Adenosine Receptor Expression Research Articles

A1 adenosine receptor attenuates intracerebral hemorrhage-induced secondary brain injury in rats by activating the P38-MAPKAP2-Hsp27 pathway.

BackgroundThis study was designed to determine the role of the A1 adenosine receptors in intracerebral hemorrhage (ICH)-induced secondary brain injury and the underlying mechanisms.MethodsA collagenase-induced ICH model was established in Sprague–Dawley rats, and cultured primary rat cortical neurons were exposed to oxyhemoglobin at a concentration of 10 μM to mimic ICH in vitro. The A1 adenosine receptor agonist N(6)-cyclohexyladenosine and antagonist 8-phenyl-1,3-dipropylxanthine were used to study the role of A1 adenosine receptor in ICH-induced secondary brain injury, and antagonists of P38 and Hsp27 were used to study the underlying mechanisms of A1 adenosine receptor actions.ResultsThe protein level of A1 adenosine receptor was significantly increased by ICH, while there was no significant change in protein levels of the other 3 adenosine receptors. In addition, the A1 adenosine receptor expression could be increased by N(6)-cyclohexyladenosine and decreased by 8-phenyl-1,3-dipropylxanthine under ICH conditions. Activation of the A1 adenosine receptor attenuated neuronal apoptosis in the subcortex, which was associated with increased phosphorylation of P38, MAPK, MAPKAP2, and Hsp27. Inhibition of the A1 adenosine receptor resulted in opposite effects. Finally, the neuroprotective effect of the A1 adenosine receptor agonist N(6)-cyclohexyladenosine was inhibited by antagonists of P38 and Hsp27.ConclusionsThis study demonstrates that activation of the A1 adenosine receptor by N(6)-cyclohexyladenosine could prevent ICH-induced secondary brain injury via the P38-MAPKAP2-Hsp27 pathway.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48h. Accordingly, caffeine (30mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6h after injection, but an increase after 18 and 24h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an invivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.

CD39 mediated regulation of Th17-cell effector function is impaired in juvenile autoimmune liver disease

Background & aimsT-helper-type 17 (Th17) cells are involved in autoimmune tissue damage. CD39 is an ectonucleotidase that catalyzes extracellular ATP/ADP hydrolysis, culminating in the generation of immunosuppressive adenosine. Functional CD39 expression confers immunosuppressive properties upon immune cells. As the proportion of CD39 lymphocytes is decreased in juvenile autoimmune liver disease (AILD), we have explored whether decreased CD39 expression is present on Th17 cells and whether this phenomenon is associated with heightened effector function and inflammation. MethodsThirty-eight patients with juvenile AILD (22 autoimmune hepatitis and 16 autoimmune sclerosing cholangitis), 8 disease controls (DC) and 16 healthy subjects (HS) were studied. Peripheral blood cell phenotype was determined by flow cytometry; ability to suppress by inhibition of cell proliferation/effector cytokine production; ectoenzymatic activity by thin layer chromatography; expression of adenosine receptor, adenosine deaminase (ADA) and phosphodiesterases (PDE) by quantitative real-time PCR or by Western Blot. ResultsCD39+ Th17 (Th17CD39+) cells from HS appear activated and contain high frequencies of lymphocytes producing regulatory cytokines. In AILD, however, Th17CD39+ cells are markedly diminished and fail to generate AMP/adenosine, thereby limiting control of both target cell proliferation and IL-17 production. When compared to HS, Th17 cells from AILD patients also show lower A2A adenosine receptor expression while displaying similar levels of PDE4A, PDE4B and ADA. Only rare Th17CD39+ cells are observed by liver immunohistochemistry. ConclusionsTh17CD39+ cells in juvenile AILD are both quantitatively decreased and qualitatively deficient. Low levels CD39 and A2A expression may contribute to the perpetuation of Th17 cell effector properties and unfettered inflammation in this disease.

