Adenosine Receptor Expression Research Articles

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

Effect of emulsified isoflurane preconditioning on adenosine A1 receptor expression in hippocampus during focal cerebral ischemia-reperfusion in rats

目的 探讨乳化异氟醚预处理对大鼠局灶性脑缺血再灌注时海马腺苷A1(AA1)受体表达的影响.方法 健康雄性SD大鼠88只,体重280～320 g,采用随机数字表法,将其随机分为7组:假手术组(S组,n=16)、缺血再灌注组(I/R组,n=16)、乳化异氟醚预处理组(EI组,n=16)、乳化异氟醚溶剂组(LE组,n=16)、乳化异氟醚+AA1受体拮抗剂8-环戊基-1,3-二丙基黄嘌呤(DPCPX)组(ED组,n=8)、DPCPX组(DP组,n=8)和DPCPX溶剂二甲基亚砜组(DM组,n=8).采用大脑中动脉阻塞法制备局灶性脑缺血再灌注损伤模型.于缺血前24h,I/R组、EI组和LE组分别腹腔注射生理盐水、8％乳化异氟醚和脂肪乳剂10.5 ml/kg,DP组和DM组分别腹腔注射DPCPX 1 mg/kg和二甲基亚砜2.5mg/kg,S组于相应时点腹腔注射生理盐水10.5 ml/kg; ED组于腹腔注射8％乳化异氟醚前30 min腹腔注射DPCPX 1 mg/kg.各组于再灌注24h时取8只大鼠,进行神经功能缺陷评分,然后处死,取脑组织,测定脑梗死体积；S组、I/R组、EI组和LE组于再灌注24 h时处死8只大鼠,取脑组织,采用Western blot法检测海马AA1受体的表达水平.结果 与S组比较,其余各组神经功能缺陷评分和脑梗死体积百分比升高,I/R组和LE组海马AA1受体表达下调,EI组海马AA1受体表达上调(P＜0.01)；与I/R组比较,EI组神经功能缺陷评分和脑梗死体积百分比降低,海马AA1受体表达上调(P＜0.05或0.01)；与EI组比较,LE组、ED组、DP组和DM组神经功能缺陷评分和脑梗死体积百分比升高(P＜0.05或0.01).结论 乳化异氟醚预处理减轻大鼠局灶性脑缺血再灌注损伤的机制可能与上调海马AA1受体表达有关。

Expression of mRNA for Adenosine A1, A2a, A2b, and A3 Receptors in HL-60 Cells: Dependence on Cell Cycle Phases

The present studies investigated changes in expression of mRNA for adenosine A(1), A(2a), A(2b), and A(3) receptors in samples of HL-60 promyelocytic cells differing in the actual presence of cells in various phases of the cell cycle induced by the double thymidine block method. Real-time PCR technique was used for obtaining data on mRNA expression. Statistical analysis of the data revealed that the mRNA expression of adenosine A(1), A(2a), and A(3) receptors is dependent on the cell cycle phase. G(0)/G(1) and G(2)/M phases were characterized by a higher mRNA expression of adenosine A(1) receptors and a lower one of adenosine A(2a) and A(3) receptors whereas the opposite was true for the S phase. Interestingly, expression of mRNA of the adenosine A(2b) receptors was independent on the cell cycle phase. The results indicate the plasticity of mRNA expression of adenosine receptors in the investigated promyelocytic cells and its interaction with physiological mechanisms of the cell cycle.

