Adenosine Receptor Expression Research Articles

Ability of γδ T cells to modulate the Foxp3 T cell response is dependent on adenosine.

Whether γδ T cells inhibit or enhance the Foxp3 T cell response depends upon their activation status. The critical enhancing effector in the supernatant is adenosine. Activated γδ T cells express adenosine receptors at high levels, which enables them to deprive Foxp3+ T cells of adenosine, and to inhibit their expansion. Meanwhile, cell-free supernatants of γδ T cell cultures enhance Foxp3 T cell expansion. Thus, inhibition and enhancement by γδ T cells of Foxp3 T cell response are a reflection of the balance between adenosine production and absorption by γδ T cells. Non-activated γδ T cells produce adenosine but bind little, and thus enhance the Foxp3 T cell response. Activated γδ T cells express high density of adenosine receptors and have a greatly increased ability to bind adenosine. Extracellular adenosine metabolism and expression of adenosine receptor A2ARs by γδ T cells played a major role in the outcome of γδ and Foxp3 T cell interactions. A better understanding of the functional conversion of γδ T cells could lead to γδ T cell-targeted immunotherapies for related diseases.

Differential effect of adenosine on rhabdomyosarcoma migration and proliferation.

IntroductionAdenosine and its receptors are involved deeply in the regulation of tumour biology. Purine nucleotides are released from stressed cells in states of hypoxia or radiochemotherapy-induced cell damage. Adenosine exerts its effect through the P1 family of selective receptors. The purpose of the study was to evaluate the exact role of extracellular role on biology of Rhabdomyosarcoma (RMS) cells.Material and methodsSeries of in vitro studies accompanied by immunohistochemical, RQ-PCR and shRNA methods have characterised adenosine receptor expression on Rhabdomyosarcoma cell lines, normal skeletal muscle and effect of adenosine on Rhabdomyosarcoma growth and migration.ResultsExtracellular adenosine (highest at 50 μM, p < 0.05) and AMP (highest at 300 μM, p < 0.05) markedly enhanced chemotaxis in the Boyden chamber assay The reaction is mostly governed by the A1 receptor, which is greatly overexpressed in Rhabdomyosarcoma as compared with normal skeletal muscle. Cell migration induced by adenosine and AMP is blocked by pertussis toxin, phospholipase C and MAP kinase inhibitor, which demonstrates the importance of these signalling pathways. High doses of adenosine have a detrimental effect on cellular proliferation, in a receptor-independent manner (≥ 500 μM; p < 0.05). The blockage of adenosine transporter by dipyridamole abolishes this effect, indicating involvement of an intrinsic pathway. Further increase of adenosine concentration, induced by deaminase inhibitors, augment the effect.ConclusionsOur results suggest that adenosine and AMP trigger cell migration by binding to P1 receptors and directing cancer cells to the sites of hypoxia or cellular damage. Specifically by A1 receptor which is overexpressed in RMS.

Adenosine decreases oxidative stress and protects H2O2-treated neural stem cells against apoptosis through decreasing Mst1 expression.

Overproduction of free radicals during oxidative stress induces damage to key biomolecules and activates programed cell death pathways. Neuronal cell death in the nervous system leads to a number of neurodegenerative diseases. The aim of the present study was to evaluate the neuroprotective effect of adenosine on inhibition of apoptosis induced by hydrogen peroxide (H2O2) in bone marrow-derived neural stem cells (B-dNSCs), with focus on its regulatory effect on the expression of mammalian sterile 20-like kinase 1 (Mst1), as a novel proapoptotic kinase. B-dNSCs were exposed to adenosine at different doses (2, 4, 6, 8 and 10 µM) for 48 h followed by 125 µM H2O2 for 30 min. Using MTT, terminal deoxynucleotidyl transferase dUTP nick-end labeling and real-time reverse transcription polymerase chain reaction assays, the effects of adenosine on cell survival, apoptosis and Mst1, nuclear factor (erythroid-derived 2)-like 2 and B-cell lymphoma 2 and adenosine A1 receptor expression were evaluated in pretreated B-dNSCs compared with controls (cells treated with H2O2 only). Firstly, results of the MTT assay indicated 6 µM adenosine to be the most protective dose in terms of promotion of cell viability. Subsequent assays using this dosage indicated that apoptosis rate and Mst1 expression in B-dNSCs pretreated with 6 µM adenosine were significantly decreased compared with the control group. These findings suggest that adenosine protects B-dNSCs against oxidative stress-induced cell death, and therefore, that it may be used to promote the survival rate of B-dNSCs and as a candidate for the treatment of oxidative stress-mediated neurological diseases.

