproduced with permission of AMERICAN SOCIETY FOR CLINICAL INVESTIGATION in the format Journal via Copyright Clearance Center. Abstract: Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5prime-nucleotidase, and adenosine production and adenosine receptor activation are known to play cri...
Adenosine A1 receptors and vascular reactivity
2010-01-01
Abstract Aim: Blood pressure is higher in A1 receptor knock-out (A1R-/-) mice than in wild type litter mates (A1R+/+) and we have examined if this could be related to altered vascular functions. Methods: Contraction of aortic rings and mesenteric arteries were examined. To examine if the adenosine A1 receptor-mediated contraction of aortic muscle was functionally important we examined pulse pressure (PP) and augmentation index (AIX) using a sensor that allows measurements of rapid pressure transients. Results: Contraction of aortic rings to phenylephrine and relaxation to acetylcholine were similar between genotypes. The non-selective adenosine receptor agonist N-ethyl carboxamido adenosine (NECA) enhanced the contractile response, and this was eliminated in aortas from A1R-/- mice. Howeve...
Adenosine receptor subtypes in airways responses of sensitized guinea-pigs to inhaled ovalbumin
2010-01-01
Endogenous adenosine is released in asthmatic patients’ lungs by inhaled allergen, however, its exact role in asthmatic responses or the receptors mediating these responses has not been determined. Our hypothesis was that adenosine released during allergen challenge contributes to the airways responses to inhaled allergen. The effects of selective antagonists of the four adenosine receptor subtypes were investigated on the airways responses of sensitized guinea-pigs to inhaled ovalbumin to ascertain the role of adenosine in these allergen responses, and compared with a corticosteroid, dexamethasone. Early (EAR) and late asthmatic responses (LAR) to inhaled ovalbumin (10 mg/ml) of sensitized, conscious guinea-pigs were recorded by whole body plethysmography following administration o...
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