Potential therapeutic effects of antagonizing adenosine A2A receptor, curcumin and niacin in rotenone-induced Parkinson's disease mice model.

Parkinson's disease (PD) is the second common age-related neurodegenerative disease. It is characterized by control loss of voluntary movements control, resting tremor, postural instability, bradykinesia, and rigidity. The aim of the present work is to evaluate curcumin, niacin, dopaminergic and non-dopaminergic drugs in mice model of Parkinson's disease through behavioral, biochemical, genetic and histopathological observations. Mice treated with rotenone rerecorded significant increase in adenosine A2A receptor (A2AR) gene expression, α synuclein, acetylcholinesterase (AchE), malondialdehyde (MDA), angiotensin-II (Ang-II), c-reactive protein (CRP), interleukin-6 (IL-6), caspase-3 (Cas-3) and DNA fragmentation levels as compared with the control group. While, significant decrease in dopamine (DA), norepinephrine (NE), serotonin (5-HT), superoxide dismutase (SOD), reduced glutathione (GSH), ATP, succinate and lactate dehydrogenases (SDH &LDH) levels were detected. Treatment with curcumin, niacin, adenosine A2AR antagonist; ZM241385 and their combination enhanced the animals' behavior and restored all the selected parameters with variable degrees of improvement. The brain histopathological features of hippocampal and substantia nigra regions confirmed our results. In conclusion, the combination of curcumin, niacin and ZM241385 recorded the most potent treatment effect in Parkinsonism mice followed by ZM241385, as a single treatment. ZM241385 succeeded to antagonize adenosine A2A receptor by diminishing its gene expression and ameliorating all biochemical parameters under investigation. The newly investigated agent; ZM241385 has almost the same pattern of improvement as the classical drug; Sinemet®. This could shed the light to the need of detailed studies on ZM241385 for its possible role as a promising treatment against PD. Additionally, food supplements such as curcumin and niacin were effective in Parkinson's disease eradication.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.

Adenosine A2A receptor plays an important role in radiation-induced dermal injury.

Ionizing radiation is a common therapeutic modality and following irradiation dermal changes, including fibrosis and atrophy, may lead to permanent changes. We have previously demonstrated that occupancy of A2A receptor (A2AR) stimulates collagen production, so we determined whether blockade or deletion of A2AR could prevent radiation-induced fibrosis. After targeted irradiation (40 Gy) of the skin of wild-type (WT) or A2AR knockout (A2ARKO) mice, the A2AR antagonist ZM241385 was applied daily for 28 d. In irradiated WT mice treated with the A2AR antagonist, there was a marked reduction in collagen content and skin thickness, and ZM241385 treatment reduced the number of myofibroblasts and angiogenesis. After irradiation, there is an increase in loosely packed collagen fibrils, which is significantly diminished by ZM241385. Irradiation also induced an increase in epidermal thickness, prevented by ZM241385, by increasing the number of proliferating keratinocytes. Similarly, in A2ARKO mice, the changes in collagen alignment, skin thickness, myofibroblast content, angiogenesis, and epidermal hyperplasia were markedly reduced following irradiation. Radiation-induced changes in the dermis and epidermis were accompanied by an infiltrate of T cells, which was prevented in both ZM241385-treated and A2ARKO mice. Radiation therapy is administered to a significant number of patients with cancer, and radiation reactions may limit this therapeutic modality. Our findings suggest that topical application of an A2AR antagonist prevents radiation dermatitis and may be useful in the prevention or amelioration of radiation changes in the skin.

Role of the Anterior Cingulate Cortex in the Control of Micturition Reflex in a Rat Model of Parkinson's Disease.