SB-334867 (an Orexin-1 Receptor Antagonist) Effects on Morphine-Induced Sensitization in Mice\u2014a View on Receptor Mechanisms

The present study focused upon the role of SB-334867, an orexin-1 receptor antagonist, in the acquisition of morphine-induced sensitization to locomotor activity in mice. Behavioral sensitization is an enhanced systemic reaction to the same dose of an addictive substance, which assumingly increases both the desire for the drug and the risk of relapse to addiction. Morphine-induced sensitization in mice was achieved by sporadic doses (five injections every 3 days) of morphine (10 mg/kg, i.p.), while a challenge dose of morphine (10 mg/kg) was injected 7 days later. In order to assess the impact of orexin system blockade on the acquisition of sensitization, SB-334867 was administered before each morphine injection, except the morphine challenge dose. The locomotor activity test was performed on each day of morphine administration. Brain structures (striatum, hippocampus, and prefrontal cortex) were collected after behavioral tests for molecular experiments in which mRNA expression of orexin, dopamine, and adenosine receptors was explored by the qRT-PCR technique. Additionally, the mRNA expression of markers, such as GFAP and Iba-1, was also analyzed by the same technique. SB-334867 inhibited the acquisition of morphine-induced sensitization to locomotor activity of mice. Significant alterations were observed in mRNA expression of orexin, dopamine, and adenosine receptors and in the expression of GFAP and Iba-1, showing a broad range of interactions in the mesolimbic system among orexin, dopamine, adenosine, and glial cells during behavioral sensitization. Summing up, the orexin system may be an effective measure to inhibit morphine-induced behavioral sensitization.

Ankylosing spondylitis monocyte-derived macrophages express increased level of A2A adenosine receptor and decreased level of ectonucleoside triphosphate diphosphohydrolase-1 (CD39), A1 and A2B adenosine receptors.

Macrophages play an important role in the ankylosing spondylitis (AS) auto-inflammatory responses and fibrocartilage destruction. Adenosine is a key modulator of inflammatory conditions. The various effects of adenosine are mediated by its interaction with adenosine receptors (AR). In this study, we investigated the mRNA expression of A1, A2A, A2B, and A3 adenosine receptors, ectonucleoside triphosphate diphosphohydrolase-1 (CD39), and ecto-5'-nucleotidase (CD73) in the monocyte-derived macrophages from AS patients in comparison to healthy controls. We also explored the correlation between analyzed gene expression and patients' clinical manifestations. Whole blood-separated monocytes from 23 healthy controls and 23 active AS patients were stimulated by macrophage colony-stimulating factor (M-CSF) for 7days and differentiated to macrophages. Monocyte and macrophage markers were analyzed by flow cytometry. Analysis of adenosine receptors (ADORA1، ADORA2A، ADORA2B، ADORA3), CD39 and CD73 gene expression was performed by SYBR green real-time PCR. Our results demonstrated monocyte-derived macrophages from AS patients expressed increased level of A2AAR and reduced level of A1, A2BAR, and CD39 mRNA compared to healthy controls. We found an inverse correlation between A2AAR mRNA expression and Bath Ankylosing Spondylitis Functional Index (BASFI) score in AS patients. According to our results, altered expression level of adenosine-relying system would be involved in AS macrophage dysfunction and inflammation and correlated with functional status in AS patients.

Unveiling the Role of Ecto-5'-Nucleotidase/CD73 in Astrocyte Migration by Using Pharmacological Tools.