Mechanisms of caffeine-induced diuresis

Caffeine is an alkaloid which belongs to the family of methylxanthines and is present in beverages, food and drugs. Caffeine competitively antagonizes the adenosine receptors (AR), which are G protein-coupled receptors largely distributed throughout the body, including brain, heart, vessels and kidneys. Caffeine consumption has a well-known diuretic effect. The homeostasis of salt and water involves different segments of the nephron, in which adenosine plays complex roles depending on the differential expression of AR. Hence, caffeine increases glomerular filtration rate by opposing the vasoconstriction of renal afferent arteriole mediated by adenosine via type 1 AR during the tubuloglomerular feedback. Caffeine also inhibits Na(+) reabsorption at the level of renal proximal tubules. In addition, caffeine perturbs the hepatorenal reflex via sensory nerves in Mall's intrahepatic spaces. Here, we review the physiology of caffeine-induced natriuresis and diuresis, as well as the putative pathological implications.

Expression of calcitonin gene-related peptide, adenosine A2a receptor and adenosine A1 receptor in experiment rat migraine models.

A migraine is a disabling neurovascular disorder characterized by a unilateral throbbing headache that lasts from 4 to 72 h. The headache is often accompanied by nausea, vomiting, phonophobia and photophobia, and may be worsened by physical exercise. The trigeminovascular system (TVS) is speculated to have an important role in migraines, although the pathophysiology of this disorder remains to be elucidated. Trigeminal ganglion (TG) and spinal trigeminal nucleus caudalis (TNC) are important components of the TVS. Several clinical cases have provided evidence for the involvement of the brainstem in migraine initiation. Electrical stimulation of the trigeminal ganglion (ESTG) in rats can activate TVS during a migraine attack. Calcitonin gene-related peptide (CGRP) is an important vasoactive compound produced following TVS activation. Numerous studies have revealed that adenosine and its receptors have an important role in pain transmission and regulation process. However, only a few studies have examined whether adenosine A2a receptor (A2aR) and adenosine A1 receptor (A1R) are involved in migraine and nociceptive pathways. In the present study, CGRP, A2aR and A1R expression levels were detected in the TG and TNC of ESTG models through reverse transcription-quantitative polymerase chain reaction and western blot analysis. Tianshu capsule (TSC), a type of Chinese medicine, was also used in the ESTG rat models to examine its influence on the three proteins. Results demonstrated that CGRP, A2aR and A1R mediated pain transmission and the regulation process during migraine and the expression of the three proteins was regulated by TSC.

Hyperglycemia alters E-NTPDases, ecto-5′-nucleotidase, and ectosolic and cytosolic adenosine deaminase activities and expression from encephala of adult zebrafish (Danio rerio)

Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.

Insulin requires A1 adenosine receptors expression to reverse gestational diabetes-increased L-arginine transport in human umbilical vein endothelium.

Gestational diabetes mellitus (GDM) associates with increased L-arginine transport and extracellular concentration of adenosine in human umbilical vein endothelial cells (HUVECs). In this study we aim to determine whether insulin reverses GDM-increased L-arginine transport requiring adenosine receptors expression in HUVECs. Primary cultured HUVECs from full-term normal (n = 38) and diet-treated GDM (n = 38) pregnancies were used. Insulin effect was assayed on human cationic amino acid transporter 1 (hCAT1) expression (protein, mRNA, SLC7A1 promoter activity) and activity (initial rates of L-arginine transport) in the absence or presence of adenosine receptors agonists or antagonists. A1 adenosine receptors (A1AR) and A2AAR expression (Western blot, quantitative PCR) was determined. Experiments were done in cells expressing or siRNA-suppressed expression of A1AR or A2AAR. HUVECs from GDM exhibit higher maximal transport capacity (maximal velocity (V max)/apparent Michaelis Menten constant (K m), V max/K m), which is blocked by insulin by reducing the V max to values in cells from normal pregnancies. Insulin also reversed the GDM-associated increase in hCAT-1 protein abundance and mRNA expression, and SLC7A1 promoter activity for the fragment -606bp from the transcription start point. Insulin effects required A1AR, but not A2AAR expression and activity in this cell type. In the absence of insulin, GDM-increased hCAT-1 expression and activity required A2AAR expression and activity. HUVECs from GDM pregnancies exhibit a differential requirement of A1AR or A2AAR depending on the level of insulin, a phenomenon that represent a condition where adenosine or analogues of this nucleoside could be acting as helpers of insulin biological effects in GDM.

