Inhibitory Effect Of Adenosine Research Articles

Presynaptic Purinergic Modulation of the Rat Neuro-Muscular Transmission.

ATP, being a well-known universal high-energy compound, plays an important role as a signaling molecule and together with its metabolite adenosine they both attenuate the release of acetylcholine in the neuro-muscular synapse acting through membrane P2 and P1 receptors, respectively. In this work, using a mechanomyographic method, we analyzed the presynaptic mechanisms by which ATP and adenosine can modulate the transduction in the rat m. soleus and m. extensor digitorum longus. N-ethylmaleimide, a G-protein antagonist, prevents the modulating effects of both ATP and adenosine. The action of ATP is abolished by chelerythrin, a specific phospholipase C inhibitor, while the inhibitory effect of adenosine is slightly increased by Rp-cAMPS, an inhibitor of protein kinase A, and by nitrendipine, a blocker of L-type Ca2+ channels. The addition of DPCPX, an A1 receptor antagonist, fully prevents the inhibitory action of adenosine in both muscles. Our data indicate that the inhibitory action of ATP involves metabotropic P2Y receptors and is mediated by phospholipase C dependent processes in rat motor neuron terminals. We suggest that the presynaptic effect of adenosine consists of negative and positive actions. The negative action occurs by stimulation of adenosine A1 receptors while the positive action is associated with the stimulation of adenosine A2A receptors, activation of protein kinase A and opening of L-type calcium channels. The combined mechanism of the modulating action of ATP and adenosine provides fine tuning of the synapse to fast changing conditions in the skeletal muscles.

Adenosine Induces Autophagy in Cholangiocarcinoma Cells.

Cholangiocarcinoma is a lethal disease with increasing incidence worldwide. New therapeutic compounds are urgently needed for this disease. Here, the inhibitory effect of adenosine on cholangiocarcinoma cells was studied. Western blot analysis was used to study autophagy and flow cytometry to analyze cell death and the cell cycle. Cholangiocarcinoma and immortalized cholangiocytes responded to adenosine differently, and adenosine inhibited cholangiocarcinoma cell growth by inducing autophagy. Adenosine failed to activate adenylyl cyclase in cholangiocarcinoma cell lines, but activated this enzyme in immortalized cholangiocytes. Adenosine treatment activated AMPK and led to phosphorylation of its downstream proteins including ULK and Raptor. In addition, autophagy induced by adenosine appeared to be a survival mechanism. The combination of adenosine with autophagy inhibitors greatly increased cell death, as compared to the use of either agent alone. Interestingly, immortalized cholangiocytes were more resistant to adenosine. Adenosine may have potential for application in cholangiocarcinoma treatment.

Adenosine Suppresses Cholangiocarcinoma Cell Growth and Invasion in Equilibrative Nucleoside Transporters-Dependent Pathway

Cholangiocarcinoma (CCA) is a lethal disease with increasing incidence worldwide. Previous study showed that CCA was sensitive to adenosine. Thereby, molecular mechanisms of CCA inhibition by adenosine were examined in this study. Our results showed that adenosine inhibited CCA cells via an uptake of adenosine through equilibrative nucleoside transporters (ENTs), instead of activation of adenosine receptors. The inhibition of ENTs by NBTI caused the inhibitory effect of adenosine to subside, while adenosine receptor antagonists, caffeine and CGS-15943, failed to do so. Intracellular adenosine level was increased after adenosine treatment. Also, a conversion of adenosine to AMP by adenosine kinase is required in this inhibition. On the other hand, inosine, which is a metabolic product of adenosine has very little inhibitory effect on CCA cells. This indicates that a conversion of adenosine to inosine may reduce adenosine inhibitory effect. Furthermore, there was no specific correlation between level of proinflammatory proteins and CCA responses to adenosine. A metabolic stable analog of adenosine, 2Cl-adenosine, exerted higher inhibition on CCA cell growth. The disturbance in intracellular AMP level also led to an activation of 5′ AMP-activated protein kinase (AMPK). Accordingly, we proposed a novel adenosine-mediated cancer cell growth and invasion suppression via a receptor-independent mechanism in CCA.

Adenosine inhibits human astrocyte proliferation independently of adenosine receptor activation.