PURPOSE: In the current study we examined dynamic changes in neural activity of the anterior cingulate cortex and the midbrain periaqueductal gray during the micturition reflex in a Parkinson's disease model as well as the effects of direct stimulation of the anterior cingulate cortex on the micturition reflex. MATERIALS AND METHODS: Electrodes were inserted in the anterior cingulate cortex or the periaqueductal gray. The effects of intravenous administration of the adenosine A2A receptor antagonist ZM24138 on pelvic nerve evoked field potentials were examined. The effect of electrical stimulation of the anterior cingulate cortex was also examined. RESULTS: Rats with Parkinson's disease showed bladder overactivity as evidenced by a significant decrease in the intercontraction interval compared with sham operated rats. Intravenous administration of ZM24138 increased the intercontraction interval in both groups with the inhibitory effects greater in rats with Parkinson's disease. It dose dependently increased the amplitude of evoked potentials in the anterior cingulate cortex of rats with Parkinson's disease but not in sham operated rats. Intravenous administration of ZM24138 decreased evoked potential amplitude in the periaqueductal gray of both groups with the inhibitory effects greater in Parkinson's disease vs sham operated rats. Electrical stimulation of the anterior cingulate cortex significantly increased the intercontraction interval. CONCLUSIONS: These results suggest that anterior cingulate cortex neurons have an inhibitory role in bladder control. Neural activity in the anterior cingulate cortex was significantly increased along with suppression of bladder overactivity after ZM241385 administration in the Parkinson's disease model and the stimulation of the anterior cingulate cortex inhibited the micturition reflex. Understanding the roles of the anterior cingulate cortex in the modulation of micturition could provide further insights into the pathophysiology of overactive bladder.

Chronic sleep restriction induces long-lasting changes in adenosine and noradrenaline receptor density in the rat brain.

Although chronic sleep restriction frequently produces long-lasting behavioural and physiological impairments in humans, the underlying neural mechanisms are unknown. Here we used a rat model of chronic sleep restriction to investigate the role of brain adenosine and noradrenaline systems, known to regulate sleep and wakefulness, respectively. The density of adenosine A1 and A2a receptors and ß-adrenergic receptors before, during and following 5 days of sleep restriction was assessed with autoradiography. Rats (n = 48) were sleep-deprived for 18 h day(-1) for 5 consecutive days (SR1-SR5), followed by 3 unrestricted recovery sleep days (R1-R3). Brains were collected at the beginning of the light period, which was immediately after the end of sleep deprivation on sleep restriction days. Chronic sleep restriction increased adenosine A1 receptor density significantly in nine of the 13 brain areas analysed with elevations also observed on R3 (+18 to +32%). In contrast, chronic sleep restriction reduced adenosine A2a receptor density significantly in one of the three brain areas analysed (olfactory tubercle which declined 26-31% from SR1 to R1). A decrease in ß-adrenergic receptors density was seen in substantia innominata and ventral pallidum which remained reduced on R3, but no changes were found in the anterior cingulate cortex. These data suggest that chronic sleep restriction can induce long-term changes in the brain adenosine and noradrenaline receptors, which may underlie the long-lasting neurocognitive impairments observed in chronic sleep restriction.

Synthesis and biological evaluation of novel 2-amino-3-aroyl-4-neopentyl-5-substituted thiophene derivatives as allosteric enhancers of the A1 adenosine receptor.

2-Amino-3-benzoyl thiophenes have been widely reported to act as allosteric enhancers at the A1 adenosine receptor. Their activity can be increased considerably by appropriate substitutions at the 4- and 5-positions of the thiophene ring. Substituent size at the thiophene C-4 position seemed to be a factor closely related to activity, with the 4-neopentyl (2,2-dimethylpropyl) substitution showing the greatest enhanced activity. A wide series of 2-amino-3-aroyl-4-neopentylthiophene derivatives with general structure 3, characterized by the presence of different substituents (bromine, aryl and heteroaryl) at the 5-position of the thiophene ring, have been identified as potent AEs at the A1AR. With only one exception, all of the synthesized compounds proved to be superior to the reference compound PD 81,723 in a functional assay. Derivatives 3p, 3u, 3am, 3ap and 3ar were the most active compounds in binding (saturation and competition) and functional cAMP studies, being able to potentiate agonist [(3)H]CCPA binding to the A1 receptor.

Roles of adenosine A1 and A2A receptors in the control of micturition in rats.

