A2A Expression Research Articles

Rosmarinic acid modulates purinergic signaling and induces apoptosis in melanoma cells.

Cancer cases have increased worldwide. Cutaneous melanoma (CM), a highly metastatic skin cancer, largely contributes to global statistical cancer death data. Research has shown that rosmarinic acid (RA) is a promising phenolic compound with antineoplastic properties. Thus, we investigated the effects of RA on apoptosis-inducing in melanoma cells, purinergic signaling modulation, and cytokine levels. We treated SK-MEL-28 cells for 24h with different concentrations of RA and assessed the apoptosis, CD39, CD73, and A2A expression, and cytokine levels. We found RA-induced apoptosis in melanoma cells. Regarding the purinergic system, we verified that RA downregulated the expression of CD73 and A2A, specially at high concentrations of treatment. Additionally, RA increased IL-6, IL-4, IL-10, IFN-γ, and TNF-α levels. Our in vitro results confirm RA's potential to be used to induce melanoma cell apoptosis, having CD73 and A2A as targets when reversion of immune suppression is desired. Further studies in animal models and clinical trials focusing on RA's modulation of purinergic signaling in melanoma are required.

Reduced Proportion of CD39+ Treg Cells Correlates with the Development of Primary Biliary Cholangitis in Patients and a Murine Model

Primary Biliary Cholangitis (PBC) is a chronic autoimmune cholestatic liver disease. This study investigated changes of adenosine metabolism pathway related molecules and their contribution to inflammatory injury in PBC. Clinical results showed that the concentrations of alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), Alkaline Phosphatase (ALP) and g-Glutamyl Transferase (GGT) in serum of 64 PBC subjects increased compared with 60 Healthy Controls (HC). The proportion of CD39+CD4+ cells was decreased in PBMCs from PBC patients in comparsion to HC. Moreover, the proportion of CD39+ Treg cells in PBMCs from PBC patients also significantly decreased in comparsion to HC. A PBC murine model results showed that, compared with the normal mice, the concentrations of ALT, AST and GGT in serum significantly increased induced by polyinosinic-polycytidylic acid (poly I:C). Histopathology result revealed that poly I:C induced liver inflammation in mice. The proportion of CD39+ cells in PBMCs from poly I:C-treated mice was decreased compared with the normal mice. Meanwhile, the proportions of CD39+CD4+ cells and CD39+Treg cells were markedly reduced in poly I:C-induced mouse spleen. Finally, poly I:C significantly induced a global reduction of CD39 and adenosine receptor A2a expression in mouse liver. In addition, poly I:C treatment didn’t alter the proportions of CD39+CD8+ and CD39+CD19+ cells in mouse liver. In conclusion, our results suggest that the proportion of CD39+ Treg cells decreasing in PBC patients and animal model are closely associated with the disease progression. Our study offers a novel perspective on the role of adenosine metabolism pathway in PBC pathogenesis.

A2A Receptor Dysregulation in Dystonia DYT1 Knock-Out Mice.

We aimed to investigate A2A receptors in the basal ganglia of a DYT1 mouse model of dystonia. A2A was studied in control Tor1a+/+ and Tor1a+/− knock-out mice. A2A expression was assessed by anti-A2A antibody immunofluorescence and Western blotting. The co-localization of A2A was studied in striatal cholinergic interneurons identified by anti-choline-acetyltransferase (ChAT) antibody. A2A mRNA and cyclic adenosine monophosphate (cAMP) contents were also assessed. In Tor1a+/+, Western blotting detected an A2A 45 kDa band, which was stronger in the striatum and the globus pallidus than in the entopeduncular nucleus. Moreover, in Tor1a+/+, immunofluorescence showed A2A roundish aggregates, 0.3–0.4 μm in diameter, denser in the neuropil of the striatum and the globus pallidus than in the entopeduncular nucleus. In Tor1a+/−, A2A Western blotting expression and immunofluorescence aggregates appeared either increased in the striatum and the globus pallidus, or reduced in the entopeduncular nucleus. Moreover, in Tor1a+/−, A2A aggregates appeared increased in number on ChAT positive interneurons compared to Tor1a+/+. Finally, in Tor1a+/−, an increased content of cAMP signal was detected in the striatum, while significant levels of A2A mRNA were neo-expressed in the globus pallidus. In Tor1a+/−, opposite changes of A2A receptors’ expression in the striatal-pallidal complex and the entopeduncular nucleus suggest that the pathophysiology of dystonia is critically dependent on a composite functional imbalance of the indirect over the direct pathway in basal ganglia.