Adenosine A2A receptor contributes to the anti-inflammatory effect of the fixed herbal combination STW 5 (Iberogast®) in rat small intestinal preparations

STW 5 (Iberogast®), an established herbal combination, was effective in randomized, double blind clinical studies in functional dyspepsia and irritable bowel syndrome. Since STW 5 was found to influence intestinal motility and has anti-inflammatory properties, this study investigated the expression of adenosine receptors and characterized their role in the control of the anti-inflammatory action of STW 5 and its fresh plant component STW 6 in inflammation-disturbed rat small intestinal preparations. The inflammation was induced by intraluminal instillation of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 0.01M). The effects of coincubation with selective receptor agonists and antagonists, STW 5, STW 6, or combinations of these compounds on acetylcholine (ACh)-evoked contraction of ileum/jejunum preparations were tested. Adenosine receptor mRNA expression was examined by reverse transcription-polymerase chain reaction (RT-PCR). In untreated preparations, RT-PCR revealed the presence of all adenosine receptor subtypes. Suppressed expression was detected for all subtypes in inflamed tissues, except for A(2B)R mRNA, which was unaffected. STW 5 reversed these effects and enhanced A(2A)R expression above control levels. Radioligand binding assays confirm the affinity of STW 5 to the A(2A)R, and the A(2A)R antagonist was able to prevent the effect of STW 5 on TNBS-induced attenuation of the ACh contraction. Our findings provide evidence that STW 5, but not STW 6 interacts with A(2A)R, which is involved in the anti-inflammatory action of STW 5. STW 6 did not contribute to adenosine A(2A)R-mediated anti-inflammatory effect of STW 5. Other signaling pathways could be involved in the mechanism of action of STW 6.

Caffeine Acts via A1 Adenosine Receptors to Disrupt Embryonic Cardiac Function

BackgroundEvidence suggests that adenosine acts via cardiac A1 adenosine receptors (A1ARs) to protect embryos against hypoxia. During embryogenesis, A1ARs are the dominant regulator of heart rate, and A1AR activation reduces heart rate. Adenosine action is inhibited by caffeine, which is widely consumed during pregnancy. In this study, we tested the hypothesis that caffeine influences developing embryos by altering cardiac function.Methodology/Principal FindingsEffects of caffeine and adenosine receptor-selective antagonists on heart rate were studied in vitro using whole murine embryos at E9.5 and isolated hearts at E12.5. Embryos were examined in room air (21% O2) or hypoxic (2% O2) conditions. Hypoxia decreased heart rates of E9.5 embryos by 15.8% and in E12.5 isolated hearts by 27.1%. In room air, caffeine (200 µM) had no effect on E9.5 heart rates; however, caffeine increased heart rates at E12.5 by 37.7%. Caffeine abolished hypoxia-mediated bradycardia at E9.5 and blunted hypoxia-mediated bradycardia at E12.5. Real-time PCR analysis of RNA from isolated E9.5 and E12.5 hearts showed that A1AR and A2aAR genes were expressed at both ages. Treatment with adenosine receptor-selective antagonists revealed that SCH-58261 (A2aAR-specific antagonist) had no affects on heart function, whereas DPCPX (A1AR-specific antagonist) had effects similar to caffeine treatment at E9.5 and E12.5. At E12.5, embryonic hearts lacking A1AR expression (A1AR−/−) had elevated heart rates compared to A1AR+/− littermates, A1AR−/− heart rates failed to decrease to levels comparable to those of controls. Caffeine did not significantly affect heart rates of A1AR−/− embryos.Conclusions/SignificanceThese data show that caffeine alters embryonic cardiac function and disrupts the normal cardiac response to hypoxia through blockade of A1AR action. Our results raise concern for caffeine exposure during embryogenesis, particularly in pregnancies with increased risk of embryonic hypoxia.

Enhanced actions of adenosine in medial entorhinal cortex layer II stellate neurons in temporal lobe epilepsy are mediated via A1‐receptor activation