AIMS: Adenosine is a neurotransmitter that exerts numerous physiological effects in many organs. However, few studies have focused on the role of adenosine receptors in the control of micturition. Therefore, we examined the role of adenosine A1 and A2A receptors in the control of bladder activity in rats with normal or acetic acid (AA) irritated bladders. METHODS: Cystometrograms during saline or 0.2% AA infusion were recorded under urethane anesthesia in female Sprague-Dawley rats. After a stabilization period, CCPA (A1 receptor agonist) and/or ZM24138 (A2A receptor antagonist) were administered intravenously (i.v.), intrathecally (i.t.), intracerebroventricularly (i.c.v.), or intravesically. Micturition parameters were recorded and compared before and after drug administration. RESULTS: I.v., i.t., or i.c.v. administration of CCPA or ZM24138 significantly increased intercontraction intervals (ICIs) in both saline and AA infusion groups. During AA infusion, the inhibitory effects induced by i.c.v. CCPA or i.t. ZM24138 were significantly greater than those by i.t. or i.c.v. administration, respectively. Intravesical administration of CCPA, but not ZM24138, significantly increased ICI. CONCLUSIONS: These results indicated that: (1) when nociceptive signals from the bladder increase, adenosine A1 receptor-mediated inhibition of micturition is enhanced in the brain, compared to the normal condition, (2) A1 receptor activation also exerts a peripheral inhibitory effect on micturition, and (3) adenosine A2A receptor-mediated excitatory mechanisms are enhanced in the spinal cord following C-fiber bladder afferent stimulation. Thus adenosine A1 receptor agonists and A2A receptor antagonists might be effective for the treatment of overactive bladder and/or bladder hypersensitive disorders, in which C-fiber afferent function is enhanced.

A novel A2a adenosine receptor inhibitor effectively mitigates hepatic fibrosis in a metabolic dysfunction-associated steatohepatitis mouse model.

Hepatic fibrosis exacerbates mortality and complications in progressive metabolic dysfunction-associated steatohepatitis (MASH). The role of the adenosine 2A receptor (A2aAR) in hepatic fibrosis within the context of MASH remains uncertain. This study aims to elucidate the involvement of the A2aAR signaling pathway and the efficacy of a novel potent A2aAR antagonist in treating hepatic fibrosis in MASH-induced mice fed a chlorine-deficient, L-amino acid-defined, high fat diet (CDAHFD). A2aAR overexpression in LX-2 cells increased fibrosis markers, whereas the known A2aAR antagonist, ZM241385, decreased these markers. A novel A2aAR antagonist, RAD11, not only attenuated fibrosis progression but also exhibited greater inhibition of the A2aAR signaling pathway compared to ZM241385 in mice with MASH, activated primary hepatocytes, and LX-2 cells. RAD11 exhibited a dual antifibrotic mechanism by targeting both activated HSCs and hepatocytes. Its superior antifibrotic efficacy over ZM241385 in the MASH condition stems from its ability to suppress A2aAR-mediated signaling, inhibit HSC activation, reduce hepatic lipogenesis in hepatocytes, and mitigate lipid accumulation-induced oxidative stress-mediated liver damage. This study has shed light on the relationship between A2aAR signaling and hepatic fibrosis, presenting RAD11 as a potent therapeutic agent for managing MASH and hepatic fibrosis.

Antagonist binding and induced conformational dynamics of GPCR A2A adenosine receptor.

The A2A adenosine receptor (A2AAR) is a unique G-protein coupled receptor (GPCR), because besides agonist, its antagonist could also lead to therapeutic relevance. Based on A2AAR-antagonist crystal structure, we have studied the binding mechanism of two distinct antagonists, ZM241385 and KW6002, and dynamic behaviors of A2AAR induced by antagonist binding. Key residues interacting with both antagonists and residues specifically binding to one of them are identified. ZM241385 specifically bound to S67(2.65), M177(5.38), and N253(6.55), while KW6002 binds to F62(2.60), A81(3.29), and H264(7.29). Moreover, interactions with L167(5.28) are found for both antagonists, which were not reported in agonist binding. The dynamic behaviors of antagonist bound holo-A2AARs were found to be different from the apo-A2AAR in three typical functional switches, (i) the "ionic lock" was in equilibrium between formation and breakage in apo-A2AAR, but stayed broken in holo-A2AARs; (ii) the "rotamer toggle switch," T88(3.36)/F242(6.44)/W246(6.48), adopted different rotameric conformations in apo-A2AAR and holo-A2AARs; (iii) apo-A2AAR preferred α-helical intracellular loop (IC)2 and flexible IC3, while holo-A2AARs had a flexible IC2 and α-helical IC3. Our results indicated that antagonist binding induced different conformational rearrangements of these characteristic functional switches in apo-A2AAR and holo-A2AARs.

