A2A Agonist Research Articles

Regional Haemodynamic responses to Adenosine A2A‐Receptor Agonists in Conscious Freely Moving Rats

Endogenous adenosine acts as a local mediator in the cardiovascular system to protect against the influences of stress (including ischaemia and reperfusion injury). In highly metabolic tissues, such as skeletal muscle and the coronary circulation, adenosine A1 and A2 receptors help to mediate arteriolar vasodilatation [1]. The present study compared the effects of adenosine or specific A2A agonists on regional haemodynamics in the presence or absence of an A2A‐specific antagonist (SCH58261). As described previously [2], rats were instrumented with miniature pulsed Doppler flow probes (for assessment of regional haemodynamics) sutured around the renal and mesenteric arteries, and the descending abdominal aorta. Animals were also instrumented with intra‐arterial and intra‐venous catheters for measurement of heart rate (HR) and mean arterial pressure (MAP), and drug administration, respectively. Studies were conducted in conscious, freely‐moving rats. All procedures were carried out with approval of the University of Nottingham Animal Welfare Ethical Review Board, under Home Office Project and Personal License Authority. All compounds were prepared in a propylene glycol (PEG) ‐ based buffer (5% PEG, 2% Tween, 0.9% saline) or saline. Agonists were evaluated in the presence or absence of SCH58261 or vehicle (0.1 or 1.0 mg/kg; given as a bolus of 0.1 ml). For example, CGS21680 was administered as 3 min infusions of 0.1, 0.3 and 1 μg/kg/min administered over a total of 9 min at an infusion rate of 0.1ml/min, at least 10 min following the bolus injection of SCH58261 or vehicle. CGS21680 produced tachycardic and depressor effects, which were associated with renal and hindlimb vasodilatations. There was little effect in the mesenteric vascular bed. Responses were antagonised by SCH58261 at 1.0 mg/kg. Responses to adenosine and other modulators of adenosine receptors (e.g. VCP746) will also be presented.Support or Funding InformationWe thank the British Heart Foundation for financial support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The effect of an adenosine A2A agonist on intra-tumoral concentrations of temozolomide in patients with recurrent glioblastoma

BackgroundThe blood–brain barrier (BBB) severely limits the entry of systemically administered drugs including chemotherapy to the brain. In rodents, regadenoson activation of adenosine A2A receptors causes transient BBB disruption and increased drug concentrations in normal brain. This study was conducted to evaluate if activation of A2A receptors would increase intra-tumoral temozolomide concentrations in patients with glioblastoma.MethodsPatients scheduled for a clinically indicated surgery for recurrent glioblastoma were eligible. Microdialysis catheters (MDC) were placed intraoperatively, and the positions were documented radiographically. On post-operative day #1, patients received oral temozolomide (150 mg/m2). On day #2, 60 min after oral temozolomide, patients received one intravenous dose of regadenoson (0.4 mg). Blood and MDC samples were collected to determine temozolomide concentrations.ResultsSix patients were enrolled. Five patients had no complications from the MDC placement or regadenoson and had successful collection of blood and dialysate samples. The mean plasma AUC was 16.4 ± 1.4 h µg/ml for temozolomide alone and 16.6 ± 2.87 h µg/ml with addition of regadenoson. The mean dialysate AUC was 2.9 ± 1.2 h µg/ml with temozolomide alone and 3.0 ± 1.7 h µg/ml with regadenoson. The mean brain:plasma AUC ratio was 18.0 ± 7.8 and 19.1 ± 10.7% for temozolomide alone and with regadenoson respectively. Peak concentration and Tmax in brain were not significantly different.ConclusionsAlthough previously shown to be efficacious in rodents to increase varied size agents to cross the BBB, our data suggest that regadenoson does not increase temozolomide concentrations in brain. Further studies exploring alternative doses and schedules are needed; as transiently disrupting the BBB to facilitate drug entry is of critical importance in neuro-oncology.