Zika virus exposure affects neuron-glia communication in the hippocampal slices of adult rats

Zika virus (ZIKV) infection during pregnancy was associated with microcephaly in neonates, but clinical and experimental evidence indicate that ZIKV also causes neurological complications in adults. However, the changes in neuron-glial communication, which is essential for brain homeostasis, are still unknown. Here, we report that hippocampal slices from adult rats exposed acutely to ZIKV showed significant cellular alterations regarding to redox homeostasis, inflammatory process, neurotrophic functions and molecular signalling pathways associated with neurons and glial cells. Our findings support the hypothesis that ZIKV is highly neurotropic and its infection readily induces an inflammatory response, characterized by an increased expression and/or release of pro-inflammatory cytokines. We also observed changes in neural parameters, such as adenosine receptor A2a expression, as well as in the release of brain-derived neurotrophic factor and neuron-specific enolase, indicating plasticity synaptic impairment/neuronal damage. In addition, ZIKV induced a glial commitment, with alterations in specific and functional parameters such as aquaporin 4 expression, S100B secretion and glutathione synthesis. ZIKV also induced p21 senescence-associated gene expression, indicating that ZIKV may induce early senescence. Taken together, our results indicate that ZIKV-induced neuroinflammation, involving nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear factor κB (NFκB) pathways, affects important aspects of neuron-glia communication. Therefore, although ZIKV infection is transient, long-term consequences might be associated with neurological and/or neurodegenerative diseases.

Chemoresistance in Breast Cancer Patients Associated With Changes in P2X7 and A2A Purinergic Receptors in CD8+ T Lymphocytes.

Breast cancer (BRCA) is the most frequent cancer type that afflicts women. Unfortunately, despite all the current therapeutic strategies, many patients develop chemoresistance hampering the efficacy of treatment. Hence, an early indicator of therapy efficacy might aid in the search for better treatment and patient survival. Although emerging evidence indicates a key role of the purinergic receptors P2X7 and A2A in cancer, less is known about their involvement in BRCA chemoresistance. In this sense, as the chemotherapeutic treatment stimulates immune system response, we evaluated the expression and function of P2X7 and A2A receptors in CD8+ T cells before and four months after BRCA patients received neoadjuvant chemotherapy. The results showed an increase in the levels of expression of P2X7 and a decrease in the expression of A2A in CD8+ T cells in non-chemoresistant (N-CHR) patients, compared to chemoresistant (CHR) patients. Interestingly, in CHR patients, reduced expression of P2X7 occurs along with a decrease in the CD62L shedding and the production of IFN-γ. In the case of the A2A function, the inhibition of IFN-γ production was not observed after chemotherapy in CHR patients. A possible relationship between the modulation of the expression and function of the P2X7 and A2A receptors was found, according to the molecular subtypes, where the patients that were triple-negative and human epidermal growth factor receptor 2 (HER2)-enriched presented more alterations. Comorbidities such as overweight/obesity and type 2 diabetes mellitus (T2DM) participate in the abnormalities detected. Our results demonstrate the importance of purinergic signaling in CD8+ T cells during chemoresistance, and it could be considered to implement personalized therapeutic strategies.

A 2 Adenosine Receptors Mediate Whole-Body Insulin Sensitivity in a Prediabetes Animal Model: Primary Effects on Skeletal Muscle.