Adenosine A1 Receptor-Mediated Endocytosis of AMPA Receptors Contributes to Impairments in Long-Term Potentiation (LTP) in the Middle-Aged Rat Hippocampus

Aging causes multiple changes in the mammalian brain, including changes in synaptic signaling. Previous reports have shown increased extracellular adenosine in the aging brain, and we recently reported that activation of adenosine A1 receptors (A1Rs) induces AMPA receptor (AMPAR) internalization in rat hippocampus. This study investigated whether aging-related changes in the rat hippocampus include altered surface expression of adenosine A1 and A2A receptors, and whether these changes correspond to changes in AMPAR surface expression and altered synaptic plasticity. We found reduced A1R surface expression in middle-aged rat hippocampus, and also reduced GluA1 and GluA2 AMPAR subunit surface expression. Using a chemically-induced LTP (cLTP) experimental protocol, we recorded fEPSPs in young (1month old) and middle-aged (7-12month old) rat hippocampal slices. There were significant impairments in cLTP in middle-aged slices, suggesting impaired synaptic plasticity. Since we previously showed that the A1R agonist N(6)-cyclopentyladenosine (CPA) reduced both A1Rs and GluA2/GluA1 AMPARs, we hypothesized that the observed impaired synaptic plasticity in middle-aged brains is regulated by A1R-mediated AMPAR internalization by clathrin-mediated endocytosis. Following cLTP, we found a significant increase in GluA1 and GluA2 surface expression in young rats, which was blunted in middle-aged brains or in young brains pretreated with CPA. Blocking A1Rs with 8-cyclopentyl-1,3-dipropylxanthine or AMPAR endocytosis with either Tat-GluA2-3Y peptide or dynasore (dynamin inhibitor) similarly enhanced AMPAR surface expression following cLTP. These data suggest that age-dependent alteration in adenosine receptor expression contributes to increased AMPAR endocytosis and impaired synaptic plasticity in aged brains.

Adenosine A1 receptor activation attenuates cardiac hypertrophy and fibrosis in response to α1 -adrenoceptor stimulation in vivo.

Adenosine has been proposed to exert anti-hypertrophic effects. However, the precise regulation and the role of the different adenosine receptor subtypes in the heart and their effects on hypertrophic signalling are largely unknown. We aimed to characterize expression and function of adenosine A1 receptors following hypertrophic stimulation in vitro and in vivo. Pro-hypertrophic stimuli and adenosine A1 receptor stimulation of neonatal rat cardiomyocytes and male C57/Bl6 mice, sc. drug administration, real-time PCR, (3) [H]-leucine-incorporation assay, immunostaining, tissue staining, Western blots, gravimetric analyses and echocardiography were applied in this study. In neonatal rat cardiomyocyte cultures, phenylephrine, but not angiotensin II or insulin-like growth factor 1 (IGF1), up-regulated adenosine A1 receptors concentration-dependently. The hypertrophic phenotype (cardiomyocyte size, sarcomeric organization, total protein synthesis, c-fos expression) mediated by phenylephrine (10 μM), but not that by angiotensinII (1 μM) or IGF1 (20 ng·mL(-1) ), was counteracted by the selective A1 receptor agonist, N6-cyclopentyladenosine. In C57/BL6 mice, continuous N6-cyclopentyladenosine infusion (2 mg·kg(-1) ·day(-1) ; 21 days) blunted phenylephrine (120 mg·kg(-1) ·day(-1) ; 21 days) induced hypertrophy (heart weight, cardiomyocyte size and fetal genes), fibrosis, MMP 2 up-regulation and generation of oxidative stress - all hallmarks of maladaptive remodelling. Concurrently, phenylephrine administration increased expression of adenosine A1 receptors. We have presented evidence for a negative feedback mechanism attenuating pathological myocardial hypertrophy following α1 -adrenoceptor stimulation. Our results suggest adenosine A1 receptors as potential targets for therapeutic strategies to prevent transition from compensated myocardial hypertrophy to decompensated heart failure due to chronic cardiac pressure overload.