Adenosine A2A Receptor Agonist, 2-p-(2-Carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine Hydrochloride Hydrate, Inhibits Inflammation and Increases Fibroblast Growth Factor-2 Tissue Expression in Carrageenan-Induced Rat Paw Edema.

Adenosine is the final product of ATP metabolism, mainly derived from the action of 5'-nucleotidase cleavage of AMP. Cellular production of adenosine is greatly enhanced in inflamed tissues, ischemic tissues, and under hypoxia, where ATP is released from damaged cells. Much evidence has been accumulated on adenosine anti-inflammatory effects mediated through A2A receptor activation; A2A adenosine receptor has also been shown to play a role in matrix deposition and wound healing in a damaged tissue, contributing to dermal tissue protection and repair. Fibroblast growth factor-2 (FGF-2) is a powerful mitogen for fibroblast; it is expressed by several inflammatory cell types and plays a pivotal role in angiogenesis, wound healing, gastric ulcer protection. Human recombinant FGF-2 has been shown to have anti-inflammatory effects. The purpose of the present work was to investigate on the anti-inflammatory effect of systemic administration of the adenosine A2A agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride hydrate (CGS21680) in the rat model of carrageenan-induced paw edema. We found that CGS21680 inhibits inflammation induced by carrageenan injection into the rat paw, and this effect is associated to the local reduction of cytokine levels and dermal increase of FGF-2 expression. Our results suggest that FGF-2 might be involved in the anti-inflammatory and tissue protective effect due to A2A receptor activation.

Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603

Endogenous adenosine A2B receptors (A2BAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A2BAR antagonist PSB 603 in HEK 293 cells. The A2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A2AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA EMAX with no significant effect on EC50 values. Kinetics analysis of the effect of PSB 603 on the A2BAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A2BAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.

Abstract 1683: A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity

Abstract High levels of extracellular adenosine in the tumor microenvironment are known to play a significant role in tumor immune evasion and promote tumor growth and metastasis. We defined the receptor(s) required for mediating the effect of adenosine on immune cells within the tumor microenvionment and report the characterization of a novel Immuno-Oncology-dedicated adenosine receptor 2A antagonist that functions in the high adenosine concentration found in tumors. We first explored the expression of the four adenosine receptors in primary human immune cells. A2A receptor was the main adenosine receptor expressed by CD4 and CD8 T lymphocytes and monocytes, and the only one in mature monocyte-derived dendritic cells and NK cells. A2B receptor was poorly detected in T cells and monocytes, while A1 and A3 receptors were never detected. Given these expression patterns, we further studied A2A functions in primary human T lymphocytes and monocytes. Selective A2A agonists such as CGS-21680 strongly suppressed cytokine production by activated primary human T lymphocytes, thus highlighting that A2A is the main effector receptor of the sensing of adenosine in tumors. We further confirmed the elevated extracellular adenosine level in the tumor microenvironment in several mouse and human tumors. High adenosine levels correlated with strong tumoral expression of CD73, the enzyme that converts AMP to adenosine. Interestingly, we showed that A2A receptor antagonists designed for Parkinson’s disease dramatically lost potency in a high adenosine environment ; our data indicated that a 30-fold dose increase may be required for full target inhibition within tumors. Therefore we developed a novel and potent A2A blocker with sub-nanomolar Ki and IC50 in a cAMP assay and a more than 100-fold selectivity over other adenosine receptors. Our lead compound kept a high potency in an adenosine-rich environment and restored cytokine production even in the presence of high concentrations of A2A agonists. iTeos inhibitor also efficiently reversed AMP-mediated T cell suppression. Furthermore, our compound rescued A2A receptor agonist-induced decrease of TNFα production by primary human monocytes, and was able to potently increase CD8 T cell cytotoxicity in a cytotoxicity assay with CD8 T cells as effectors and cancer cells as targets. These results suggest that iTeos new generation of A2A receptor antagonist, designed to keep a high potency in the adenosine-rich tumor microenvironment, may offer a new therapeutic opportunity in Immuno-Oncology. Citation Format: Erica Houthuys, Margreet Brouwer, Florence Nyawouame, Romain Pirson, Reece Marillier, Theo Deregnaucourt, Joao Marchante, Jakub Swiercz, Charlotte Moulin, Vanesa Bol, Gregory Driessens, Michel Detheux, Christophe Quéva, Stefano Crosignani, Bruno Gomes. A novel adenosine A2A receptor antagonist optimized for high potency in adenosine-rich tumor microenvironment boosts antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1683. doi:10.1158/1538-7445.AM2017-1683