Epidemiological studies showed that chronic caffeine intake decreased the risk of type 2 diabetes. Previously, we described that chronic caffeine intake prevents and reverses insulin resistance induced by hypercaloric diets and aging, in rats. Caffeine has several cellular mechanisms of action, being the antagonism of adenosine receptors the only attained with human coffee consumption. Here, we investigated the subtypes of adenosine receptors involved on the effects of chronic caffeine intake on insulin sensitivity and the mechanisms and sex differences behind this effect. Experiments were performed in male and female Wistar rats fed either a chow or high-sucrose (HSu) diet (35% of sucrose in drinking water) during 28 days, to induce insulin resistance. In the last 15 days of diet the animals were submitted to DPCPX (A1 antagonist, 0.4 mg/kg), SCH58261 (A2A antagonist, 0.5 mg/kg), or MRS1754 (A2B antagonist, 9.5 μg/kg) administration. Insulin sensitivity, fasting glycaemia, blood pressure, catecholamines, and fat depots were assessed. Expression of A1, A2A, A2B adenosine receptors and protein involved in insulin signaling pathways were evaluated in the liver, skeletal muscle, and visceral adipose tissue. UCP1 expression was measured in adipose tissue. Paradoxically, SCH58261 and MRS1754 decreased insulin sensitivity in control animals, whereas they both improved insulin response in HSu diet animals. DPCPX did not alter significantly insulin sensitivity in control or HSu animals, but reversed the increase in total and visceral fat induced by the HSu diet. In skeletal muscle, A1, A2A, and A2B adenosine receptor expression were increased in HSu group, an effect that was restored by SCH58261 and MRS1754. In the liver, A1, A2A expression was increased in HSu group, while A2B expression was decreased, being this last effect reversed by administration of MRS1754. In adipose tissue, A1 and A2A block upregulated the expression of these receptors. A2 adenosine antagonists restored impaired insulin signaling in the skeletal muscle of HSu rats, but did not affect liver or adipose insulin signaling. Our results show that adenosine receptors exert opposite effects on insulin sensitivity, in control and insulin resistant states and strongly suggest that A2 adenosine receptors in the skeletal muscle are the majors responsible for whole-body insulin sensitivity.

Effect of low androgen status on the expression of adenosine A 2A and A 2B receptors in rat penile corpus cavernosum

To investigate whether low androgen status affects erectile function by regulating the expression of adenosine A2A and A2B receptors in rat penile corpus cavernosum. Thirty-six 8-week-old male Sprague-Dawley rats were randomly divided into six groups: sham-operated group (4w-sham, 8w-sham), castration group (4w-cast, 8w-cast) and androgen replacement group (4w-cast+T, 8w-cast+T). The rats in the androgen replacement groups were subcutaneously injected with testosterone propionate (3mg/kg) every other day after castration. The maximum intracavernous pressure/mean arterial pressure (ICPmax/MAP), the expression of A2A , A2B , AKT and eNOS and the concentrations of cAMP and cGMP in the corpus cavernosum were detected at the 4th and 8th weeks after the operation. The serum testosterone level and the ratio of ICPmax/MAP decreased significantly in the castration group as compared to other groups (p<0.01). There was no significant difference in the expression of A2A receptor among groups, while the expression of A2B , AKT and eNOS and the concentrations of cAMP and cGMP in the castration group were significantly lower than in other groups (p<0.01). Low androgen status inhibits the AKT/eNOS/cGMP signalling pathways and the production of cAMP in the corpus cavernosum of castrated rats by down-regulating the expression of A2B receptor, and results in decreased of ICPmax/MAP.

Nucleotide ecto-enzyme metabolic pattern and spatial distribution in calcific aortic valve disease; its relation to pathological changes and clinical presentation