Brain adenosine concentrations can reach micromolar concentrations in stressful situations such as stroke, neurodegenerative diseases or hypoxic regions of brain tumours. Adenosine can act by receptor-independent mechanism by reversing the reaction catalysed by S-adenosylhomocysteine (SAH) hydrolase, leading to SAH accumulation and inhibition of S-adenosylmethionine (SAM)-dependent methyltransferases. Astrocytes are essential in maintaining brain homeostasis but their pathological activation and uncontrolled proliferation plays a role in neurodegeneration and glioma. Adenosine can affect cell proliferation, but the effect of increased adenosine concentration on proliferation of astrocytes is not clarified and was addressed in present work. Human astrocytes (HA) were treated for 3days with test drugs. Cell proliferation/viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium assay and by cell counting. Cell death was evaluated by assessing lactate dehydrogenase release and by western blot analysis of αII-Spectrin cleavage. 30µM-Adenosine caused a 40%±3% (p<.05, n=5) reduction in cell proliferation/viability, an effect reversed by 2U/ml-adenosine deaminase, but unchanged in the presence of antagonists of any of the adenosine receptors. Adenosine alone did not induce cell death. 100µM-Homocysteine alone caused 16%±3% (p<.05) decrease in HA proliferation. Combined action of adenosine and homocysteine decreased HA proliferation by 76%±4%, an effect higher (p<.05) than the sum of the effects of adenosine and homocysteine alone (56%±5%). The inhibitory effect of adenosine on HA proliferation/viability was mimicked by two adenosine kinase inhibitors and attenuated in the presence of folate (100µM) or SAM (50-100µM). The results suggest that adenosine reduces HA proliferation by a receptor-independent mechanism probably involving reversal of SAH hydrolase-catalysed reaction.

Adenosine, Via A2B Receptors, Inhibits Human (P-SMC) Progenitor Smooth Muscle Cell Growth.

c-Kit+ progenitor smooth muscle cells (P-SMCs) can develop into SMCs that contribute to injury-induced neointimal thickening. Here, we investigated whether adenosine reduces P-SMC migration and proliferation and whether this contributes to adenosine's inhibitory actions on neointima formation. In human P-SMCs, 2-chloroadenosine (stable adenosine analogue) and BAY60-6583 (A2B agonist) inhibited P-SMC proliferation and migration. Likewise, increasing endogenous adenosine by blocking adenosine metabolism with erythro-9-(2-hydroxy-3-nonyl) adenine (inhibits adenosine deaminase) and 5-iodotubercidin (inhibits adenosine kinase) attenuated P-SMC proliferation and migration. Neither N6-cyclopentyladenosine (A1 agonist), CGS21680 (A2A agonist), nor N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (A3 agonist) affected P-SMC proliferation or migration. 2-Chloroadenosine increased cyclic AMP, reduced Akt phosphorylation (activates cyclin D expression), and reduced levels of cyclin D1 (promotes cell-cycle progression). Moreover, 2-chloroadenosine inhibited expression of Skp2 (promotes proteolysis of p27Kip1) and upregulated levels of p27Kip1 (negative cell-cycle regulator). A2B receptor knockdown prevented the effects of 2-chloroadenosine on cyclic AMP production and P-SMC proliferation and migration. Likewise, inhibition of adenylyl cyclase and protein kinase A rescued P-SMCs from the inhibitory effects of 2-chloroadenosine. The inhibitory effects of adenosine were similar in male and female P-SMCs. In vivo, peri-arterial (rat carotid artery) 2-chloroadenosine (20 μmol/L for 7 days) reduced neointimal hyperplasia by 64.5% (P<0.05; intima/media ratio: control, 1.4±0.02; treated, 0.53±0.012) and reduced neointimal c-Kit+ cells. Adenosine inhibits P-SMC migration and proliferation via the A2B receptor/cyclic AMP/protein kinase A axis, which reduces cyclin D1 expression and activity via inhibiting Akt phosphorylation and Skp2 expression and upregulating p27kip1 levels. Adenosine attenuates neointima formation in part by inhibiting infiltration and proliferation of c-Kit+ P-SMCs.