Adenosine A2A receptor agonists with potent antiplatelet activity

Selected adenosine A2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC50 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC50 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC50 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC50 0.97±0.07 µmol/L) and equipotent to NECA (EC50 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, Ki values determined in radioligand-binding studies were not predictive of the A2A agonists’ antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A2A receptor agonists.

Hypoxic postconditioning attenuates apoptosis via activation of adenosine A2a receptors on dermal microvascular endothelial cells of human flaps

BackgroundIn recent years, many studies have demonstrated that endogenous adenosine induced by ischemia postconditioning reduces apoptosis in various models, but no study has clearly elucidated the effects of hypoxic postconditioning (HPC) in human dermal microvascular endothelial cells (HDMECs) of flaps, and the subtype of adenosine receptors involved remains unknown. In our study, we sought to identify the role of adenosine A2a receptor in the antiapoptotic effects of HPC. Materials and methodsThe HDMECs were isolated from human upper eyelid tissue. After hypoxia for 6 h, the HDMECs were either abruptly reperfused with medium for 12 h (reoxygenation) or postconditioned by three cycles of 5 min of transitory reoxygenation and 5 min of rehypoxia followed by 11.5 h of reoxygenation. CGS-21680 was used as an adenosine A2a agonist. ResultsAdenosine A2a receptors were found in the cytoplasm of HDMECs. Hypoxia/reoxygenation (H/R) resulted in severe injury in HDMECs as evidenced by an increase in apoptosis percentage and an increase in expressions of apoptosis-related proteins (the ratio of Bax/Bcl-2 and caspase-3), which can be significantly attenuated by HPC treatment or exposure to CGS-21680, a selective adenosine A2a receptor agonist (all P values < 0.05). Meanwhile, HPC treatment or CGS-21680 significantly activated A2a receptors compared with the hypoxia/reoxygenation group (P < 0.05 versus P < 0.05, respectively). Statistical analysis showed that the increase of cell apoptosis closely correlated inversely with the increase of relative protein level of adenosine A2a receptors (r2 = 0.8177, P < 0.0001). ConclusionsHPC protects against apoptosis induced by reoxygenation via activation of adenosine A2a receptors on HDMECs.

Role and Function of A2A and A₃ Adenosine Receptors in Patients with Ankylosing Spondylitis, Psoriatic Arthritis and Rheumatoid Arthritis.

Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are chronic inflammatory rheumatic diseases that affect joints, causing debilitating pain and disability. Adenosine receptors (ARs) play a key role in the mechanism of inflammation, and the activation of A2A and A3AR subtypes is often associated with a reduction of the inflammatory status. The aim of this study was to investigate the involvement of ARs in patients suffering from early-RA (ERA), RA, AS and PsA. Messenger RNA (mRNA) analysis and saturation binding experiments indicated an upregulation of A2A and A3ARs in lymphocytes obtained from patients when compared with healthy subjects. A2A and A3AR agonists inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation and reduced inflammatory cytokines release, such as tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6. Moreover, A2A and A3AR activation mediated a reduction of metalloproteinases (MMP)-1 and MMP-3. The effect of the agonists was abrogated by selective antagonists demonstrating the direct involvement of these receptor subtypes. Taken together, these data confirmed the involvement of ARs in chronic autoimmune rheumatic diseases highlighting the possibility to exploit A2A and A3ARs as therapeutic targets, with the aim to limit the inflammatory responses usually associated with RA, AS and PsA.

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP-dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier.