Adenosine receptors as potential targets in melanoma.

Melanoma is one of the most aggressive types of cancer, that is difficult to manage clinically. A major feature of melanoma cells is their ability to escape immune surveillance. Adenosine receptors play a pivotal role in host immune-surveillance. A2a (A2aR) and, partially, A2bR receptors mediate the adenosine-induced immune-suppression, which markedly facilitates tumor development/progression. On the contrary, A3R stimulation enhances the anti-tumor immune response and thus limits tumor growth. A3R also inhibits the proliferation of many cancer cells. Given that A2aR and A3R have profound effects on tumor growth and metastasis, they are attractive targets for novel therapeutic anti-cancer agents. Here, we review the role played by A2aR and A3R in regulating cancer pathogenesis, with a focus on melanoma, and the therapeutic potential of adenosine receptors pharmacological modulation.

The role of the adenosinergic system in lung fibrosis.

Adenosine (ADO) is a retaliatory metabolite that is expressed in conditions of injury or stress. During these conditions ATP is released at the extracellular level and is metabolized to adenosine. For this reason, adenosine is defined as a "danger signal" for cells and organs, in addition to its important role as homeostatic regulator. Its physiological functions are mediated through interaction with four specific transmembrane receptors called ADORA1, ADORA2A, ADORA2B and ADORA3. In the lungs of mice and humans all four adenosine receptors are expressed with different roles, having pro- and anti-inflammatory roles, determining bronchoconstriction and regulating lung inflammation and airway remodeling. Adenosine receptors can also promote differentiation of lung fibroblasts into myofibroblasts, typical of the fibrotic event. This last function suggests a potential involvement of adenosine in the fibrotic lung disease processes, which are characterized by different degrees of inflammation and fibrosis. Idiopathic pulmonary fibrosis (IPF) is the pathology with the highest degree of fibrosis and is of unknown etiology and burdened by lack of effective treatments in humans.

Effects of caffeine and phosphodiesterase inhibitors on activation of neonatal T lymphocytes.

Caffeine and selective PDE inhibitors are widely used in clinical management of preterm and term neonates. However, little is known about how these compounds interact with the neonatal adaptive immune system. We aimed to describe the effects of caffeine, milrinone and sildenafil on the activation and cytokine production of T cells from umbilical cord blood (UCB) compared to adult peripheral blood (APB). We isolated mononuclear cells from 10 APB and 6 UCB samples. We assessed intracellular cytokine production (IFN-γ, IL-2, IL-4, IL-6, IL-17) of stimulated CD4 cells and parameters of calcium influx and ROS production following treatment with caffeine, milrinone, sildenafil, dbcAMP or a specific A2A receptor antagonist, ZM241385 using flow cytometry. In ABP, only ZM241385 caused a 1.14-fold increase in calcium influx, while all compounds increased calcium influx in UCB. This effect was more pronounced in case of caffeine (1.41-fold) and dbcAMP (1.3-fold) compared to milrinone (1.22-fold), sildenafil (1.23-fold) or ZM241385 (1.23-fold). Intracellular levels of the studied cytokines were unaffected by the applied compounds in both APB and UCB samples. Caffeine increases calcium influx upon activation in neonatal T lymphocytes to a larger extent than milrinone or sildenafil. This effect appears to be mediated primarily via increased cAMP levels rather than A2A receptor inhibition. Overall, the application of caffeine, sildenafil or milrinone does not appear to have immunosuppressive effects on neonatal T cells.

Behavioral changes induced through adenosine A2A receptor ligands in a rat depression model induced by olfactory bulbectomy.

Background: Major depressive disorders are characterized by their severity and long-lasting symptoms, which make such disorders highly disabling illnesses. Unfortunately, 50% of major depressive patients experience relapses, perhaps partly because drug research has been performed only in animal models that screen for antidepressant drugs that appear to only ameliorate acute depression symptoms. The bilateral olfactory bulbectomy (OBX) animal model presents the advantage of mimicking the symptoms of chronic depression by means of brain surgery. Adenosine purinergic receptors A2A (A2AR) have been the target of interest in the field of psychiatric diseases. This study aimed to show which A2A receptor ligands exert antidepressive-like effects in the OBX rat model. Methods: Forty Sprague-Dawley male rats were divided into four groups: control, OBX + vehicle, OBX + ZM 241385, and OBX + adenosine groups. Pharmacological treatment was administered for 14 days, and the rats were examined via the forced swim test (FST), open field test (OFT), and sucrose preference test (SPT). Results: The OBX + ZM 241385 group exhibited decreased immobility time in the FST, decreased isolation time in the OFT, and reversed anhedonia behavior in the SPT compared to the vehicle group. However, no significant differences for adenosine treatment were found. Conclusions: ZM 241385 administration (2 mg/kg i.p.) restored behavioral changes associated with OBX-induced depression.