&lt;p&gt;Effects Of Adenosine On Apoptosis Of Ovarian Cancer A2780 Cells Via ROS And Caspase Pathways&lt;/p&gt;

BackgroundOvarian cancer is the second most common malignant tumor of the female genital tract and the main cause of death of gynecological malignant tumors. Exogenous adenosine has been shown to induce apoptosis in tumor cells.Materials and methodsThe current study aimed to investigate the inhibitory effect and underlying mechanism of adenosine on the proliferation of ovarian cancer cells. The inhibitory effects of adenosine on ovarian cancer cells were assessed through MTT assay. The adenosine-induced apoptosis was determined by Hoechst 33342 staining and flow cytometry. The effect of adenosine on the intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) was assessed according to the DCFH-DA and JC-1 methods, respectively. Expression of apoptosis-related proteins and mitochondrial proteins was measured using Western blotting.ResultsMTT suggested that adenosine inhibited A2780 and SKOV3 cells viability in a dose- and time-dependent manner (P<0.05). Hoechst 33342 staining had demonstrated pronounced changes in cell morphology; to be specific, cells treated with 0 mmol/L adenosine showed a light blue color, while those in treatment groups had nuclear pyknosis and apoptotic body formation. Besides, the apoptosis rate was positively correlated with adenosine concentration (P<0.05). Flow cytometry results revealed that adenosine increased the intracellular ROS level and decreased MMP. Western blotting indicated that, the expression of Bax, cleaved-caspase-3 and cleaved-poly (ADP-ribose) polymerase was up-regulated with the increase in adenosine concentration, while that of Bcl-2 protein and apoptosis-related protein caspase-3 was down-regulated.ConclusionWith the increase in drug concentration, the CytoC expression in mitochondria was gradually reduced, while that in the cytoplasm was gradually increased. In conclusion, Ado may inhibit the proliferation and induce the apoptosis of ovarian cancer cells by increasing ROS, up-regulating the pro-apoptotic protein Bax and activating the caspase-3 expression in vitro.

Effects of selective agonists of adenosine, P2X, and P2Y receptors on motility of isolated fallopian tube

Background: During reproductive life of women, adenosine causes both contraction (with low concentrations) of fallopian tubes and inhibition of their spontaneous motor activity (with high concentrations). Objective: The aim of this study was to investigate effects of natural agonists of adenosine, P2X and P2Y receptors on motility of isolated fallopian tubes taken from postmenopausal women. Materials and Methods: Isolated preparations of isthmus and ampoule were made from fallopian tubes of 21 women in post-menopause, and then tested for reactivity on increasing concentrations of adenosine and P2X/P2Y selective agonists. Results: Adenosine showed concentration-dependent inhibitory effect on spontaneous contraction of both isthmic and ampullary segments of fallopian tubes, while P2X and P2Y agonists (adenosine-5-diphosphate, adenosine-5-triphosphate, uridine-5-diphosphate, and uridine-5-triphosphate) did not influence motility of the isolated preparations. Contractile effect of adenosine was not observed throughout the concentration range used in the experiments. Conclusions: Fallopian tubes of postmenopausal women are unresponsive to P2X and P2Y agonists, unlike those of women in reproductive period. Only an inhibitory effect of adenosine on spontaneous contractions of fallopian tubes is maintained in post-menopause, while a contractile effect is observed in younger women at low concentrations is lost.

The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.