Polydeoxyribonucleotide, an Adenosine-A2A Receptor Agonist, Preserves Blood Testis Barrier from Cadmium-Induced Injury.

Cadmium (Cd) impairs blood-testis barrier (BTB). Polydeoxyribonucleotide (PDRN), an adenosine A2A agonist, has positive effects on male reproductive system. We investigated the effects of PDRN on the morphological and functional changes induced by Cd in mice testes. Adult Swiss mice were divided into four groups: controls administered with 0.9% NaCl (1 ml/kg, i.p., daily) or with PDRN (8 mg/kg, i.p. daily), animals challenged with Cd chloride (CdCl2; 2 mg/kg, i.p, daily) and animals challenged with CdCl2 (2 mg/kg, i.p., daily) and treated with PDRN (8 mg/kg, i.p., daily). Experiments lasted 14 days. Testes were processed for biochemical, structural, and ultrastructural evaluation and hormones were assayed in serum. CdCl2 increased pERK 1/2 expression and Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels; it decreased testosterone (TE) and inhibin-B levels and induced structural damages in extratubular compartment and in seminiferous epithelium, with ultrastructural features of BTB disruption. Many TUNEL-positive germ cells were present. CdCl2 increased tubular TGF-β3 immunoreactivity and reduced claudin-11, occludin, and N-cadherin immunoreactivity. PDRN administration reduced pERK 1/2 expression, FSH, and LH levels; it increased TE and inhibin-B levels, ameliorated germinal epithelium changes and protected BTB ultrastructure. Few TUNEL-positive germ cells were present and the extratubular compartment was preserved. Furthermore, PDRN decreased TGF-β3 immunoreactivity and enhanced claudin-11, occludin, and N-cadherin immunoreactivity. We demonstrate a protective effect of PDRN on Cd-induced damages of BTB and suggest that PDRN may play an important role against Cd, particularly against its harmful effects on gametogenesis.

Retinal A2A and A3 adenosine receptors modulate the components of the rat electroretinogram.

Adenosine is a neuromodulator present in various areas of the central nervous system, including the retina. Adenosine may serve a neuroprotective role in the retina, based on electroretinogram (ERG) recordings from the rat retina. Our purpose was to assess the role of A2A and A3 adenosine receptors in the generation and modulation of the rat ERG. The flash ERG was recorded with corneal electrodes from Sprague Dawley rats. Agonists and antagonists for A2A and A3 receptors, and adenosine were injected (5 µl) into the vitreous. The effects on the components of the single flash scotopic and photopic ERGs were examined, and ERG flicker. Adenosine (0.5 mM) increased the mean amplitudes of the scotopic ERG a-waves (68 ± 8 to 97 ± 14 µV, P = 0.042), and b-waves (236 ± 38 µV to 305 ± 42 µV). A2A agonist CGS21680 (2 mM) reduced the mean amplitude of the ERG b-wave, from 298 ± 21 µV in response to the brightest stimulus to 212 ± 19 µV (P = 0.005), and mean scotopic oscillatory potentials (OPs) from 100 ± 9 µV to 47 ± 11 µV (P = 0.023). ZM241385 [4 mM], an A2A antagonist, decreased the scotopic b-wave of the ERG. A3 agonist 2-CI-IB-MECA (0.5 mM) increased the a-wave, while decreasing the scotopic and photopic ERG b-waves, and the scotopic OPs. A3 antagonist VUF5574 (1 mM) increased the mean amplitude of the scotopic a-wave (66 ± 8 to 140 ± 29 µV, P = 0.046) and b-wave (224 ± 20 to 312 ± 39 µV, P = 0.0037). No significant effects on ERG flicker were found. We conclude that retinal neurons containing A2A and/or A3 adenosine receptors contribute to the generation of the ERG a- and b-waves and OPs.

Myocardial stress perfusion magnetic resonance: initial experience in a pediatric and young adult population using regadenoson.