CD73 is a bifunctional glycosylphosphatidylinositol (GPI)-anchored membrane protein which functions as ecto-5′-nucleotidase and a membrane receptor for extracellular matrix protein (ECM). A large body of evidence demonstrates a critical involvement of altered purine metabolism and particularly, increased expression of CD73 in a number of human disorders, including cancer and immunodeficiency. Massive up-regulation of CD73 was also found in reactive astrocytes in several experimental models of human neuropathologies. In all the pathological contexts studied so far, the increased expression of CD73 has been associated with the altered ability of cells to adhere and/or migrate. Thus, we hypothesized that increased expression of CD73 in reactive astrocytes has a role in the process of astrocyte adhesion and migration. In the present study, the involvement of CD73 in astrocyte migration was investigated in the scratch wound assay (SW), using primary astrocyte culture prepared from neonatal rat cortex. The cultures were treated with one of the following pharmacological inhibitors which preferentially target individual functions of CD73: (a) α,β-methylene ADP (APCP), which inhibits the catalytic activity of CD73 (b) polyclonal anti-CD73 antibodies, which bind to the internal epitope of CD73 molecule and mask their surface exposure and (c) small interfering CD73-RNA (siCD73), which silences the expression of CD73 gene. It was concluded that approaches that reduce surface expression of CD73 increase migration velocity and promote wound closure in the scratch wound assay, while inhibition of the enzyme activity by APCP induces redistribution of CD73 molecules at the cell surface, thus indirectly affecting cell adhesion and migration. Application of anti-CD73 antibodies induces a decrease in CD73 activity and membrane expression, through CD73 molecules shedding and their release to the culture media. In addition, all applied pharmacological inhibitors differentially affect other aspects of astrocyte function in vitro, including reduced cell proliferation, altered expression of adenosine receptors and increased expression of ERK1/2. Altogether these data imply that CD73 participates in cell adhesion/migration and transmits extracellular signals through interactions with ECM.

Effects of electroacupuncture on the expression of adenosine receptors in the heart tissue of myocardial ischemia rats

To explore the impact of electroacupuncture (EA) on the protein expression of adenosine receptors in the heart of the rats with myocardial ischemia (MI). Thirty healthy male SD rats were divided randomly into a control group (n=6), a model group (n=12) and an EA group (n=12). We ligated the left anterior descending artery (LAD) for MI model in the model group and EA group, and exposed the heart after opening the chest without ligation in the control group. EA, 2 Hz /15 Hz and 1.5-2 mA, was applied at bilateral"Neiguan"(PC 6) in the EA group for 20 min, once a day for continuous 5 days. No intervention except grabbing and fixation was used in the control group and model group. We applied 2% TTC staining to observe the infarct size of myocardium, colorimetry to analyze serum lactic dehydrogenase (LDH), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), radio-immunity assessment to detect cardiac troponin T (cTnT), Western blot to evaluate the adenosine A1 receptor (A1AR), A2aAR, A2bAR and A3AR. After treatment, myocardial infarction of (27.56±3.24)% was obvious in the model group; the myocardial infarction in the EA group was (21.04±3.61)%, with statistical significance (P<0.05). The expressions of serum LDH, CK, CK-MB and cTnT levels in the model group increased compared with those in the control group (all P<0.01), and the expressions of LDH, CK, CK-MB and cTnT levels in the EA group decreased compared with those in the model group (P<0.05，P<0.01). The A1AR expression in the model group was not different from that in the control group (P>0.05), and A2aAR、A2bAR、A3AR expressions decreased (P<0.05, P<0.01). A2aAR and A2bAR expressions in the EA group increased compared with those in the model group (both P<0.01), and there was no statistical significance between A1AR and A3AR expressions (both P>0.05). . EA may achieve cardioprotective effect by regulating the expressions of A2aAR and A2bAR in myocardial tissue, which induce the corresponding signal cascade for reducing myocardial infarction area.

A novel combination of astilbin and low-dose methotrexate respectively targeting A2AAR and its ligand adenosine for the treatment of collagen-induced arthritis

Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) treatment with frequently serious adverse effects. Therefore, combination of low-dose MTX with other drugs is often used in clinic. In this study, we investigated the improvement of astilbin and low-dose MTX combination on collagen-induced arthritis in DBA/1J mice. Results showed that the clinic score, incidence rate, paw swelling, pathological changes of joints and rheumatoid factors were more alleviated in combination therapy than MTX or astilbin alone group. Elevated antibodies (IgG, IgG1, IgG2a, IgM and anti-collagen IgG) and pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, IFN-γ and IL-17A) in serum were significantly inhibited, while anti-inflammatory cytokine, IL-10, was enhanced by combination therapy. Further studies indicated that combination therapy significantly decreased Th1 and Th17 cell differentiation and increased Treg cell differentiation. Mechanisms analysis demonstrated combination therapy greatly inhibited Con A-activated MAPK and inflammatory transcriptional signals. Moreover, MTX activated adenosine release and astilbin specifically up-regulated A2A adenosine receptor (A2AAR) expression simultaneously, which most probably contributed to the synergistic efficacy of combination therapy. ZM241385, a specific antagonist of A2AAR, greatly blocked the effects of combination therapy on T cell functions and downstream pathways. All these findings suggest that astilbin is a valuable candidate for low-dose MTX combined therapy in RA via increasing A2AAR/adenosine system and decreasing ERK/NFκB/STATs signals.