Event Abstract Back to Event The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines. Helena Marcelino1, 2, Cecilia R. Santos2, Vanda S. Nogueira2, 3, 4, João F. Fonseca-Gomes3, 4, Joana Tomás2, Maria José Diógenes3, 4, Ana M. Sebastião3, 4 and José F. Cascalheira1, 2* 1 Department of Chemistry, Faculty of Sciences, University of Beira Interior, Portugal 2 Center for Research in Health Sciences, Faculty of Health Sciences, University of Beira Interior, Portugal 3 Institute of Pharmacology and Neurosciences, Faculty of Medicine, University of Lisbon, Portugal 4 Institute of Molecular Medicine João Lobo Antunes, Faculty of Medicine, University of Lisbon, Portugal Adenosine has been implicated in the proliferation and survival of various tumour cells, namely glioblastoma cells. Adenosine usually produces its cellular effects in the nervous system by activating G protein-coupled adenosine receptors. Increased adenosine concentrations are found in stressful conditions such as hypoxic regions in brain tumours. However it isn’t known if there any difference between the effects of increased adenosine concentration on cell proliferation in normal astrocytes when compared with malign transformed cells. This issue will be addressed in present work. Human astrocytes (HA), and human glioblastoma cell lines U-87MG, U-373 MG, SNB19, were treated for 3 days with 30µM-Adenosine, with or without the selective adenosine kinase inhibitor 5-idotubercidin (25µM). Cell proliferation was evaluated using MTT assay while cell death was assessed by measuring the released LDH activity and αII-Spectrin cleavage. 30µM-Adenosine produced a 40%±4% (P<0.005) reduction in HA cell proliferation but had no effect in any of the 3 glioblastoma cell lines (p<0.01, when compared with HA). Adenosine alone did not induce cell death, assessed by LDH release and αII-Spectrin cleavage. Curiously, incubation with 5-iodotubercidin+adenosine produced a strong and similar decrease (ranging from 86±5% for HA, to 75%±4% for U87, P<0.01) on cell proliferation in both HA and glioblastoma cell lines. The results show a strong attenuation of the antiproliferative effect of adenosine in glioblastoma cells compared to HA, probably resulting from an increased adenosine kinase activity in glioblastoma cells, suggesting an adaptation of the tumour cells to the antiproliferative effect of adenosine. Acknowledgements The present work was funded by a CENTRO 2020 and Lisboa 2020 (POCI-01-0145-FEDER-016822) and FCT – Fundação para a Ciência e Tecnologia, I.P. (PTDC/BIM¬ONC/7121/2014) research Grant. Keywords: Adenosine, Astrocytes, Cell Proliferation, Glioblastoma, Homocysteine Conference: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019), Lisboa, Portugal, 30 May - 1 Jun, 2019. Presentation Type: Poster presentation Topic: Glia / Neuroinflammation Citation: Marcelino H, Santos CR, Nogueira VS, Fonseca-Gomes JF, Tomás J, Diógenes M, Sebastião AM and Cascalheira JF (2019). The inhibitory effect of adenosine on human astrocyte proliferation is absent in glioblastoma cell lines.. Front. Cell. Neurosci. Conference Abstract: XVI Meeting of the Portuguese Society for Neuroscience (SPN2019). doi: 10.3389/conf.fncel.2019.01.00004 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 16 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Prof. José F Cascalheira, Department of Chemistry, Faculty of Sciences, University of Beira Interior, Covilhã, Portugal, jfcascalheira@yahoo.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Google Scholar Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira PubMed Helena Marcelino Cecilia R Santos Vanda S Nogueira João F Fonseca-Gomes Joana Tomás Maria José Diógenes Ana M Sebastião José F Cascalheira Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Inhibition of the hypercapnic ventilatory response by adenosine in the retrotrapezoid nucleus in awake rats

The brain regulates breathing in response to changes in tissue CO2/H+ via a process called central chemoreception. Neurons and astrocytes in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors. The role of astrocytes in this process appears to involve CO2/H+-dependent release of ATP to enhance activity of chemosensitive RTN neurons. Considering that in most brain regions extracellular ATP is rapidly broken down to adenosine by ectonucleotidase activity and since adenosine is a potent neuromodulator, we wondered whether adenosine signaling contributes to RTN chemoreceptor function. To explore this possibility, we pharmacologically manipulated activity of adenosine receptors in the RTN under control conditions and during inhalation of 7–10% CO2 (hypercapnia). In urethane-anesthetized or unrestrained conscious rats, bilateral injections of adenosine into the RTN blunted the hypercapnia ventilatory response. The inhibitory effect of adenosine on breathing was blunted by prior RTN injection of a broad spectrum adenosine receptor blocker (8-PT) or a selective A1-receptor blocker (DPCPX). Although RTN injections of 8PT, DPCPX or the ectonucleotidase inhibitor ARL67156 did not affected baseline breathing in either anesthetized or awake rats. We did find that RTN application of DPCPX or ARL67156 potentiated the respiratory frequency response to CO2, suggesting a portion of ATP released in the RTN during high CO2/H+ is converted to adenosine and serves to limit chemoreceptor function. These results identify adenosine as a novel purinergic regulator of RTN chemoreceptor function during hypercapnia.

Inhibitory effects of adenosine and adenosine A1 receptors stimulation on catecholamine-induced increase in camp level and force of contraction in atrial and ventricular myocyte

Inhibitory effects of adenosine and adenosine A1 receptors stimulation on catecholamine-induced increase in camp level and force of contraction in atrial and ventricular myocyte

ATP-induced changes in rat skeletal muscle contractility.