The adenosinergic system is known to exert an inhibitory affect in the brain, and as such adenosine has been considered an endogenous anticonvulsant. Entorhinal cortex (EC) layer II neurons, which serve as the primary input to the hippocampus, are spared in temporal lobe epilepsy (TLE) and become hyperexcitable. Because these neurons also express adenosine receptors, the activity of these neurons may be controlled by adenosine, specifically during seizure activity when adenosine levels are thought to rise. In light of this, we determined if the actions of adenosine on medial EC (mEC) layer II stellate neurons are augmented in TLE and by which receptor subtype. Horizontal brain slices were prepared from rats exhibiting spontaneous seizures (TLE) induced by electrical stimulation and compared with age-matched control rats. mEC layer II stellate neurons were visually identified, and action potentials (APs) were evoked either by a series of depolarizing current injection steps or via presynaptic stimulation of mEC deep layers. The effects of adenosine were compared with actions of adenosine A(1) and A(2A) receptor-specific agonists (CPA and CGS-21680) and antagonists (DPCPX and ZM-241385), respectively. Immunohistochemical and qPCR techniques were also employed to assess relative adenosine A(1)-receptor message and expression. mEC layer II stellate neurons were hyperexcitable in TLE, evoking a higher frequency of APs when depolarized and generating bursts of APs when synaptically stimulated. Adenosine reduced AP frequency and synaptically evoked APs in a dose-dependent manner (500 nM-100 μM); however, in TLE, the inhibitory actions of adenosine occurred at concentrations that were without affect in control neurons. In both cases, the inhibitory actions of adenosine were mediated via activation of the A(1)- and not the A(2A)-receptor subtype. Quantitative polymerase chain reaction (qPCR) and immunohistochemical experiments revealed an upregulation of the adenosine A(1) mRNA and an increase in A(1)-receptor staining in TLE neurons compared to control. Our data indicate that the actions of adenosine on mEC layer II stellate neurons is accentuated in TLE due to an upregulation of adenosine A(1)-receptors. Because adenosine levels are thought to rise during seizure activity, activation of adenosine A(1)-receptors could provide a possible endogenous mechanism to suppress seizure activity and spread within the temporal lobe.

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.

A holistic view of adenosine in the control of intestinal neuromuscular functions: the enteric ‘purinome’ concept

Adenosine is involved in the modulation of enteric neuromuscular functions, operating a fine tuning of smooth muscle contractility, peristaltic reflex and transit. In this issue of the BJP, Zizzo et al. report novel findings on the expression of adenosine receptors in mouse duodenum, extending our knowledge of their involvement in the control of spontaneous and neurogenic intestinal motility. In this study, particular attention was paid to the differential activation of adenosine receptors, as a result of their interplay with regulatory systems, modulating the availability of endogenous adenosine in a compartmentalised manner. This evidence will contribute to the holistic evaluation of the role played by adenosine in the regulation of intestinal motility, in accordance with the novel concept of the enteric 'purinome'. This commentary discusses the role of the 'purinome' in the modulation of enteric neuromuscular activity, pointing out its involvement in the intestinal neuroplasticity associated with bowel dysmotility.

IL-4 Amplifies the Pro-Inflammatory Effect of Adenosine in Human Mast Cells by Changing Expression Levels of Adenosine Receptors

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A2BsiRNA and selective A2B receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A2B and reduced expression of A2A adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine.

Early exposure to caffeine affects gene expression of adenosine receptors, DARPP-32 and BDNF without affecting sensibility and morphology of developing zebrafish ( Danio rerio)

Adenosine receptors are the most important biochemical targets of caffeine, a common trimethylxanthine found in food and beverages. Adenosine plays modulatory action during the development through adenosine receptors and their intracellular pathways activation. In this study, we aimed to evaluate if caffeine gave to zebrafish in the very first steps of development is able to affect its direct targets, through the adenosine receptors mRNA expression evaluation, and latter indirect targets, through evaluation of the pattern of dopamine and cAMP-regulated phosphoprotein and brain-derived neurotrophic factor (BDNF) mRNA expression. Here, we demonstrate that zebrafish express adenosine receptor subtypes (A1, A2A1, A2A2 and A2B) since 24 h post-fertilization (hpf) and that caffeine exposure is able to affect the expression of these receptors. Caffeine exposure from 1 hpf is able to increase A1 expression at 72–96 hpf and A2A1 expression at 72 hpf. No alterations occurred in A2A2 and A2B expression after caffeine treatment. DARPP-32, a phosphoprotein involved in adenosine intracellular pathway is also expressed since 24 hpf and early exposure to caffeine increased DARPP-32 expression at 168 hpf. We also evaluate the expression of BDNF as one of the targets of adenosine intracellular pathway activation. BDNF was also expressed since 24 hpf and caffeine treatment increased its expression at 48 and 72 hpf. No morphological alterations induced by caffeine treatment were registered by the check of general body features and total body length. Assessment of tactile sensibility also demonstrated no alterations by caffeine treatment. Altogether, these results suggest that caffeine is able to affect expression of its cellular targets since early phases of development in zebrafish without affect visible features. The up-regulation of direct and indirect targets of caffeine presents as a compensatory mechanism of maintenance of adenosinergic modulation during the developmental phase.