Inhibition of cytokine-mediated JNK signalling by purinergic P2Y11 receptors, a novel protective mechanism in endothelial cells.

Previous research from our laboratory has demonstrated a novel phenomenon whereby GPCRs play a role in inhibiting cytokine-mediated c-Jun N-terminal kinase (JNK) signalling. So far this novel phenomenon seems to have been vastly overlooked, with little research in the area. Therefore, in this study we explored this further; by assessing the potential of P2YRs to mediate inhibition of cytokine-mediated JNK signalling and related functional outcomes in human endothelial cells. We utilised primary endothelial cells, and employed the use of endogenous activators of P2YRs and well characterised pharmacological inhibitors, to assess signalling parameters mediated by P2YRs, Interleukin-1ß (IL-1ß), TNFα and JNK. Activation of P2YRs with adenosine tri-phosphate (ATP) resulted in a time- and concentration-dependent inhibition of IL-1ß-mediated phosphorylation of JNK and associated kinase activity. The effect was specific for cytokine-mediated JNK signalling, as ATP was without effect on JNK induced by other non-specific activators (e.g. sorbitol, anisomycin), nor effective against other MAPK pathways such as p38 and the canonical NFκB cascade. Pharmacological studies demonstrated a role for the P2Y11 receptor in mediating this effect, but not the P2Y1 nor the adenosine receptors (A1, A2A, A2B & A3). The novel Gαq/11 inhibitor YM254890 and a protein kinase A (PKA) inhibitor H89 both partially reversed ATP-mediated inhibition of IL-1ß-stimulated JNK indicating involvement of both Gαq/11 and Gαs mediated pathways. ATP also partially reversed IL-1ß-mediated induction of cyclo­oxygenase-2 (COX-2) and E-selectin. Collectively, these studies indicate the potential for activation of purinergic receptors to protect the endothelium from inflammatory driven JNK activation and may be a new target for inflammatory disease therapy.

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.

Validation of a Na+-shift binding assay for estimation of the intrinsic efficacy of ligands at the A2A adenosine receptor.

INTRODUCTION: Determination of the intrinsic efficacy of ligands at the A2A receptor is important for selecting drug candidates, e.g. in the case of inflammatory diseases where agonists are searched for or in Parkinson disease (antagonists). METHODS: Three functional binding assays were compared with up to seven ligands with different efficacies: the GTP-shift method based on the decrease of affinity observed with agonists when GTP is added to the competition binding assay; the Ki ratio method based on the different affinity states of the receptor when using an agonist or antagonist radioligand and the Na+-shift assay based on the difference of affinity of agonists when tested in a medium containing a divalent cation (50mM MgCl2) favoring the G protein coupled agonist-receptor complex or sodium (100mM NaCl) as negative allosteric modulator. RESULTS: The Na+-shift assay proposed herein successfully discriminated the full agonists CGS21680, NECA and adenosine (IC50 ratio=13-14) from the weak inverse agonists ZM241385 and IBMX (IC50 ratio=0.85) and the partial agonists LUF5834 and regadenoson (IC50 ratios equal to 3 and 10, respectively). DISCUSSION: We conclude that the Na+-shift assay proposed herein for the A2A receptors has been validated and represents a rapid, economic and efficient functional binding assay to be used in a drug development program for early estimation of the intrinsic efficacy of hits.

Getting personal: Endogenous adenosine receptor signaling in lymphoblastoid cell lines.