Extracellular purine compounds, adenosine triphosphate (ATP) and adenosine, are involved in regulation of many cell functions, engaging in rapid and long-term cellular processes. The nucleotides, including ATP, exert their extracellular effects by influencing membrane P2 receptors. ATP outside of the cell rapidly is metabolized by the ecto-enzyme system to produce adenosine, which acts on separate adenosine (P1) receptors. Since adenosine and ATP often are functional antagonists, ATP degradation not only limits its effect, but also brings new ligand with different, often opposing, properties. Great variety and widespread of P2 and adenosine receptors in the body emphasize the important physiological and pathophysiological significance of these receptors, and make them very attractive as targets for potential drug action.The existence of several subtypes of P2 and adenosine receptors has been shown in the skeletal muscles. ATP as a co-transmitter is densely packed together with classical neurotransmitters in the presynaptic vesicles of vertebral motor units but until recently ATP was refused to have its own functional role there and was recognized only as a source of adenosine. However, on the eve of the third millennium there appeared data that ATP, released from the nerve ending and acting on presynaptic P2 receptors, suppresses subsequent quantum release of acetylcholine. The final product of its degradation, adenosine, performs a similar inhibitory effect acting on presynaptic adenosine receptors.Despite the fact that the mechanisms of presynaptic inhibitory action of ATP and other purines were studied earlier, the object of those studies was usually neuromuscular synapse of cold-blooded animals. The few studies, in which experiments were carried out on preparations of warm-blooded animals, described the basic effects of purines. These often were guided by the convenience of preparation of the synapses of the diaphragm. We think that those results cannot be considered as typical effects of ATP and other purines on skeletal muscles and could not be extrapolated to all warm-blooded animals. Furthermore the role of ATP and its derivatives in the accumulation of vertebrate muscular effort has not been investigated.It is known that in physiological conditions vertebrates may mobilize only up to a third of the maximum muscle force. Why the two-thirds of muscular strength are not used normally but may be used at stress, remains unknown.It is known that the body's adaptive response to stress is a change in the activity of the endocrine system. The leading role in this is given to catechol amines and glucocorticoids, mobilized in significant quantities in blood under stress.We have found previously that incubation of frog sartorius muscle with hydrocortisone resulted in a decrease of contraction amplitude. However, when hydrocortisone was used in combination with ATP, its inhibitory effect on contractile responses disappeared. It is interesting that hydrocortisone had no effect on the inhibitory effect of adenosine. In the following experiments, assessing the effect of hydrocortisone on rat soleus muscle, it was established that hydrocortisone and purines had similar inhibitory effect. When ATP and hydrocortisone were given together the same oppression occurred.To study the effects of ATP and adenosine on contraction parameters of rat skeletal muscle and assess the impact of the catechol amines on these processes.Contractions of rat soleus muscles were recorded isometrically by mechanical sensor Linton FSG-01 (UK) according to standard procedures. The average of muscle parameters received within 30 seconds (30 responses) was treated as one result. Amplitude and time characteristics of the curve reductions were estimated. During all experiments standard Krebs solution flowed through the bath continuously to which agents were added at necessary concentrations. All experimental animals were maintained and prepared for dissection under the European Convention for the Protection of Vertebrate Animals used in scientific experiments. All agents used in the study were supplied by Sigma Chemical Company Ltd. (UK), Tocris Cookson and Research Biochemicals International (USA).The concentration of 100 μM for adenosine is close to saturation [1], and for its predecessor ATP this concentration is created after the passage of a pulse through the synapse [2]. We used this concentration of purines to study the mechanism of action of adenosine and ATP on neuromuscular synapse.The effect of adenosine was partially inhibited in the presence of 100 μM 8-SPT, an antagonist of adenosine receptors. The contraction force of "fast" and "slow" rat skeletal muscles was raised by half in the presence of norepinephrine. In the presence of norepinephrine adenosine exerted its effect fully, but ATP by half reduced its depressor effect on the contraction force of both muscles.1. Norepinephrine increases half times of the reduction of "fast" and "slow" skeletal muscle.2. In the presence of norepinephrine, inhibitory effect of adenosine on contraction force is maintained.3. Inhibitory effect of ATP on contraction force of studied skeletal muscles becomes twice less pronounced in the presence of norepinephrine.We think that reduction of ATP depressive effect on the skeletal muscle by norepinephrine may be an adaptive response to acute stress.

Inhibition of platelet activation and thrombus formation by adenosine and inosine: studies on their relative contribution and molecular modeling.