Ecto-5'-Nucleotidase Overexpression Reduces Tumor Growth in a Xenograph Medulloblastoma Model.

BackgroundEcto-5’-nucleotidase/CD73 (ecto-5’-NT) participates in extracellular ATP catabolism by converting adenosine monophosphate (AMP) into adenosine. This enzyme affects the progression and invasiveness of different tumors. Furthermore, the expression of ecto-5’-NT has also been suggested as a favorable prognostic marker, attributing to this enzyme contradictory functions in cancer. Medulloblastoma (MB) is the most common brain tumor of the cerebellum and affects mainly children.Materials and MethodsThe effects of ecto-5’-NT overexpression on human MB tumor growth were studied in an in vivo model. Balb/c immunodeficient (nude) 6 to 14-week-old mice were used for dorsal subcutaneous xenograph tumor implant. Tumor development was evaluated by pathophysiological analysis. In addition, the expression patterns of adenosine receptors were verified.ResultsThe human MB cell line D283, transfected with ecto-5’-NT (D283hCD73), revealed reduced tumor growth compared to the original cell line transfected with an empty vector. D283hCD73 generated tumors with a reduced proliferative index, lower vascularization, the presence of differentiated cells and increased active caspase-3 expression. Prominent A1 adenosine receptor expression rates were detected in MB cells overexpressing ecto-5’-NT.ConclusionThis work suggests that ecto-5’-NT promotes reduced tumor growth to reduce cell proliferation and vascularization, promote higher differentiation rates and initiate apoptosis, supposedly by accumulating adenosine, which then acts through A1 adenosine receptors. Therefore, ecto-5’-NT might be considered an important prognostic marker, being associated with good prognosis and used as a potential target for therapy.

Alveolar Epithelial A2B Adenosine Receptors in Pulmonary Protection during Acute Lung Injury.

Acute lung injury (ALI) is an acute inflammatory lung disease that causes morbidity and mortality in critically ill patients. However, there are many instances where ALI resolves spontaneously through endogenous pathways that help to control excessive lung inflammation. Previous studies have implicated the extracellular signaling molecule adenosine and signaling events through the A2B adenosine receptor in lung protection. In this context, we hypothesized that tissue-specific expression of the A2B adenosine receptor is responsible for the previously described attenuation of ALI. To address this hypothesis, we exposed mice with tissue-specific deletion of Adora2b to ALI, utilizing a two-hit model where intratracheal LPS treatment is followed by injurious mechanical ventilation. Interestingly, a head-to-head comparison of mice with deletion of Adora2b in the myeloid lineage (Adora2b(loxP/loxP) LysM Cre(+)), endothelial cells (Adora2b(loxP/loxP) VE-cadherin Cre(+)), or alveolar epithelial cells (Adora2b(loxP/loxP) SPC Cre(+)) revealed a selective increase in disease susceptibility in Adora2b(loxP/loxP) SPC Cre(+) mice. More detailed analysis of Adora2b(loxP/loxP) SPC Cre(+) mice confirmed elevated lung inflammation and attenuated alveolar fluid clearance. To directly deliver an A2B adenosine receptor-specific agonist to alveolar epithelial cells, we subsequently performed studies with inhaled BAY 60-6583. Indeed, aerosolized BAY 60-6583 treatment was associated with attenuated pulmonary edema, improved histologic lung injury, and dampened lung inflammation. Collectively, these findings suggest that alveolar epithelial A2B adenosine receptor signaling contributes to lung protection, and they implicate inhaled A2B adenosine receptor agonists in ALI treatment.