Dipyridamole and adenosine are traditional pharmacological stressors for myocardial perfusion. Regadenoson, a selective adenosine A2A agonist, has a lower side effect profile with lower incidence of bronchospasm and bradycardia. There is a growing need for myocardial perfusion assessment within pediatrics. There is no report on the utility of regadenoson as a stress agent in children. To observe the safety and feasibility of regadenoson as a pharmacologic stressor for perfusion cardiac MR in a pilot cohort of pediatric patients weighing more than 40kg who have congenital heart disease and pediatric acquired heart disease. We reviewed our initial experience with regadenoson stress cardiac MR in 31 pediatric patients 15.8 ± 1.7years (range 12-22years) with congenital heart disease and acquired heart disease. Mean patient weight was 60 ± 15kg (range of 40-93kg). All patients underwent cardiac MR because of concern for ischemia. The cohort included a heterogeneous group of patients at a pediatric institution with potential risk for ischemia. Subjects' heart rate and blood pressure were monitored and pharmacologic stress was induced by injection of 400mcg of regadenoson. We evaluated their hemodynamic response and adverse effects using changes in vital signs and onset of symptoms. A pediatric cardiologist and radiologist qualitatively assessed myocardial perfusion and viability images. One child was unable to complete the stress perfusion portion of the examination, but did complete the remaining portion of the CMR. Resting heart rate was 72 ± 14 beats per minute (bpm) and rose to peak of 124 ± 17bpm (95 ± 50% increase, P < 0.005) with regadenoson. Image quality was considered good or diagnostic in all cases. Three patients had irreversible perfusion defects. Four patients had reversible perfusion defects. Nine of the patients underwent cardiac catheterization with angiography and the findings showed excellent agreement. Regadenoson might be a safe and feasible pharmacologic stress agent for use in cardiac MR in older pediatric patients with congenital heart disease and acquired heart disease. The ease of use as a bolus and the advantage of a prolonged hyperemia make its use appealing in pediatrics. In a limited number of cases, regadenoson stress perfusion showed excellent agreement with cardiac catheterization. Regadenoson might be a viable pharmacologic stress agent in this population.

Deregulation of Adenosine Receptors in Psoriatic Epidermis: An Option for Therapeutic Treatment

Purinergic signaling is involved in psoriasis, a chronic skin disease characterized by increased epidermis cell growth. In particular, Andrés etal. focus on the keratinocyte biology modulated by adenosine receptors providing evidence that the A2B subtype plays a prominent role in the reduction of keratinocyte proliferation whereas A2A and A2B agonists have antiinflammatory effects independent of adenosine receptors. The authors report that psoriatic epidermis presents a deregulated adenosine receptor expression profile with reduced A2B and increased A2A.

Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics.

The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy.

Adenosine A2A receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies.

We aim to investigate whether A2A/nitric oxide-mediated regulation of vascular endothelial growth factor (VEGF) expression is impaired in feto-placental endothelial cells from late-onset pre-eclampsia. Cultures of human umbilical vein endothelial cells (HUVECs) and human placental microvascular endothelial cells (hPMECs) from normal and pre-eclamptic pregnancies were used. Assays by using small interference RNA (siRNA) for A2A were performed, and transfected cells were used for estimation of messenger RNA (mRNA) levels of VEGF, as well as for cell proliferation and angiogenesis in vitro. CGS-21680 (A2A agonist, 24h) increases HUVEC and hPMEC proliferation in a dose response manner. Furthermore, similar to CGS-21680, the nitric oxide donor, S-nitroso-N-acetyl-penicillamine oxide (SNAP), increased cell proliferation in a dose response manner (logEC50 10-9.2M). In hPMEC, CGS-21680 increased VEGF protein levels in both normal (∼1.5-fold) and pre-eclamptic pregnancies (∼1.2-fold), an effect blocked by the A2A antagonist, ZM-241385 (10-5M) and the inhibitor of NO synthase, N ω-nitro-L-arginine methyl ester hydrochloride (L-NAME). Subsequently, SNAP partially recovered cell proliferation and in vitro angiogenesis capacity of cells from normal pregnancies exposed to siRNA for A2A. CGS-21680 also increased (∼1.5-fold) the level of VEGF mRNA in HUVEC from normal pregnancies, but not in pre-eclampsia. Additionally, transfection with siRNA for A2A decrease (∼30%) the level of mRNA for VEGF in normal pregnancy compared to untransfected cells, an effect partially reversed by co-incubation with SNAP. The A2A-NO-VEGF pathway is present in endothelium from microcirculation and macrocirculation in both normal and pre-eclamptic pregnancies. However, NO signaling pathway seems to be impaired in HUVEC from pre-eclampsia.