BackgroundThe inhibitory effect of adenosine on platelet aggregation is abrogated after the addition of adenosine-deaminase. Inosine is a naturally occurring nucleoside degraded from adenosine.ObjectivesThe mechanisms of antiplatelet action of adenosine and inosine in vitro and in vivo, and their differential biological effects by molecular modeling were investigated.ResultsAdenosine (0.5, 1 and 2 mmol/L) inhibited phosphatidylserine exposure from 52±4% in the control group to 44±4 (p<0.05), 29±2 (p<0.01) and 20±3% (p<0.001). P-selectin expression in the presence of adenosine 0.5, 1 and 2 mmol/L was inhibited from 32±4 to 27±2 (p<0.05), 14±3 (p<0.01) and 9±3% (p<0.001), respectively. At the concentrations tested, only inosine to 4 mmol/L had effect on platelet P-selectin expression (p<0.05). Adenosine and inosine inhibited platelet aggregation and ATP release stimulated by ADP and collagen. Adenosine and inosine reduced collagen-induced platelet adhesion and aggregate formation under flow. At the same concentrations adenosine inhibited platelet aggregation, decreased the levels of sCD40L and increased intraplatelet cAMP. In addition, SQ22536 (an adenylate cyclase inhibitor) and ZM241385 (a potent adenosine receptor A2A antagonist) attenuated the effect of adenosine on platelet aggregation induced by ADP and intraplatelet level of cAMP. Adenosine and inosine significantly inhibited thrombosis formation in vivo (62±2% occlusion at 60 min [n = 6, p<0.01] and 72±1.9% occlusion at 60 min, [n = 6, p<0.05], respectively) compared with the control (98±2% occlusion at 60 min, n = 6). A2A is the adenosine receptor present in platelets; it is known that inosine is not an A2A ligand. Docking of adenosine and inosine inside A2A showed that the main difference is the formation by adenosine of an additional hydrogen bond between the NH2 of the adenine group and the residues Asn253 in H6 and Glu169 in EL2 of the A2A receptor.ConclusionTherefore, adenosine and inosine may represent novel agents lowering the risk of arterial thrombosis.

Inhibitory effect of adenosine on intimal hyperplasia and proliferation of smooth muscle cells in a carotid arterial anastomosis animal model.

The effect of adenosine (9-β-0-ribifuranosyladenine) on the endothelial cell proliferation and neointimal hyperplasia is investigated in the rabbit carotid artery anastomosis model. Twenty-eight New Zealand white rabbits were arranged in four groups of seven animals each. The right carotid arteries of each animal were transsected and re-anastomosed. The left sides remained as control. In Group A, no medication was used. In Group B, subcutaneous Adenosine was applied for 3 days. In Group C, the same dose was applied for 7 days, and in Group D for 21 days. After 28 days, the luminal diameters, luminal areas, intima/media ratios were all measured by using histopathological evaluation. The mean luminal diameters and areas of the four groups were smaller than the control ones. Massive thickening of smooth muscle cell proliferation and dense intensifying in the connecting tissues were observed most prominently in Group A, in decreasing degrees within other groups. Intima/media ratio was highest in Group A. Scoring the quantity of e-NOS positive staining also revealed a significant difference between the experimental groups and their control associates. The process of endothelial cell proliferation and neointimal hyperplasia can be significantly reduced by the use of adenosine.

The Imbalanced Expression of Adenosine Receptors in an Epilepsy Model Corrected Using Targeted Mesenchymal Stem Cell Transplantation

Adenosine inhibits epileptic episodes by interacting with G-protein-coupled receptors. This study examined the mechanism by which the inhibitory effect of adenosine becomes impaired during epileptogenesis. Dynamic changes in adenosine A1 receptors (A1Rs) and A2a receptors (A2aRs) were investigated in a kindling model of epilepsy. RT-PCR, Western blotting, and immunofluorescence results indicated that expression of A1Rs was increased in the hippocampus 24 h after kindling, but progressively decreased 1 and 6 months after kindling. Opposite changes were seen in the expression of A2aRs. This bidirectional change resulted in an imbalance between A1Rs and A2aRs and dysregulation of the adenosine system. Autologous mesenchymal stem cell (MSC) transplantation was used to correct this disorder and avoid side effects of systematic adenosine therapy. Paramagnetic iron oxide particles were used to mark and track the MSCs in vivo using MRI. The results indicated that the transplanted cells migrated along the callosum and settled at the ependymal layer. The MSCs displayed a relatively long survival time, at least 3 months. The improved AR expression and EEG findings suggested that MSC transplantation was a potentially effective means of treating refractory epilepsy.