Chronic caffeine exposure attenuates blast-induced memory deficit in mice

ObjectiveTo investigate the effects of three different ways of chronic caffeine administration on blast-induced memory dysfunction and to explore the underlying mechanisms. MethodsAdult male C57BL/6 mice were used and randomly divided into five groups: control: without blast exposure, con-water: administrated with water continuously before and after blast-induced traumatic brain injury (bTBI), con-caffeine: administrated with caffeine continuously for 1 month before and after bTBI, pre-caffeine: chronically administrated with caffeine for 1 month before bTBI and withdrawal after bTBI, post-caffeine: chronically administrated with caffeine after bTBI. After being subjected to moderate intensity of blast injury, mice were recorded for learning and memory performance using Morris water maze (MWM) paradigms at 1, 4, and 8 weeks post-blast injury. Neurological deficit scoring, glutamate concentration, proinflammatory cytokines production, and neuropathological changes at 24 h, 1, 4, and 8 weeks post-bTBI were examined to evaluate the brain injury in early and prolonged stages. Adenosine A1 receptor expression was detected using qPCR. ResultsAll of the three ways of chronic caffeine exposure ameliorated blast-induced memory deficit, which is correlated with the neuroprotective effects against excitotoxicity, inflammation, astrogliosis and neuronal loss at different stages of injury. Continuous caffeine treatment played positive roles in both early and prolonged stages of bTBI; pre-bTBI and post-bTBI treatment of caffeine tended to exert neuroprotective effects at early and prolonged stages of bTBI respectively. Up-regulation of adenosine A1 receptor expression might contribute to the favorable effects of chronic caffeine consumption. ConclusionSince caffeinated beverages are widely consumed in both civilian and military personnel and are convenient to get, the results may provide a promising prophylactic strategy for blast-induced neurotrauma and the consequent cognitive impairment.

CD56brightCD16- NK Cells Produce Adenosine through a CD38-Mediated Pathway and Act as Regulatory Cells Inhibiting Autologous CD4+ T Cell Proliferation.

Recent studies suggested that human CD56(bright)CD16(-) NK cells may play a role in the regulation of the immune response. Since the mechanism(s) involved have not yet been elucidated, in the present study we have investigated the role of nucleotide-metabolizing enzymes that regulate the extracellular balance of nucleotides/nucleosides and produce the immunosuppressive molecule adenosine (ADO). Peripheral blood CD56(dim)CD16(+) and CD56(bright)CD16(-) NK cells expressed similar levels of CD38. CD39, CD73, and CD157 expression was higher in CD56(bright)CD16(-) than in CD56(dim)CD16(+) NK cells. CD57 was mostly expressed by CD56(dim)CD16(+) NK cells. CD203a/PC-1 expression was restricted to CD56(bright)CD16(-) NK cells. CD56(bright)CD16(-) NK cells produce ADO and inhibit autologous CD4(+) T cell proliferation. Such inhibition was 1) reverted pretreating CD56(bright)CD16(-) NK cells with a CD38 inhibitor and 2) increased pretreating CD56(bright)CD16(-) NK cells with a nucleoside transporter inhibitor, which increase extracellular ADO concentration. CD56(bright)CD16(-) NK cells isolated from the synovial fluid of juvenile idiopathic arthritis patients failed to inhibit autologous CD4(+) T cell proliferation. Such functional impairment could be related to 1) the observed reduced CD38/CD73 expression, 2) a peculiar ADO production kinetics, and 3) a different expression of ADO receptors. In contrast, CD56(bright)CD16(-) NK cells isolated from inflammatory pleural effusions display a potent regulatory activity. In conclusion, CD56(bright)CD16(-) NK cells act as "regulatory cells" through ADO produced by an ectoenzymes network, with a pivotal role of CD38. This function may be relevant for the modulation of the immune response in physiological and pathological conditions, and it could be impaired during autoimmune/inflammatory diseases.