Adenosine A2A and A2B Receptors Differentially Modulate Keratinocyte Proliferation: Possible Deregulation in Psoriatic Epidermis

Adenosine is a potent regulator of inflammation and immunity, but the role of adenosine receptors in keratinocytes remains controversial. We determined that in addition to A2B receptors, human epidermal keratinocytes also express A2A receptors, although to a lower extent. Through the use of selective adenosine receptor agonists and antagonists, we showed that physiological concentrations of adenosine activate A2B receptors in normal human keratinocytes, inducing cell cycle arrest through the increase of intracellular calcium but not through cAMP signaling. In contrast, the selective activation of A2A receptors by CGS-21680 induces keratinocyte proliferation via p38-mitogen-activated protein kinase activation. Adenosine and selective A2A and A2B agonists presented anti-inflammatory profiles independent of adenosine receptors but mediated by membrane phosphatase activation. Finally, keratinocyte exposure to diverse inflammatory cytokines altered adenosine receptor expression by reducing A2B and increasing A2A, a pattern also observed in psoriatic epidermis. Because increased epidermal turnover and inflammatory response are characteristics of psoriatic disease, further studies are needed to assess the role and consequences of the altered adenosine receptor expression in lesional and nonlesional psoriatic keratinocytes.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5′-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5′-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5′-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73-derived adenosine acting on A2A receptors.

Multiple D2 heteroreceptor complexes: new targets for treatment of schizophrenia.

The dopamine (DA) neuron system most relevant for schizophrenia is the meso-limbic-cortical DA system inter alia densely innervating subcortical limbic regions. The field of dopamine D2 receptors and schizophrenia changed markedly with the discovery of many types of D2 heteroreceptor complexes in subcortical limbic areas as well as the dorsal striatum. The results indicate that the D2 is a hub receptor which interacts not only with many other G protein-coupled receptors (GPCRs) including DA isoreceptors but also with ion-channel receptors, receptor tyrosine kinases, scaffolding proteins and DA transporters. Disturbances in several of these D2 heteroreceptor complexes may contribute to the development of schizophrenia through changes in the balance of diverse D2 homo- and heteroreceptor complexes mediating the DA signal, especially to the ventral striato-pallidal γ-aminobutyric acid (GABA) pathway. This will have consequences for the control of this pathway of the glutamate drive to the prefrontal cortex via the mediodorsal thalamic nucleus which can contribute to psychotic processes. Agonist activation of the A2A protomer in the A2A-D2 heteroreceptor complex inhibits D2 Gi/o mediated signaling but increases the D2 β-arrestin2 mediated signaling. Through this allosteric receptor-receptor interaction, the A2A agonist becomes a biased inhibitory modulator of the Gi/o mediated D2 signaling, which may the main mechanism for its atypical antipsychotic properties especially linked to the limbic A2A-D2 heterocomplexes. The DA and glutamate hypotheses of schizophrenia come together in the signal integration in D2-N-methyl-d-aspartate (NMDA) and A2A-D2-metabotropic glutamate receptor 5 (mGlu5) heteroreceptor complexes, especially in the ventral striatum. 5-Hydroxytryptamine 2A (5-HT2A)-D2 heteroreceptor complexes are special targets for atypical antipsychotics with high potency to block their 5-HT2A protomer signaling in view of the potential development of pathological allosteric facilitatory 5-HT2A-D2 interaction increasing D2 protomer signaling. Neurotensin (NTS1)-D2 heterocomplexes also exist in the ventral and dorsal striatum, and likely also in midbrain DA nerve cells as NTS1-D2 autoreceptor complexes where neurotensin produces antipsychotic and propsychotic actions, respectively.