Abstract 213: Ticagrelor Inhibits Human Platelet Aggregation via Adenosine in Addition to P2Y12 Antagonism

Introduction. Ticagrelor, a P2Y 12 antagonist, inhibits adenosine uptake by erythrocytes and increases adenosine-induced coronary blood flow. We hypothesiszed that ticagrelor inhibits platelet function not only by antagonizing P2Y 12 , but also by increasing the levels of adenosine, which inhibits platelets by interacting with the platelet A 2A receptor. Methods. Collagen-induced platelet aggregation (PA) was measured in citrate-anticoagulated whole blood (WB) (Multiplate) or platelet-rich plasma (PRP) (light-transmission aggregometry) from 12 healthy controls and 2 patients with congenital deficiency of P2Y 12 , in the presence or absence of adenosine (7.1 μM), which marginally affected PA in WB, and the A 2A inhibitor ZM241385. The effects on PA of ticagrelor, the active metabolite of prasugrel (PAM) (P2Y 12 antagonist) and dipyridamole (DYP) (ENT-1 inhibitor) were compared. The effects of these drugs on the clearance of adenosine after its in vitro addition to WB were also studied. Results. PA in WB: Adenosine contributed to drug-induced inhibition of PA in healthy controls. The magnitude of the contribution (absolute difference in % aggregation) was greater in the presence of ticagrelor (20%, p &lt;0.01) or DYP (18%, p &lt;0.01) than in the presence of PAM (6%, p 0.02). PA in PRP: Adenosine markedly contributed to inhibition of PA in healthy controls in the presence of all tested drugs. In both WB and PRP ZM241385 reversed the inhibitory effect of adenosine. Data from P2Y 12 -deficient patients was in agreement, but due to low numbers no statistical analysis was performed. Adenosine, added at 7.1μM to WB in vitro, was detectable up to 0.5 min in the presence of vehicle or PAM, up to 3 min in the presence of ticagrelor, and up to 60 min in the presence of DYP. These results are compatible with the hypothesis that ticagrelor and DYP, but not PAM, increase the adenosine levels by inhibiting its uptake by erythrocytes. Conclusion. This study provides the first evidence of an additional anti-platelet mechanism by ticagrelor, mediated by the induced increase of adenosine levels in WB. It remains to be established if and to what extent this additional mechanism of action contributes to the strong clinical benefit observed with ticagrelor in patients with acute coronary syndromes.

Presynaptic inhibition of GABAergic synaptic transmission by adenosine in mouse hypothalamic hypocretin neurons

Hypocretin neurons in the lateral hypothalamus, a new wakefulness-promoting center, have been recently regarded as an important target involved in endogenous adenosine–regulating sleep homeostasis. The GABAergic synaptic transmissions are the main inhibitory afferents to hypocretin neurons, which play an important role in the regulation of excitability of these neurons. The inhibitory effect of adenosine, a homeostatic sleep-promoting factor, on the excitatory glutamatergic synaptic transmissions in hypocretin neurons has been well documented, whether adenosine also modulates these inhibitory GABAergic synaptic transmissions in these neurons has not been investigated. In this study, the effect of adenosine on inhibitory postsynaptic currents (IPSCs) in hypocretin neurons was examined by using perforated patch-clamp recordings in the acute hypothalamic slices. The findings demonstrated that adenosine suppressed the amplitude of evoked IPSCs in a dose-dependent manner, which was completely abolished by 8-cyclopentyltheophylline (CPT), a selective antagonist of adenosine A1 receptor but not adenosine A2 receptor antagonist 3,7-dimethyl-1-(2-propynyl) xanthine. A presynaptic origin was suggested as following: adenosine increased paired-pulse ratio as well as reduced GABAergic miniature IPSC frequency without affecting the miniature IPSC amplitude. Further findings demonstrated that when the frequency of electrical stimulation was raised to 10 Hz, but not 1 Hz, a time-dependent depression of evoked IPSC amplitude was detected in hypocretin neurons, which could be partially blocked by CPT. However, under a higher frequency at 100 Hz stimulation, CPT had no action on the depressed GABAergic synaptic transmission induced by such tetanic stimulation in these hypocretin neurons. These results suggest that endogenous adenosine generated under certain stronger activities of synaptic transmissions exerts an inhibitory effect on GABAergic synaptic transmission in hypocretin neurons by activation of presynaptic adenosine A1 receptors, which may finely regulate the excitability of these neurons as well as eventually modulate the sleep–wakefulness.