Expression of A1 and A3 adenosine receptors in human breast tumors.

Adenosine receptors (A1, A2A, A2B, A3) play an important role in the regulation of growth, proliferation and death of cancer and normal cells. We recently showed the expression profile of A2A and A2B receptors in normal and tumor breast tissues. In the present study, we used semiquantitative RT-PCR to measure the A1 and A3 gene expression levels in normal and tumor breast tissues. Breast tumors (n = 18) and non-neoplastic mammary tissues (n = 10) were collected and histologically confirmed to be neoplastic or non-neoplastic, respectively. Total RNA was extracted and reverse transcribed into cDNA, and PCR was performed under optimized condition for each receptor subtype. Amplification of beta-actin mRNA served as control for RT-PCR. The PCR products were separated on 1.7% agarose gels. The intensity of the bands was quantitated with ImageJ software after normalization against beta-actin expression. All breast tumor and normal tissue specimens expressed A1 and A3 adenosine receptor transcripts. However, we observed that the expression level of the A3 receptor in tumor tissues was 1.27-fold that of normal tissues, whereas there was no significant difference between the expression levels of A1 in normal and tumor tissues. Interestingly, the results of the present study indicate that breast tumors exhibit a higher level of A3 transcripts (than normal tissues) and support the possible key role of A3 adenosine receptor in tumor development. However, further studies based on real-time quantitative RT-PCR are needed to identify the exact gene expression levels.

Elevated Pressure Changes the Purinergic System of Microglial Cells.

Glaucoma is the second cause of blindness worldwide and is characterized by the degeneration of retinal ganglion cells (RGCs) and optic nerve atrophy. Increased microglia reactivity is an early event in glaucoma that may precede the loss of RGCs, suggesting that microglia and neuroinflammation are involved in the pathophysiology of this disease. Although global changes of the purinergic system have been reported in experimental and human glaucoma, it is not known if this is due to alterations of the purinergic system of microglial cells, the resident immune cells of the central nervous system. We now studied if elevated hydrostatic pressure (EHP), mimicking ocular hypertension, changed the extracellular levels of ATP and adenosine and the expression, density and activity of enzymes, transporters and receptors defining the purinergic system. The exposure of the murine microglial BV-2 cell line to EHP increased the extracellular levels of ATP and adenosine, increased the density of ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) and decreased the density of the equilibrative nucleotide transporter 2 as well as the activity of adenosine deaminase. The expression of adenosine A1 receptor also decreased, but the adenosine A3 receptor was not affected. Notably, ATP and adenosine selectively control migration rather than phagocytosis, both bolstered by EHP. The results show that the purinergic system is altered in microglia in conditions of elevated pressure. Understanding the impact of elevated pressure on the purinergic system will help to unravel the mechanisms underlying inflammation and neurodegeneration associated with glaucoma.

Targeting expression of adenosine receptors during hypoxia induced angiogenesis – A study using zebrafish model