Increased Signaling via Adenosine A1 Receptors, Sleep Deprivation, Imipramine, and Ketamine Inhibit Depressive-like Behavior via Induction of Homer1a

Major depressive disorder is among the most commonly diagnosed disabling mental diseases. Several non-pharmacological treatments of depression upregulate adenosine concentration and/or adenosine A1 receptors (A1R) in the brain. To test whether enhanced A1R signaling mediates antidepressant effects, we generated a transgenic mouse with enhanced doxycycline-regulated A1R expression, specifically in forebrain neurons. Upregulating A1R led to pronounced acute and chronic resiliencetoward depressive-like behavior in various tests. Conversely, A1R knockout mice displayed an increased depressive-like behavior and were resistant to the antidepressant effects of sleep deprivation (SD). Various antidepressant treatments increase homer1a expression in medial prefrontal cortex (mPFC). Specific siRNA knockdown of homer1a in mPFC enhanced depressive-like behavior and prevented the antidepressant effects of A1R upregulation, SD, imipramine, and ketamine treatment. In contrast, viral overexpression of homer1a in the mPFC had antidepressant effects. Thus, increased expression of homer1a is a final common pathway mediating the antidepressant effects of different antidepressant treatments.

Adenosine A2b receptor promotes progression of human oral cancer.

BackgroundAdenosine A2b receptor (ADORA2B) encodes an adenosine receptor that is a member of the G protein-coupled receptor superfamily. This integral membrane protein stimulates adenylate cyclase activity in the presence of adenosine. Little is known about the relevance of ADORA2B to human malignancy including oral squamous cell carcinoma (OSCC). We aimed to characterize the expression state and function of ADORA2B in OSCC.MethodsThe ADORA2B expression levels in nine OSCC-derived cells were analyzed by quantitative reverse transcriptase-polymerase chain reaction and immunoblotting analyses. Using an ADORA2B knockdown model, we assessed cellular proliferation and expression of hypoxia-inducible factor1α (HIF-1α). We examined the adenosine receptor expression profile under both normoxic and hypoxic conditions in the OSCC-derived cells. In addition to in vitro data, the clinical correlation between the ADORA2B expression levels in primary OSCCs (n = 100 patients) and the clinicopathological status by immunohistochemistry (IHC) also was evaluated.ResultsADORA2B mRNA and protein were up-regulated significantly (p < 0.05) in seven OSCC-derived cells compared with human normal oral keratinocytes. Suppression of ADORA2B expression with shRNA significantly (p < 0.05) inhibited cellular proliferation compared with the control cells. HIF-1α also was down-regulated in ADORA2B knockdown OSCC cells. During hypoxia, ADORA2B expression was induced significantly (p < 0.05) in the mRNA and protein after 24 hours of incubation in OSCC-derived cells. IHC showed that ADORA2B expression in primary OSCCs was significantly (p < 0.05) greater than in the normal oral counterparts and that ADORA2B-positive OSCCs were correlated closely (p < 0.05) with tumoral size.ConclusionOur results suggested that ADORA2B controls cellular proliferation via HIF-1α activation, indicating that ADORA2B may be a key regulator of tumoral progression in OSCCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1577-2) contains supplementary material, which is available to authorized users.