Disparate purinergic modulation of respiration in rats and mice

Disparate purinergic modulation of respiration in rats and mice

The interactions of nitric oxide and adenosine on penicillin-induced epileptiform activity in rats.

In this study, the influence of nitric oxide (NO) and adenosine systems on penicillin-induced epileptiform activity was examined in rats. NO donor, sodium nitroprusside (SNP, 50 micrograms per rat, i.c.v.) reduced the frequency but not the amplitude of epileptiform discharges. Non-selective NOS inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME, 100 micrograms per rat, i.c.v.) practically did not exert any effect on the spike frequency and amplitude. Adenosine (100 micrograms per rat, i.c.) reduced spike frequency but not the amplitude, whereas theophylline (100 micrograms per rat, i.c.v.) increased the mean spike frequency and amplitude of penicillin-induced epileptiform discharges. Co-injection of theophylline and L-NAME did not cause a further increase in the epileptiform activity compared with theophylline. When NO production was blocked with L-NAME, the inhibitory effects of adenosine were lost. The obtained results suggest that NO and adenosine may decrease penicillin-induced epileptiform activity in rats and that NO, at least in part, may mediate the anticonvulsant effect of adenosine.

Actions of ATP on Guinea-Pig SA Node

ATP is well established as a co-transmitter in sympathetic nerves supplying smooth muscle. In the heart, ATP has been shown to increase pacemaker activity in amphibian preparations but little is known about its effects on pacemaker activity in mammals. The aim of these experiments was to investigate the actions of ATP and its analogues on guinea-pig SA node. Apha,beta methylene ATP (abMeATP) caused a concentration dependent increase in the rate of beating of isolated atrial preparations over the range 0.3 to 30 micro M. ATP also caused a concentration-dependent increase in the rate of beating in isolated atrial preparations over the range 3 to 100 micro M, provided that adenosine receptors were antagonised by 1,3-dipropyl-8-cyclopentylxanthine. In myocytes isolated from guinea-pig SA node, abMeATP (1 and 10 micro M) caused an increase in the rate of firing of spontaneous action potentials. In the same range of concentrations, abMeATP increased the I(f) current activated by hyperpolarization in voltage-clamped SA node myocytes. This effect of abMeATP was prevented when SA node myocytes were loaded with the calcium chelator BAPTA (by exposure to the AM ester). These observations are consistent with an action of abMeATP on pacemaker activity in guinea-pig SA node mediated by calcium entry via P2X receptors. This calcium entry is expected to activate calcium-stimulated adenylyl cyclase (which we have shown to be present in these cells) leading to increased levels of cAMP and enhanced activation of I(f). Calcium entry via P2X receptors may also have additional effects on other pathways involved in pacemaker activity. Actions on P2X receptors are expected to contribute to the observed effects of ATP in SA node when the inhibitory effects of adenosine (a possible breakdown product of ATP) are antagonised.

STIMULATION OF ADENOSINE A2A RECEPTOR INHIBITS LPS-INDUCED EXPRESSION OF INTERCELLULAR ADHESION MOLECULE 1 AND PRODUCTION OF TNF-α IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS

LPS stimulates CD14/Toll-like receptor (TLR) 4, leading to induce TNF-alpha production. Cell-to-cell interaction through the engagement between intercellular adhesion molecule (ICAM) 1 on monocytes and its ligand on T cells has been suggested to play a role in the TNF-alpha production by LPS-treated human peripheral blood mononuclear cells (PBMCs). Adenosine is reported to inhibit LPS-induced TNF-alpha production. However, little is known about the mechanism of the inhibitory effects induced by adenosine on the LPS-induced immune responses. We found that adenosine inhibited the expression of ICAM-1 and the production of TNF-alpha by human PBMC via adenosine A2A receptor in the presence of LPS. However, the stimulation of A1R or A3R enhanced the actions of adenosine. Adenosine had no effect on the expression of CD14 and TLR-4, suggesting that the inhibitory effects of adenosine on the LPS actions might be independent of the expression of CD14 and TLR-4. Thus, adenosine differentially regulates the expression of ICAM-1 and the production of TNF-alpha through plural subtypes of receptors.