Genetic differences between individuals that affect drug action form a challenge in drug therapy. Many drugs target G protein-coupled receptors (GPCRs), and a number of receptor variants have been noted to impact drug efficacy. This, however, has never been addressed in a systematic way, and, hence, we studied real-life genetic variation of receptor function in personalized cell lines. As a showcase we studied adenosine A2A receptor (A2AR) signaling in lymphoblastoid cell lines (LCLs) derived from a family of four from the Netherlands Twin Register (NTR), using a non-invasive label-free cellular assay. The potency of a partial agonist differed significantly for one individual. Genotype comparison revealed differences in two intron SNPs including rs2236624, which has been associated with caffeine-induced sleep disorders. While further validation is needed to confirm genotype-specific effects, this set-up clearly demonstrated that LCLs are a suitable model system to study genetic influences on A2AR response in particular and GPCR responses in general.

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-ß. 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-ß-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,ß-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.

Would calcium or potassium channels be responsible for cardiac arrest produced by adenosine and ATP in the right atria of Wistar rats?

Autonomic nerves release ATP, which is processed into adenosine in the synaptic cleft. Adenosine and ATP exert a negative chronotropic effect in the heart. This study aims to evaluate adenosine and P2 receptors and cellular signalling in cardiac arrest produced by purines in the heart. Right atria of adult Wistar rats were used to evaluate the effects of adenosine, ATP and CPA (an adenosine A1 receptor agonist), in the presence and absence of DPCPX, an adenosine A1 receptor antagonist. Effects of adenosine A2 and A3 receptors agonists and antagonists were also investigated. Finally, involvement of calcium and potassium channels in these responses was assessed using BayK 8644 and 4-Aminopyridine. Cumulative concentration-effect curves of adenosine and CPA resulted in a negative chronotropic effect culminating in cardiac arrest at 1000µM (adenosine) and 1µM (CPA). Furthermore, ATP produced a negative chronotropic effect at 1-300µM and cardiac arrest at 1000µM in the right atrium. ATPγS (a non-hydrolysable analogue of ATP) reduced chronotropism only. The effects of adenosine, CPA and ATP were inhibited by DPCPX, a selective adenosine A1 receptor antagonist. The selective adenosine A2 and A3 receptors antagonists did not alter the chronotropic response of adenosine. 4-Aminopyridine, a blocker of potassium channels at 10mM, prevented the cardiac arrest produced by adenosine and ATP, while BayK 8644, activator of calcium channels, did not prevent cardiac arrest. Adenosine A1 receptor activation by adenosine and ATP produces cardiac arrest in the right atrium of Wistar rats predominantly through activation of potassium channels.

Thymoquinone, the main constituent of Nigella sativa, affects adenosine receptors in asthmatic guinea pigs.

OBJECTIVES: For determining the mechanism of anti-asthmatic effect of thymoquinone, this investigation evaluated the effect of thymoquinone in the presence of selective A2A and A2B adenosine receptor antagonists (ZM241385 and MRS1706, respectively). MATERIALS AND METHODS: Seventy guinea pigs were randomly divided to 7 groups; control (C), sensitized with ovalbumin (S), sensitized groups pretreated with thymoquinone (S+TQ), ZM241385 (S+Anta A2A), MRS1706 (S+Anta A2B), thymoquinone and antagonists (S+Anta A2A+TQ and S+Anta A2B+TQ). Thymoquinone and each of these antagonists with 3 mg/kg dose were injected i.p. on 10(th) day of sensitization protocol. Tracheal responsiveness (TR) to methacholine and ovalbumin (OA), and total and differential cell count in lung lavage fluid (LLF) in different groups were measured. RESULTS: Increased EC50 and LLF neutrophil count and decreased TR to methacholine and OA, LLF eosinophil and basophil counts were observed in S+TQ group compared to S group (P<0.001 to P<0.05). Significant decrease in EC50 (P<0.01), LLF neutrophil, lymphocyte and monocyte count (P<0.001 for all) and significant increase in TR to OA (P<0.01), LLF total WBC (P<0.01) and eosinophil count (P<0.001) were observed in S+A2A group compared to S+TQ group. There was significant increase in LLF eosinophil and monocyte counts in S+Anta A2B group compared with S+TQ group (P<0.001 for both). In S+TQ+Anta A2A group, there was significant increase in LLF eosinophil (P<0.001) and significant decrease in LLF neutrophil (P<0.01) and monocyte (P<0.001) counts compared with S+TQ group. CONCLUSION: Thymoquinone affects adenosine receptors, which suggest that some of its anti-inflammatory effects may be mediated by these receptors.