BackgroundExtracellular nucleotide metabolism contributes to chronic inflammation, cell differentiation, and tissue mineralization by controlling nucleotide and adenosine concentrations and hence its purinergic effects. This study investigated location-specific changes of extracellular nucleotide metabolism in aortic valves of patients with calcific aortic valve disease (CAVD). Individual ecto-enzymes and adenosine receptors involved were analyzed together with correlation with CAVD severity and risk factors.ResultsNucleotide and adenosine degradation rates were adversely modified on the aortic surface of stenotic valve as compared to ventricular side, including decreased ATP removal (1.25 ± 0.35 vs. 2.24 ± 0.61 nmol/min/cm2) and adenosine production (1.32 ± 0.12 vs. 2.49 ± 0.28 nmol/min/cm2) as well as increased adenosine deamination (1.28 ± 0.31 vs. 0.67 ± 0.11 nmol/min/cm2). The rates of nucleotide to adenosine conversions were lower, while adenosine deamination was higher on the aortic sides of stenotic vs. non-stenotic valve. There were no differences in extracellular nucleotide metabolism between aortic and ventricular sides of non-stenotic valves. Furthermore, nucleotide degradation rates, measured on aortic side in CAVD (n = 62), negatively correlated with echocardiographic and biochemical parameters of disease severity (aortic jet velocity vs. ATP hydrolysis: r = − 0.30, p < 0.05; vs. AMP hydrolysis: r = − 0.44, p < 0.001; valvular phosphate concentration vs. ATP hydrolysis: r = − 0.26, p < 0.05; vs. AMP hydrolysis: r = − 0.25, p = 0.05) while adenosine deamination showed positive correlation trend with valvular phosphate deposits (r = 0.23, p = 0.07). Nucleotide and adenosine conversion rates also correlated with CAVD risk factors, including hyperlipidemia (AMP hydrolysis vs. serum LDL cholesterol: r = − 0.28, p = 0.05; adenosine deamination vs. total cholesterol: r = 0.25, p = 0.05; LDL cholesterol: r = 0.28, p < 0.05; triglycerides: r = 0.32, p < 0.05), hypertension (adenosine deamination vs. systolic blood pressure: r = 0.28, p < 0.05) and thrombosis (ATP hydrolysis vs. prothrombin time: r = − 0.35, p < 0.01). Functional assays as well as histological and immunofluorescence, flow cytometry and RT-PCR studies identified all major ecto-enzymes engaged in nucleotide metabolism in aortic valves that included ecto-nucleotidases, alkaline phosphatase, and ecto-adenosine deaminase. We have shown that changes in nucleotide-converting ecto-enzymes were derived from their altered activities on valve cells and immune cell infiltrate. We have also demonstrated a presence of A1, A2a and A2b adenosine receptors with diminished expression of A2a and A2b in stenotic vs. non-stenotic valves. Finally, we revealed that augmenting adenosine effects by blocking adenosine deamination with deoxycoformycin decreased aortic valve thickness and reduced markers of calcification via adenosine-dependent pathways in a mouse model of CAVD.ConclusionsThis work highlights profound changes in extracellular nucleotide and adenosine metabolism in CAVD. Altered extracellular nucleotide hydrolysis and degradation of adenosine in stenotic valves may affect purinergic responses to support a pro-stenotic milieu and valve calcification. This emphasizes a potential mechanism and target for prevention and therapy.Graphic abstract.

Physical exercise prevents memory impairment in an animal model of hypertension through modulation of CD39 and CD73 activities and A2A receptor expression.

Herein, we investigated the effect of chronic swimming training on the purinergic system in cortex and hippocampus of L-NAME-induced hypertensive rats. Male Wistar rats were divided into four groups: Control, Exercise, L-NAME and Exercise L-NAME. Inhibitory avoidance test was used to assess memory status. NTPDase, CD73 and adenosine deaminase activities and expression, and P2 receptors expression were analyzed. Data were analyzed using two-way ANOVA and Kruskal-Wallis tests, considering P less than 0.05. Physical exercise reduced the blood pressure and prevented memory impairment induced by L-NAME model of hypertension. L-NAME treatment promoted an increase in NTPDase1, NTPDase3 and CD73 expression and activity in the cortex. A2A expression is increased in hippocampus and cortex in the hypertension group and exercise prevented this overexpression. These changes suggest that hypertension increases adenosine generation, which acts through A2A receptors, and exercise prevents these effects. These data may indicate a possible mechanism by which exercise may prevent memory impairment induced by L-NAME.