Adenosine Receptor mRNA Expression in Normal and Failing Minipig Hearts

Chronic heart failure (HF) is associated with increased systemic (plasma) and reduced local (myocardial) adenosine levels. The final biological action of adenosine in a particular organ or cell population may depend on the relative degree of expression and signaling efficiency of individual adenosine receptor (AR) subtypes. The aim of this study was to determine the myocardial expression of ARs, in the different chambers of failing versus normal minipig hearts. Cardiac tissue was collected from minipigs without (n = 5) and with HF (n = 5). ARs, adenosine deaminase, and tumor necrosis factor-α (TNF-α) mRNA expression were evaluated by real time-polymerase chain reaction. ARs were expressed in all cardiac regions. After 3 weeks of pacing, the only significant change was observed in A2BR mRNA expression in the left ventricle (P = 0.02), with a similar trend for A3R, A2AR, and A1R. A trend toward higher expression of mRNA adenosine deaminase in the myocardium of pigs with HF was observed. TNF-α mRNA expression was higher after HF in all cardiac chambers (left ventricle: P = 0.009), and a significant correlation was observed between TNF-α and A2BR (r = 0.80, P < 0.0001). In this study, A2BR mRNA resulted in being overexpressed in the left ventricle of pigs with HF as well as TNF-α expression possibly testifying a link between AR expression, inflammation, and HF.

Pro-Fibrotic Role of the A2B Adenosine Receptor in Human Cardiac Fibroblasts

Background and Aims: Adenosine is a signaling nucleoside and the interstitial levels of this nucleoside are elevated in certain pathophysiological conditions. Results of our previous studies suggest that adenosine play an important role in pulmonary fibrosis. The goal of the current study is to determine the potential role of adenosine and its receptor subtypes in cardiac fibrosis using the primary human cardiac fibroblasts (HCF) as a model system. Methods: Expression of adenosine receptors (AdoR), a-smooth muscle actin and a-1 pro-collagen was determined using real-time RT-PCR. The concentrations of IL-6, soluble ST-2 and PAPPA in the cell supernatants were measured using ELISA. The concentration of soluble collagen was determined using Sircol collagen assay. Results: Among the four subtypes of AdoRs, the A2B AdoR was expressed at the highest level in HCF. NECA, a stable analog of adenosine, significantly increased the release of IL-6 in a concentration-dependent manner, with a maximal increase of 2.4 6 0.1 fold over the basal level. In addition, NECA (10mM) increased the expression of a-smooth muscle actin and a-1 pro-collagen, and the production of collagen (1.8 6 0.1 fold induction, from 3.4 6 0.2 to 6.0 6 0.4 mg/ml, p!0.05) from HCF. Furthermore, NECA increased the release of two novel biomarkers of cardiovascular diseases (CVD), soluble ST-2 (1.7 6 0.1 fold induction, from 1.5 6 0.1 to 2.6 6 0.1 ng/ml, p!0.05) and PAPPA (4.46 0.6 fold induction, from 1.46 0.5 to 6.26 0.8 ng/ml, p!0.05). The effects of NECA on release of IL-6, collagen, ST-2, and PAPPA and expression of a-smooth muscle actin and a-1 pro-collagen were completely abolished by a selective A2B AdoR antagonist, GS-6201. Conclusions: This study indicates that the A2B AdoR is the predominant subtype of AdoRs expressed in primary human HCF. Activation of this receptor increases the release of IL-6, production of collagen, expression of fibrotic markers and release of biomarkers of CVD. These findings suggest that A2B AdoR might mediate the fibrotic response in heart diseases.