Hypoxia is known to be a major player during pathological angiogenesis and adenosine as a negative feedback signaling to maintain oxygen delivery in pathological ischemic condition. We mimicked hypoxic condition and studied angiogenesis by inducing adenosine receptors using forskolin, a plant compound and NECA analogue of adenosine using zebrafish model. Vascular endothelial growth factor (VEGF) is known to play a key role during pathological angiogenesis and regulated by the factors HIF1a under hypoxic condition and recently Notch is proposed to play a negative feedback loop mechanism along with VEGF signaling but the role of adenosine receptor during the process is not known. We evaluated the mRNA expression of adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH 1a and DLL4 and the phenotypic variations of zebrafish embryos when treated with DAPT, γ-secretase inhibitor of Notch in addition to treating the embryos with SU5416, a VEGF receptor inhibitor. Upregulation of adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH1a and DLL4 was observed embryos were when treated with forskolin and NECA could possibly mimic hypoxic condition. Hatching and heart rate also increased with NECA and forskolin. SU5416 showed decreases in blood vessel formation and decreased adenosine receptors, VEGF, VEGFR2, HIF1a and NRP1a expression and DAPT, exhibited decreases in blood vessels and decreased NRP1a, NOTCH1a, DLL4 expression. These embryos developed with poor vasculature, tail bending, abnormal phenotypes and developmental delay. Forskolin treated with inhibitors showed increased blood vessel formation, normal phenotype, development and adenosine receptors (A1, A2a.1, A2a.2, A2b), HIF1a, VEGF A, VEGF R2, NRP1a, NOTCH 1a and DLL4 gene expression suggesting that adenosine mediated Notch and VEGF could play an important role during development and angiogenesis. Targeting VEGF and Notch signaling with adenosine receptors inhibitors which might have a therapeutic significance during hypoxia and abnormal angiogenesis.

Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

Toll-like receptor 2 induces adenosine receptor A2a and promotes human squamous carcinoma cell growth via extracellular signal regulated kinases ½.

Patient treatment for oral squamous cell carcinoma (OSCC) not associated with Human papillomavirus remains problematic. OSCC microenvironment is typically inflamed and colonized by microorganisms, providing ligands for toll-like receptors (TLR). In immune cells TLR2 and TLR4 activate NF-kB and extracellular signal regulated kinase (ERK)1/2 pathways, leading to upregulation of inhibitory adenosine receptors A2a and A2b, and reduction in stimulatory A1 and A3. How TLR and adenosine receptors function in SCC cells is not understood. To address this gap, we evaluated TLR and adenosine receptor expression and function in human OSCC cells and keratinocytes. TLR2 and A2a were co-expressed in pre-cancer and SCC cells of 17 oral specimens. In vitro, 5/6 OSCC lines expressed more TLR2 than TLR1, 4 or 6 mRNA. TLR2 ligands stimulated A2a expression in TLR2-high cell lines, but no A1 or A3 was detected with or without stimuli. In TLR2-high OSCC, TLR2/1, 2/6 and adenosine receptor agonists activated ERK1/2. TLR2-mediated ERK1/2 phosphorylation resulted in accumulation of c-FOS, ERK-dependent cell proliferation and reduced caspase-3 activity. Similar ERK1/2-dependent proliferation and decreased caspase-3 activity were caused by combined TLR2 and adenosine receptor stimuli. We conclude that TLR2 and adenosine receptor agonists, known to be present in the tumor microenvironment, may contribute to OSCC progression in part via direct effects on the ERK1/2 pathway in squamous carcinoma cells.

Anti-inflammatory Effects of Heme Oxygenase-1 Depend on Adenosine A2A- and A2B-Receptor Signaling in Acute Pulmonary Inflammation.

Acute pulmonary inflammation is still a frightening complication in intensive care units. In our previous study, we determined that heme oxygenase (HO)-1 had anti-inflammatory effects in pulmonary inflammation. Recent literature has emphasized a link between HO-1 and the nucleotide adenosine. Since adenosine A2A- and A2B-receptors play a pivotal role in pulmonary inflammation, we investigated their link to the enzyme HO-1. In a murine model of pulmonary inflammation, the activation of HO-1 by hemin significantly decreased polymorphonuclear leukocyte (PMN) migration into the lung. This anti-inflammatory reduction of PMN migration was abolished in A2A- and A2B-knockout mice. Administration of hemin significantly reduced chemokine levels in the BAL of wild-type animals but had no effects in A2A-/- and A2B-/- mice. Microvascular permeability was significantly attenuated in HO-1-stimulated wild-type mice, but not in A2A-/- and A2B-/- mice. The activity of HO-1 rose after LPS inhalation in wild-type animals and, surprisingly, also in A2A-/- and A2B-/- mice after the additional administration of hemin. Immunofluorescence images of animals revealed alveolar macrophages to be the major source of HO-1 activity in both knockout strains—in contrast to wild-type animals, where HO-1 was also significantly augmented in the lung tissue. In vitro studies on PMN migration further confirmed our in vivo findings. In conclusion, we linked the anti-inflammatory effects of HO-1 to functional A2A/A2B-receptor signaling under conditions of pulmonary inflammation. Our findings may explain why targeting HO-1 in acute pulmonary inflammation has failed to prove effective in some patients, since septic patients have altered adenosine receptor expression.