Lumbar sympathectomy reduces vascular permeability, possibly through decreased adenosine receptor A2a expression in the hind plantar skin of rats

The effect of lumbar sympathectomy for the treatment of lower limb ischemia remains a matter of controversy. Sprague-Dawley rats were subjected to lumbar sympathectomy, after which Evans blue dye was injected into the hind plantar skin. Extravasation of dye was measured and compared with rats undergoing sham operation. Hind plantar skin was processed for HE staining, immunohistochemistry, and Western blot. In sympathectomized rats, blue stained areas in hind plantar skin and concentrations of Evans blue were significantly less than that of sham sympathectomy (control) rats, both 2 weeks and 3 months after surgery. Expression of prostaglandin E2, bradykinin, bradykinin B2 receptor, and adenosine triphosphate were not significantly different between the sympathectomized and control groups. Adenosine receptor A2a expression was significantly reduced in the sympathectomized group both 2 weeks and 3 months after surgery. Vascular permeability in the hind plantar skin of rats decreases following lumbar sympathectomy, possibly via reduced expression of adenosine receptor A2a.

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.

Altered purinergic signaling in uridine adenosine tetraphosphate-induced coronary relaxation in swine with metabolic derangement

We previously demonstrated that uridine adenosine tetraphosphate (Up4A) induces potent and partially endothelium-dependent relaxation in the healthy porcine coronary microvasculature. We subsequently showed that Up4A-induced porcine coronary relaxation was impaired via downregulation of P1 receptors after myocardial infarction. In view of the deleterious effect of metabolic derangement on vascular function, we hypothesized that the coronary vasodilator response to Up4A is impaired in metabolic derangement, and that the involvement of purinergic receptor subtypes and endothelium-derived vasoactive factors (EDVFs) is altered. Coronary small arteries, dissected from the apex of healthy swine and swine 6 months after induction of diabetes with streptozotocin and fed a high-fat diet, were mounted on wire myographs. Up4A (10−9–10−5 M)-induced coronary relaxation was maintained in swine with metabolic derangement compared to normal swine, despite impaired endothelium-dependent relaxation to bradykinin and despite blunted P2X7 receptor and NO-mediated vasodilator influences of Up4A. Moreover, a thromboxane-mediated vasoconstrictor influence was unmasked. In contrast, an increased Up4A-mediated vasodilator influence via P2Y1 receptors was observed, while, in response to Up4A, cytochrome P450 2C9 switched from producing vasoconstrictor to vasodilator metabolites in swine with metabolic derangement. Coronary vascular expression of A2A and P2X7 receptors as well as eNOS, as assessed with real-time PCR, was reduced in swine with metabolic derangement. In conclusion, although the overall coronary vasodilator response to Up4A was maintained in swine with metabolic derangement, the involvement of purinergic receptor subtypes and EDVF was markedly altered, revealing compensatory mechanisms among signaling pathways in Up4A-mediated coronary vasomotor influence in the early phase of metabolic derangement. Future studies are warranted to investigate the effects of severe metabolic derangement on coronary responses to Up4A.

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.

Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1 receptor heteromers is postulated.

Expression of CD73 and A2A receptors in cells from subjects with obesity and type 2 diabetes mellitus

Regulatory T cells have various mechanisms to suppress the inflammatory response, among these, the modulation of the microenvironment through adenosine and with the participation of CD39, CD73 and A2A. The aim of this study was to assess the expression of CD73 and A2A in immune cells and the effect of activation of A2A by an adenosine analogue on apoptosis in patients with obesity and type 2 diabetes mellitus (T2D). CD73 and A2A expression were analyzed by flow cytometry in lymphocyte subpopulations from patients with obesity (n=22), T2D (n=22), and healthy subjects (n=20). Lymphocytes were treated with the selective A2A antagonist (ZM241385) or the selective A2A agonist (CGS21680), and apoptotic cells were detected by Annexin V. We found an increased expression of CD39 coupled to a decrease in CD73 in the patient groups with obesity and T2D compared to the control group in the different studied lymphocyte subpopulations. A2A expression was found to be increased in different subpopulations of lymphocytes from T2D patients. We also detected positive correlations between CD39+ cells and age and BMI. Meanwhile, CD73+ cells showed negative correlations with age, WHR, BMI, FPG, HbAc1, triglycerides and cholesterol. Moreover, an increase in the percentage of apoptotic cells from T2D patients with regard to the groups with obesity and control was observed. In addition, the CD8+ T cells of patients with T2D exhibited decreased apoptosis when treated with the A2A agonist. In conclusion, our data suggest a possible role for CD73 and A2A in inflammation observed in patients with T2D and obesity mediated via apoptosis.