Polydeoxyribonucleotide, an adenosine-A2A receptor agonist, preserves blood testis barrier from cadmium-induced injury

Cadmium (Cd) impairs the blood-testis barrier (BTB) with changes of its junctional complexes [1]. Polydeoxyribonucleotide (PDRN), an adenosine A2A agonist, has positive effects on male reproductive system [2]. We investigated the effects of PDRN on the morphological and functional Cd-induced changes in mice testes. Swiss mice were divided into four groups: control animals treated with 0.9% NaCl (1 ml/kg, i.p., daily); control animals treated with PDRN (8 mg/kg, i.p. daily), animals challenged with Cd chloride (CdCl2) (2 mg/kg i.p, daily) and animals challenged with CdCl2 and treated with PDRN. The experiments lasted 14 days. At the end of experiment, the testes were processed for biochemical, structural and ultrastructural evaluation. CdCl2 increased pERK 1/2 expression and Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) levels, decreased testosterone (TE) and inhibin-B levels and induced structural damages in the extratubular compartment and in the seminiferous epithelium, with ultrastructural features of BTB disruption. Many TUNEL-positive germ cells were present in the peripheral parts of the tubules. CdCl2 increased also tubular TGF-β3 immunoreactivity and reduced claudin-11, occludin and N-cadherin immunoreactivity. PDRN administration reduced pERK 1/2 expression, FSH and LH levels, increased TE and inhibin-B levels, ameliorated germinal epithelium changes and protected BTB ultrastructure. Only few TUNEL-positive germ cells were present and the extratubular compartment was preserved, showing only a mild edema. Furthermore PDRN decreased TGF-β3 immunoreactivity and enhanced claudin-11, occludin and N-cadherin immunoreactivity. We demonstrate, for the first time, a protective effect of PDRN on Cd-induced BTB damages in mice testes. We suggest that the A2A agonist may play an important role against environmental Cd, and in particular against its harmful effects on gametogenesis.

Evaluation of the expression and function of the P2X7 receptor and ART1 in human regulatory T-cell subsets

Regulatory T cells that express CD39 (CD39+ Treg) exhibit specific immunomodulatory properties. Ectonucleotidase CD39 hydrolyses ATP and ADP. ATP is a ligand of the P2X7 receptor and induces the shedding of CD62L and apoptosis. However, the role of ATP in CD39+ Treg cells has not been defined. Furthermore, NAD can activate the P2X7 receptor via ADP-ribosyltransferase (ART) enzymes and cause cell depletion in murine models. We evaluated the expression and function of P2X7 and ART1 in CD39+ Treg and CD39- Treg cells in the presence or absence of ATP and NAD. We isolated peripheral blood mononuclear cells from healthy subjects and purified CD4+ T cells, CD4+ CD25+ T cells and CD4+ CD25+ CD39+ T cells. P2X7 and ART1 expression was assessed by flow cytometry and real-time PCR. Our results showed low P2X7 expression on CD39+ Treg cells and higher levels of ART1 expression in CD4+ CD39+ T cells than the other subtypes studied. Neither shedding of CD62L nor cell death of CD39+ Treg or CD39- Treg cells was observed by 1mM ATP or 60μM NAD. In contrast, P2Xs receptor-dependent proliferation with 300μM ATP, was inhibited by NAD in the different cell types analysed. The NAD proliferation-inhibition was increased with P2Xs and A2a agonist and was reversed with P2Xs and A2a antagonist, therefore NAD inhibits P2Xs-dependent proliferation and A2a activation. In conclusion, our results suggest that the altered function and expression of P2X7 and ART1 in the human CD39+ Treg or CD39− Treg cells could participate in the resistance against cell death induced by ATP or NAD.