Functional changes in vascular reactivity to adenosine receptor activation in type I diabetic mice

Activation of adenosine receptors has been implicated in several biological functions, including cardiovascular and renal function. Diabetes causes morphological and functional changes in the vasculature, resulting in abnormal responses to various stimuli. Recent studies have suggested that adenosine receptor expression and signaling are altered in disease states such as hypertension, diabetes. Using a streptozotocin (STZ) mouse model of type I diabetes (T1D), we investigated the functional changes in aorta and resistance mesenteric arteries to adenosine receptor agonist activation in T1D. Organ baths and DMT wire myographs were used for muscle tension measurements in isolated vascular rings, and western blotting was used for protein analysis. Concentration response curves to selective adenosine receptor agonists, including CCPA (A1 receptor agonist), Cl-IBMECA (A3 receptor agonist), CGS-21680 (A2A receptor agonist), and BAY 60–6583 (A2B receptor agonist), were performed. We found that diabetes did not affect adenosine receptor agonist-mediated relaxation or contraction in mesenteric arteries. However, aortas from diabetic mice exhibited a significant decrease (P < 0.05) in A1 receptor-mediated vasoconstriction. In addition, the aortas from STZ-treated mice exhibited an increase in phenylephrine-mediated contraction (EC50 7.40 ± 0.08 in STZ vs 6.89 ± 0.14 in vehicle; P < 0.05), while relaxation to A2A receptor agonists (CGS-21680) tended to decrease in aortas from the STZ-treated group (not statistically significant). Our data suggest that changes in adenosine receptor(s) vascular reactivity in T1D is tissue specific, and the decrease in A1 receptor-mediated aortic contraction could be a compensatory mechanism to counterbalance the increased adrenergic vascular contractility observed in aortas from diabetic mice.

Effects of cordycepin on spontaneous alternation behavior and adenosine receptors expression in hippocampus

Cordycepin, an adenosine analogue, has been reported to improve cognitive function. Important roles on learning and memory of adenosine and its receptors, such as adenosine A1 and A2A receptors (A1R and A2AR), also have been shown. Therefore, we assume that the improvement of learning and memory induced by cordycepin is likely related to hippocampal adenosine content and adenosine receptor density. Here we investigated the effects of cordycepin on the short-term spatial memory by using a spontaneous alternation behavior (SAB) test in Y-maze, and then examined hippocampal adenosine content and A1R and A2AR densities. We found that orally administrated cordycepin (at dosages of 5 and 10mg/kg twice daily for three weeks) significantly increased the percent of relative alternation of mice in SAB but not altered body weight, hippocampus weight and hippocampal adenosine content. Furthermore, cordycepin decreased A2AR density in hippocampal subareas; however, cordycepin only reduced the A1R density in DG but not CA1 or CA3 region. Our results suggest that cordycepin exerts a nootropic role possibly through modulating A2AR density of hippocampus, which further support the concept that it is mostly A2AR rather than A1R to control the adaptive processes of memory performance. These findings would be helpful to provide a new window into the pharmacological properties of cordycepin for cognitive promotion.

Upregulation of SERT and ADORA1 in broilers with acute right ventricular failure

Pulmonary hypertension (PH), remains a challenging disease with a large impact on both humans and meat-type chickens. PH is characterized by the onset of idiopathic pulmonary arterial hypertension leading to right ventricular failure. In this experiment relative gene expression of adenosine A1 receptor (ADORA1), serotonin transporter (SERT), phosphodiesterase 5A (PDE5) and troponin T2 were compared in hearts from broilers with acute right ventricular failure and from healthy birds. There were major increases in adenosine A1 receptor (177%) and serotonin transporter (475%), and more modest but significant increases in PDE5 (146%) and troponin T2 (140%) gene expressions in broilers with right ventricular failure compared to healthy birds (P<0.01). This novel report shows that pulmonary hypertension related gene expression in broilers is similar to that in humans. This molecular similarity between PH in broilers and human patients suggests, first, that they will make a suitable animal model for study PH in humans, but also that the literature on PH in humans may be profitably applied to the study of PH in broilers.