The role of activated adenosine receptors in degranulation of human LAD2 mast cells.

Mast cell degranulation triggers hypersensitivity reactions at the body-environment interface. Adenosine modulates degranulation, but enhancement and inhibition have both been reported. Which of four adenosine receptors (ARs) mediate modulation, and how, remains uncertain. Also uncertain is whether adenosine reaches mast cell ARs by autocrine ATP release and ecto-enzymatic conversion. Uncertainties partly reflect species and cell heterogeneity, circumvented here by focusing on homogeneous human LAD2 cells. Quantitative PCR detected expression of A2A, A2B, and A3, but not A1, ARs. Nonselective activation of ARs with increasing NECA monotonically enhanced immunologically or C3a-stimulated degranulation. NECA alone stimulated degranulation slightly. Selective AR antagonists did not affect C3a-stimulated degranulation. NECA's enhancement of C3a-triggered degranulation was partially inhibited by separate application of each selective antagonist, and abolished by simultaneous addition of antagonists to the three ARs. Only the A2A antagonist separately inhibited NECA's enhancement of immunologically stimulated degranulation, which was abolished by simultaneous addition of the three selective antagonists. Immunological or C3a activation did not stimulate ATP release. NECA also enhanced immunologically triggered degranulation of mouse bone marrow derived mast cells (BMMCs), which was partially reduced only by simultaneous addition of the three antagonists or by the nonselective antagonist CGS15943. BMMCs also expressed A2A, A2B, and A3 ARs. but not A1AR detectably. We conclude that (a) A1AR is unnecessary for LAD2 degranulation or AR enhancement; (b) A2A, A2B, and A3 ARs all contribute to pharmacologic AR enhancement of LAD2 and BMMC degranulation; and (c) LAD2 cells depend on microenvironmental adenosine to trigger AR modulation.

L-DOPA disrupts adenosine A2A–cannabinoid CB1–dopamine D2 receptor heteromer cross-talk in the striatum of hemiparkinsonian rats: Biochemical and behavioral studies

Long-term therapy with l-3,4-dihydroxyphenylalanine (l-DOPA), still the most effective treatment in Parkinson's disease (PD), is associated with severe motor complications such as dyskinesia. Experimental and clinical data have indicated that adenosine A2A receptor antagonists can provide symptomatic improvement by potentiating l-DOPA efficacy and minimizing its side effects. It is known that the G-protein-coupled adenosine A2A, cannabinoid CB1 and dopamine D2 receptors may interact and form functional A2A-CB1–D2 receptor heteromers in co-transfected cells as well as in rat striatum. These data suggest that treatment with a combination of drugs or a single compound selectively acting on A2A–CB1–D2 heteromers may represent an alternative therapeutic treatment of PD. We investigated the expression of A2A–CB1–D2 receptor heteromers in the striatum of both naïve and hemiparkinsonian rats (HPD-rats) bearing a unilateral 6-hydroxydopamine (6-OHDA) lesion, and assessed how receptor heteromer expression and biochemical properties were affected by l-DOPA treatment. Radioligand binding data showed that A2A–CB1–D2 receptor heteromers are present in the striatum of both naïve and HPD-rats. However, behavioral results indicated that the combined administration of A2A (MSX-3 or SCH58261) and CB1 (rimonabant) receptor antagonists, in the presence of l-DOPA does not produce a response different from administration of the A2A receptor antagonist alone. These behavioral results prompted identification of heteromers in l-DOPA-treated animals. Interestingly, the radioligand binding results in samples from lesioned animals suggest that the heteromer is lost following acute or chronic treatment with l-DOPA.