Expression of adenosine receptors in monocytes from patients with bronchial asthma

Adenosine is generated from adenosine triphosphate, which is released by stressed and damaged cells. Adenosine levels are significantly increased in patients with bronchial asthma (BA) and mediate mast cell degranulation and bronchoconstriction. Over the last decade, increasing evidence has shown that adenosine can modulate the innate immune response during monocytes differentiation towards mature myeloid cells. These adenosine-differentiated myeloid cells, characterized by co-expression of monocytes/macrophages and dendritic cell markers such as CD14 and CD209, produce high levels of pro-inflammatory cytokines, thus contributing to the pathogenesis of BA and chronic obstructive pulmonary disease. We found that expression of ADORA2A and ADORA2B are increased in monocytes obtained from patients with BA, and are associated with the generation of CD14posCD209pos pro-inflammatory cells. A positive correlation between expression of ADORA2B and IL-6 was identified in human monocytes and may explain the increased expression of IL-6 mRNA in asthmatics. Taken together, our results suggest that monocyte-specific expression of A2 adenosine receptors plays an important role in pro-inflammatory activation of human monocytes, thus contributing to the progression of asthma.

Distinct transcriptome profiles differentiate nonsteroidal anti-inflammatory drug–dependent from nonsteroidal anti-inflammatory drug–independent food-induced anaphylaxis

Lipid transfer protein (LTP), an abundant protein in fruits, vegetables, and nuts, is a common food allergen in Mediterranean areas causing diverse allergic reactions. Approximately 40% of food-related anaphylaxis induced by LTPs requires nonsteroidal anti-inflammatory drugs (NSAIDs) as a triggering cofactor. We sought to better understand the determinants of NSAID-dependent and NSAID-independent LTP-induced anaphylaxis (LTP-A). Selection of patients was based on a proved clinical history of NSAID-dependent or NSAID-independent anaphylaxis to LTPs, positive skin prick test response to LTPs, and serum LTP IgE. Whole-transcriptome (RNA sequencing) analysis of blood cells from 14 patients with NSAID-related LTP-A (NSAID-LTP-A), 7 patients with LTP-A, and 13 healthy control subjects was performed to identify distinct gene expression signatures. Expression of genes regulating gastrointestinal epithelial renewal was altered in both patient sets, particularly in those with LTP-A, who also presented with gene expression profiles characteristic of an inflammatory syndrome. These included altered B-cell pathways, increased neutrophil activation markers, and increased reactive oxygen species levels. Increased expression of the IgG receptor (CD64) in patients with LTP-A was mirrored by the presence of LTP-specific IgG1 and IgG3. Conversely, patients with NSAID-LTP-A were characterized by reduced expression of IFN-γ-regulated genes and IFN-γ levels, as well as upregulated expression of adenosine receptor 3 (ADORA3) and genes related to adenosine metabolism. Gene ontology analysis suggests disturbances in gut epithelial homeostasis in both groups with LTP-A, with potential integrity breaches in patients with LTP-A that might explain their distinct inflammatory signatures. Differential regulation in patients with LTP-A and those with NSAID-LTP-A of the IFN-γ pathway, IgG receptors, and ADORA3 might provide the pathogenic basis of their distinct responses.

Chronic Cerebral Ischemia Induces Downregulation of A1 Adenosine Receptors During White Matter Damage in Adult Mice.

The role of A1 adenosine receptors (A1ARs) in the white matter under chronic cerebral ischemic conditions remains unclear. Here, we used right unilateral common carotid artery occlusion (rUCCAO) to construct a chronic cerebral ischemic mouse model. A1AR expression and proteolipid protein (PLP, a marker of white matter myelination) in the corpus callosum were observed by immunoreaction and immunohistochemistry, respectively. Pro-inflammatory interleukin-1β (IL-1β) and anti-inflammatory interleukin-10 (IL-10) levels were determined by ELISA. The Morris water maze test was employed to detect cognitive impairment. A1AR expression significantly decreased in the rUCCAO group as compared with the sham control group on weeks 2, 4, and 6, respectively. IL-10 levels in the rUCCAO group significantly declined on week 6, while there was no significant change in IL-1β expression. PLP expression significantly decreased in the rUCCAO group on weeks 2, 4, and 6. Moreover, latency time for the Morris water maze test significantly increased in the rUCCAO group on weeks 4 and 6, while the number of platform location crossing significantly decreased in the rUCCAO group on weeks 2, 4, and 6. In conclusion, this study provides the first evidence that chronic cerebral ischemia appears to induce A1AR downregulation and inhibition of IL-10 production, which may play key roles in the neuropathological mechanisms of ischemic white matter lesions. These data will facilitate future studies in formulating effective therapeutic strategies for ischemic white matter lesions.