Evaluation of the Effect of Mesenchymal Stem Cells pulsed with Caffeine on the Function of the Peritoneal Macrophages of Rat

Introduction: Pervious studies indicated that the mesenchymal stem cells (MSCs) produce adenosine and express adenosine receptors. Moreover, produced adenosine has biological effect on these cells. On the other hand, MSCs have a wide relationship with macrophages in tissues and bone marrow. The aim of the present study was to determine the effects of the MSCs treated with caffeine, as an adenosine antagonist, on the peritoneal macrophages isolated from rats. Materials & methods: After isolation of mesenchymal stem cells from bone marrow of rats, these cells pulsed with different concentration of caffeine (0, 0.1, 0.5 and 1 mM) for 48h. Then, MSCs were co-cultured with macrophages for 1 day. Then, the level of nitric oxide production and respiratory burst of macrophages after challenge with N-formylmethionine-leucyl-phenylalanine (f-MLP) or opsonized yeast were evaluated. Findings: It seems that MSCs pulsed with caffeine or without it could significantly decrease the respiratory burst and nitric oxide production of co-cultured macrophages more prominent than macrophages alone after unspecific challenged with fMLP. Although, pervious treatment of MSCs with caffeine could decrease the respiratory burst and nitric oxide production of macrophages challenged with f-MLP more prominent than MSCs without treatment. However, the potential of nitric oxide production and respiratory burst of various group after challenge with opsonized yeast didnt show any significant differences. Discussion & conclusions: Collectively, these data may offer new insight into the potential mechanisms underlying the immunomodulatory and anti-inflammatory effects of caffeine.

Caffeine Augments The Instruction of Anti-Inflammatory Macrophages by The Conditioned Medium of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) have been shown to produce adenosine, express adenosine receptors, and communicate with macrophages and other cells. However, there is no information about the role of caffeine, as a popular drink and adenosine antagonist, on the crosstalk between MSCs and immune cells. The aim of the current study is to evaluate the effects of the conditioned medium of MSCs treated with caffeine on macrophages. In this experimental study, MSCs were isolated from bone marrow of rats and pulsed with different concentrations of caffeine (0, 0.1, 0.5 and 1 mM) for 72 hours. The conditioned medium of MSCs was collected after 24 hours, then incubated with macrophages for 24 hours. Finally, the functions of the macrophages were evaluated. Conditioned medium of MSCs treated with caffeine significantly enhanced phagocytosis and simultaneously regressed expression of reactive oxygen species (ROS) and nitric oxide (NO) as well as IL-12 by macrophages compared to the supernatants of MSCs alone. The conditioned medium of MSCs pulsed with caffeine at low to moderate concentrations preserved the neutral red uptake by macrophages and elevated IL-10 secretion by macrophages. A high concentration of caffeine could interfere with the two latter effects of supernatants of MSCs on the macrophages. Collectively, caffeine treatment of MSCs appeared to augment the instruction of anti-inflammatory macrophages by conditioned medium of MSCs. These findings might offer new insight into the potential mechanisms that underlie the immunomodulatory and anti-inflammatory effects of caffeine.

Upregulation of Neuronal Adenosine A1 Receptor in Human Rasmussen Encephalitis.

Rasmussen encephalitis (RE) is a rare neurological disorder characterized by unilateral inflammation of cerebral cortex and other structures, most notably the hippocampus, progressive cognitive deterioration, and pharmacoresistant focal epilepsy. The pathogenesis of RE with unilateral cortical atrophy and focal seizures is still enigmatic. Activation of adenosine A1 receptors (A1R) has been proven to prevent the spatial spread of seizures. We hypothesized that the epileptogenic mechanisms underlying RE are related to changes in neuronal A1R expression. Immunnohistochemistry was used to examine the expression of A1R and adenosine kinase (ADK) in cortical specimens from RE (n = 12), and compared with control cortical tissue. The quantification of A1R and ADK expression was evaluated by Western blot. A1R was predominantly localized in perinuclear of neurons and not in astrocytes or microglia. Upregulation of neuronal A1R was observed in the lesions of RE. Reactive astrocytes and subpopulation of remaining neurons demonstrated over-expression of the ADK within the lesions of RE. Significant increase of A1R and ADK expression in RE compared with controls was confirmed by Western blot. These results suggest that over-expression of ADK is a common pathologic hallmark of RE, and that upregulation of neuronal A1R in RE is crucial in preventing the spread of seizures.