Adenosine A2A and A2B receptors differentially modulate signal transduction and collagen production in murine cardiac firboblasts

Both A2A and A2B adenosine receptors (AR) can modulate organ fibrosis, but their role in cardiac remodeling remains unclear. The aim of this study was to assess the functional expression of A2A and A2BR on cardiac fibroblasts (CF), cells that play a crucial role in collagen deposition in the post‐MI heart. CF were isolated from hearts of C57BL/6 mice and cultured for one passage. AR expression was measured by real time quantitative PCR. CF were treated for 10 min with 500 nM of A2A agonist (CGS‐21680, CGS) or A2B agonist (BAY 60–6583, BAY) to assess ERK and AKT phosphorylation. Additional CF were treated for 24 hr with 500 nM CGS, 500 nM BAY or non‐selective AR agonist NECA (5 μM) to assess collagen production. Western blotting was used to assess phosphorylation and total expression of ERK and AKT, as well as procollagen 1A (Pro‐1A) expression. The expression level of A2BR was double A2AR (in % GAPDH, 1.3 vs 0.6). CGS and BAY increased ERK phosphorylation (359±85% and 263±45% respectively, p&lt;0.05). BAY reduced AKT phosphorylation by 73±4% (p&lt;0.05), while CGS had no effect. A2BR stimulation with BAY (157±46%) and NECA (107±31%) increased Pro‐1A (p&lt;0.05 vs vehicle), more‐so than A2AR stimulation with CGS (87±21%, p&gt;;0.05). In conclusion, both A2AR and A2BR stimulation increased ERK phosphorylation, but only A2BR increased collagen production. These findings indicate that CF A2BR may contribute to post‐MI cardiac remodeling.

Cooperation of Adenosine with Macrophage Toll-4 Receptor Agonists Leads to Increased Glycolytic Flux through the Enhanced Expression of PFKFB3 Gene

Macrophages activated through Toll receptor triggering increase the expression of the A(2A) and A(2B) adenosine receptors. In this study, we show that adenosine receptor activation enhances LPS-induced pfkfb3 expression, resulting in an increase of the key glycolytic allosteric regulator fructose 2,6-bisphosphate and the glycolytic flux. Using shRNA and differential expression of A(2A) and A(2B) receptors, we demonstrate that the A(2A) receptor mediates, in part, the induction of pfkfb3 by LPS, whereas the A(2B) receptor, with lower adenosine affinity, cooperates when high adenosine levels are present. pfkfb3 promoter sequence deletion analysis, site-directed mutagenesis, and inhibition by shRNAs demonstrated that HIF1α is a key transcription factor driving pfkfb3 expression following macrophage activation by LPS, whereas synergic induction of pfkfb3 expression observed with the A(2) receptor agonists seems to depend on Sp1 activity. Furthermore, levels of phospho-AMP kinase also increase, arguing for increased PFKFB3 activity by phosphorylation in long term LPS-activated macrophages. Taken together, our results show that, in macrophages, endogenously generated adenosine cooperates with bacterial components to increase PFKFB3 isozyme activity, resulting in greater fructose 2,6-bisphosphate accumulation. This process enhances the glycolytic flux and favors ATP generation helping to develop and maintain the long term defensive and reparative functions of the macrophages.

Age dependent changes in A 2A receptor expression in PreBotzinger Complex

Adenosine is a modulator of respiratory output, and can both increase and decrease respiratory frequency in an age and receptor dependent manner. We have shown an in vivo, age dependent, effect of A2A receptor activation on apneas in rats which is attenuated from postnatal day (P)14 to P21 through adulthood. We hypothesized that the attenuated response to A2A activation was due to decreased A2A receptor expression in the PreBötzinger complex (pBc). To test this hypothesis, we used unbiased stereology to quantify changes in A2A expression in pBc over the first three weeks of life. Sprague Dawley rat pups were perfused at P0, 4, 7, 14, and 21. The brains were cryosectioned and 4 identical sets of slides made from each brain. These slides were immunostained for NK-1R and somatostatin to deliniate the pBc. The remaining slides were stained with A2A, NeuN or S100β antibodies. The number of immunopositive cells in the pBc and the pBc volume were quantified using unbiased stereology (n=5 for each antibody at each age). The number of cells immunopositive for A2A per 1X105μm3 of pBc decreased significantly between P0 and P7 (p < 0.006), and rose again by P21 (P < 0.2). The number of NeuN and S100β immunopositive cells per 1X105μm3 decreased significantly between P0 and P21 (p < 0.001 for both). We conclude that A2A expression changes with age, but these changes are not correlated with the pattern of A2A dependent apneas seen in vivo.