Contrasting effects of A1 and A2b adenosine receptors on adipogenesis

Adenosine mediates its actions through four G protein-coupled receptors, A1, A2a, A2b and A3. The A1 receptor (A1R) is dominant in adipocytes where it mediates many actions that include inhibition of lipolysis, stimulation of leptin secretion and protection against obesity-related insulin resistance. The objective of this study is to investigate whether induced expression of A1Rs stimulates adipogenesis, or whether A1R expression is a consequence of cells having an adipocyte phenotype. Human A1R and A2b receptors (A2bRs) were stably transfected into a murine osteoblast precursor cell line, 7F2. Adipogenesis was determined by lipid accumulation and expression of adipocyte and osteoblast marker molecules. Adenosine receptor expression and activation of associated signal molecules were also evaluated as 7F2 cells were induced to differentiate to adipocytes. 7F2 cells transfected with the A1R showed increased adipocyte marker mRNA expression; lipoprotein lipase and glycerol-3-phosphate dehydrogenase were both upregulated, whereas the osteoblast marker alkaline phosphatase (ALP) was downregulated. When cultured in adipocyte differentiating media, such cells also showed increased adipogenesis as judged by lipid accumulation. Conversely, A2bR transfection stimulated osteocalcin and ALP expression, and in addition, adipogenesis was inhibited in the presence of adipocyte differentiation media. Adipogenic differentiation of naive 7F2 cells also resulted in increased expression of the A1R and reduced or modified expression of the A2a and A2bR. The loss of A2 receptors after adipogenic differentiation was accompanied by a loss of cyclic adenosine monophosphate and ERK1/2 signalling. These data show that expression of A1Rs induced adipocyte differentiation, whereas A2bR expression inhibited adipogenesis and stimulated an osteoblastic phenotype. These data suggest that targeting A1 and A2bR could be considered in the management of obesity and diabetes. Targeting adenosine signal pathways may be useful in treatment strategies for diseases in which there is an imbalance between osteoblasts and adipocytes.

Adenosine induces cell‐cycle arrest and apoptosis in androgen‐dependent and ‐independent prostate cancer cell lines, LNcap‐FGC‐10, DU‐145, and PC3

Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via intrinsic and extrinsic pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the DU-145, PC3, and LNcap-FGC10 human prostate cancer cells. To observe cell viability and proliferation, MTT assay, cell counting, and BrdU assay were carried out in DU-145, PC3, and LNcap-FGC10 cells. Apoptosis was assessed with the analysis of cell cycle, Hoechst 33258 staining, propidium iodide and annexin-V staining, reactive oxygen species (ROS) formation, mitochondrial membrane potential (ΔΨM) measurement, caspase-3 activity assay, Bcl-2 and Bax protein expression. Moreover, the expression of adenosine receptors and the effects of adenosine receptor (A(1) , A(2a) , and A(3) ) antagonists were examined. Adenosine significantly reduced cell proliferation in a dose-dependent manner in DU-145, PC3, and LNcap-FGC10 cell lines. Adenosine induced arrest in the cell-cycle progression in G0/G1 phase through Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by morphological changes and increased sub-G1 population. Furthermore, increase of ROS, loss of MMP, activation of caspase-3, and down-regulation of Bcl-2 expression was observed. A(1) , A(2a) , A(2b) , and A(3) adenosine receptors mRNA are expressed in the cell lines. Moreover, adenosine-induced apoptosis was inhibited by MRS1220, A(3) adenosine receptor antagonist. Our results suggest that adenosine induced apoptosis in prostate cancer cells via the mitochondrial pathway and is related to the adenosine receptors. These data might suggest that adenosine could be used as an agent for the treatment of prostate cancer.