Age-related decrease of adenosine-mediated relaxation in rat detrusor is a result of A2B receptor downregulation.

To analyze the effect of adenosine on detrusor smooth muscle contraction and to assess age-related changes of adenosine function. Sustained contractions were induced in young (10-30 days) and old (>60 days) rat detrusor muscle strips by application of 30 mmol/L K(+) and adenosine (0.1-400 µmol/L), which was either applied before raising the K(+) concentration or added to the precontracted muscle strip. Quantitative polymerase chain reaction analyses were used to study adenosine receptor expression in rat and human detrusor specimens. Pretreatment with adenosine dose-dependently reduced subsequent K(+) -induced contraction in detrusor muscle strips from young rats (half-maximal effect = 40 µmol/L). The residual depolarization-induced contraction strength in young tissue was significantly smaller than in tissue from old animals, showing a greater potency of adenosine in young detrusor samples. Likewise, the relaxing effect of adenosine on precontracted detrusor muscle was also significantly more pronounced in young compared with older detrusor. Quantitative polymerase chain reaction showed an age-related downregulation of the adenosine A2B receptor in rat detrusor tissues, which could be confirmed in human detrusor samples. Furthermore, relaxation of both K(+) -induced as well as carbachol-induced contraction by the specific A2B receptor agonist BAY 60-6583 was significantly more pronounced in young than in old rats. Adenosine powerfully counteracts contraction of detrusor smooth muscle, which is lost in the aging bladder. This is paralleled by an age-dependent transcriptional downregulation of the low-affinity A2B receptor. Hence, this might be pathophysiologically relevant in conditions of raised adenosine concentrations, such as hyperactive bladder contractility.

Neuroinflammation after neonatal hypoxia-ischemia is associated with alterations in the purinergic system: adenosine deaminase 1 isoenzyme is the most predominant after insult.

Hypoxic-ischemic (HI) injury perinatal brain is a major contributor to morbidity and mortality to infants and children. Adenosine may play a role in the pathophysiology of HI, since it modulates the inflammatory process and the release of several neurotransmitters. Thus, the aim of this study was to identify the isoforms of adenosine deaminase (ADA) responsible for the enzymatic activity as well as the adenosine kinase (ADK) and A1 adenosine receptor (A1R) expression in the cerebral cortex eight days after HI. Myeloperoxidase (MPO) and N-acetyl-glucosaminidase (NAG) were assessed as inflammation markers. ADA activity was analyzed, in the presence or absence of a specific ADA1 inhibitor, erythro-9-(2-hydroxy-3-nonyl) adenine. The ADA1 activity (92.6%) was significantly higher than ADA2 (7.4%) activity in the cerebral cortex eight days after HI. A1Rs and ADK protein expression showed decreased 8 days after insult. Interestingly, the ADA1, MPO, and NAG activities were correlated positively. In view of this, we conclude that the inhibitor of ADA1, in in vitro conditions, was effective in decreasing the ADA activity, and that mainly ADA1 isoform is responsible for the increase in the ADA activity 8 days after HI insult. Therefore, HI neonatal was able to alter the ADK and A1R expression. Thus, due to the importance of adenosine signaling in the regulation of inflammatory and immune process and the crucial role of ADA in the postischemic homeostase of adenosine as well as during inflammatory process, we suggest that ADA1 inhibitors may play an important role in the regulation of events that follow the HI insult, favoring the increase in the adenosine in the sites of tissue injury. Together, these results highlight a role of the purinergic signaling cascade in the pathophysiology of HI neonatal.