Activation of Adenosine A2AReceptors Inhibits Neutrophil Transuroepithelial Migration

Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A(2A) receptors. In this study, we examined the role of adenosine and A(2A) receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A(1), A(2A), A(2B), and A(3) receptors), but A(3) receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A(2A) receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A(2A) receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A(2A) receptor activation, possibly through inhibition of NF-κB signaling pathways.

The protective effects of inosine against chemical hypoxia on cultured rat oligodendrocytes.

Inosine is a purine nucleoside and is considered protective to neural cells including neurons and astrocytes against hypoxic injury. However, whether oligodendrocytes (OLs) could also be protected from hypoxia by inosine is not known. Here we investigated the effects of inosine on primarily cultured rat OLs injured by rotenone-mediated chemical hypoxia, and the mechanisms of the effects using ATP assay, MTT assay, PI-Hoechst staining, TUNEL, and immunocytochemistry. Results showed that rotenone exposure for 24 h caused cell death and impaired viability in both immature and mature OLs, while pretreatment of 10 mM inosine 30 min before rotenone administration significantly reduced cell death and improved the viability of OLs. The same concentration of inosine given 120 min after rotenone exposure also improved viability of injured mature OLs. Immunocytochemistry for nitrotyrosine and cellular ATP content examination indicated that inosine may protect OLs by providing ATP and scavenging peroxynitrite for cells. In addition, immature OLs were more susceptible to hypoxia than mature OLs; and at the similar degree of injury, inosine protected immature and mature OLs differently. Quantitative real-time PCR revealed that expression of adenosine receptors was different between these two stages of OLs. These data suggest that inosine protect OLs from hypoxic injury as an antioxidant and ATP provider, and the protective effects of inosine on OLs vary with cell differentiation, possibly due to the adenosine receptors expression profile. As OLs form myelin in the central nervous system, inosine could be used as a promising drug to treat demyelination-involved disorders.

Nuclear Factor κB and Adenosine Receptors: Biochemical and Behavioral Profiling.

Adenosine is produced primarily by the metabolism of ATP and mediates its physiological actions by interacting primarily with adenosine receptors (ARs) on the plasma membranes of different cell types in the body. Activation of these G protein-coupled receptors promotes activation of diverse cellular signaling pathways that define their tissue-specific functions. One of the major actions of adenosine is cytoprotection, mediated primarily via two ARs - A1 (A1AR) and A3 (A3AR). These ARs protect cells exposed to oxidative stress and are also regulated by oxidative stress. Stress-mediated regulation of ARs involves two prominent transcription factors - activator protein-1 (AP-1) and nuclear factor (NF)-κB – that mediate the induction of genes important in cell survival. Mice that are genetically deficient in the p50 subunit of NF-κB (i.e., p50 knock-out mice) exhibit altered expression of A1AR and A2AAR and demonstrate distinct behavioral phenotypes under normal conditions or after drug challenges. These effects suggest an important role for NF-κB in dictating the level of expression of ARs in vivo, in regulating the cellular responses to stress, and in modifying behavior.

Adenosine receptor subtype expression and activation influence the differentiation of mesenchymal stem cells to osteoblasts and adipocytes

Osteoblasts and adipocytes differentiate from a common precursor cell, the mesenchymal stem cell (MSC). Adenosine is known to signal via four adenosine receptor subtypes, and significantly, recent findings indicate that these may play a role in MSC differentiation. We therefore investigated adenosine receptor expression and activation during the differentiation of MSCs to osteoblasts and adipocytes. The A(2B) R was dominant in MSCs, and its expression and activity were transiently upregulated at early stages of osteoblastic differentiation. Both activation and overexpression of A(2B) R induced the expression of osteoblast-related genes [Runx2 and alkaline phosphatase (ALP)], as well as ALP activity, and stimulation increased osteoblast mineralization. The expression of A(2A) R was upregulated during later stages of osteoblastic differentiation, when its activation stimulated ALP activity. Differentiation of MSCs to adipocytes was accompanied by significant increases in A(1) R and A(2A) R expression, and their activation was associated with increased adipogenesis. Enhanced A(2A) R expression was sufficient to promote expression of adipocyte-related genes (PPARγ and C/EBPα), and its activation resulted in increased adipocytic differentiation and lipid accumulation. In contrast, the A(1) R was involved mainly in lipogenic activity of adipocytes rather than in their differentiation. These results show that adenosine receptors are differentially expressed and involved in lineage-specific differentiation of MSCs. We conclude, therefore, that fruitful strategies for treating diseases associated with an imbalance in the differentiation and function of these lineages should include targeting adenosine receptor signal pathways. Specifically, these research avenues will be useful in preventing or treating conditions with insufficient bone or excessive adipocyte formation.

Mechanisms involved in the adenosine-induced vasorelaxation to the pig prostatic small arteries

Benign prostatic hypertrophy has been related with glandular ischemia processes and adenosine is a potent vasodilator agent. This study investigates the mechanisms underlying the adenosine-induced vasorelaxation in pig prostatic small arteries. Adenosine receptors expression was determined by Western blot and immunohistochemistry, and rings were mounted in myographs for isometric force recording. A(2A) and A(3) receptor expression was observed in the arterial wall and A(2A)-immunoreactivity was identified in the adventitia-media junction and endothelium. A(1) and A(2B) receptor expression was not obtained. On noradrenaline-precontracted rings, P1 receptor agonists produced concentration-dependent relaxations with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA) = CGS21680 > 2-Cl-IB-MECA = 2-Cl-cyclopentyladenosine = adenosine. Adenosine reuptake inhibition potentiated both NECA and adenosine relaxations. Endothelium removal and ZM241385, an A(2A) antagonist, reduced NECA relaxations that were not modified by A(1), A(2B), and A(3) receptor antagonists. Neuronal voltage-gated Ca(2+) channels and nitric oxide (NO) synthase blockade, and adenylyl cyclase activation enhanced these responses, which were reduced by protein kinase A inhibition and by blockade of the intermediate (IK(Ca))- and small (SK(Ca))-conductance Ca(2+)-activated K(+) channels. Inhibition of cyclooxygenase (COX), large-conductance Ca(2+)-activated-, ATP-dependent-, and voltage-gated-K(+) channel failed to modify these responses. These results suggest that adenosine induces endothelium-dependent relaxations in the pig prostatic arteries via A(2A) purinoceptors. The adenosine vasorelaxation, which is prejunctionally modulated, is produced via NO- and COX-independent mechanisms that involve activation of IK(Ca) and SK(Ca) channels and stimulation of adenylyl cyclase. Endothelium-derived NO playing a regulatory role under conditions in which EDHF is non-functional is also suggested. Adenosine-induced vasodilatation could be useful to prevent prostatic ischemia.

Modulation of Adenosine Receptors by [60]Fullerene Hydrosoluble Derivative in SK-N-MC Cells

The most known fullerenes are spherical carbon compounds composed of 60 carbon atoms. C(60) fullerenes have shown biochemical and biomedical properties in the last years such as as blockade of apoptosis and neuroprotection. The nucleoside adenosine has a neuroprotective role mainly due to inhibition of glutamate release, which is a neurotransmitter related to excitotoxicity and cell death. In the present work, we have determined the presence of adenosine receptors in SK-N-MC cells, a neuroepithelioma human cell line, and analyzed the effect of fullerenes in these receptors by using radioligand binding, immunoblotting, and quantitative real time PCR assays. Results demonstrated that SK-N-MC cells endogenously express adenosine receptors. Fullerene exposure of these cells did not affect cell viability measured by MTT reduction assay. However, adenosine A(1) and A(2A) receptors were both increased in SK-N-MC cells after treatment. These results suggest for the first time the modulation of adenosine receptors after C(60) fullerenes exposure.