A2A Receptor Antagonist ZM241385 Research Articles

Rapid Eye Movement Sleep Deprivation Enhances Adenosine Receptor Activation and the CREB1/YAP1/c-Myc Axis to Alleviate Depressive-like Behaviors in Rats.

As neuromodulators, adenosine and its receptors are mediators of sleep-wake regulation. A putative correlation between CREB1 and depression has been predicted in our bioinformatics analyses, and its expression was also predicted to be upregulated in response to sleep deprivation. Therefore, this study aims to elaborate the A1 and A2A adenosine receptors and CREB1-associated mechanism underlying the antidepressant effect of rapid eye movement sleep deprivation (REMSD) in rats with chronic unpredictable mild stress (CUMS)-induced depressive-like behaviors. The modeled rats were injected with adenosine A1 receptor antagonist DPCPX or adenosine A2A receptor antagonist ZM241385 to assess the role of adenosine receptors in depression. In addition, ectopic expression and depletion experiments of CREB1 and YAP1 were also conducted in vivo and in vitro. It was found that REMSD alleviated depressive-like behaviors in CUMS rats, as shown by increased spontaneous activity, sucrose consumption and percentage, and shortened escape latency and immobility duration. Meanwhile, A1 or A2A adenosine receptor antagonists negated the antidepressant effect of REMSD. REMSD enhanced adenosine receptor activation and promoted the phosphorylation of CREB1, thus increasing the expression of CREB1. In addition, the overexpression of CREB1 activated the YAP1/c-Myc axis and consequently alleviated depressive-like behaviors. Collectively, our results provide new mechanistic insights for an understanding of the antidepressant effect of REMSD, which is associated with the activation of adenosine receptors and the CREB1/YAP1/c-Myc axis.

Suppression of adenosine A2a receptors alleviates bladder overactivity and hyperalgesia in cyclophosphamide-induced cystitis by inhibiting TRPV1

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a type of chronic bladder inflammation characterized by increased voiding frequency, urgency and pelvic pain. The sensitization of bladder afferents is widely regarded as one of the pathophysiological changes in the development of IC/BPS. There is evidence that adenosine A2a receptors are involved in regulating the sensitization of sensory afferents. However, the effect of adenosine A2a receptors on cystitis remains unknown. In the present study, a rat model of chronic cystitis was established by intraperitoneal injection with cyclophosphamide (CYP). Cystometry and behavioral tests were performed to investigate bladder micturition function and nociceptive pain. The rats with chronic cystitis showed symptoms of bladder overactivity, characterized by an increase in bladder voiding frequency and voiding pressure. CYP treatment significantly increased the expression of the A2a receptor in bladder afferent fibers and dorsal root ganglion (DRG) neurons. The A2a receptor antagonist ZM241385 prevented bladder overactivity and hyperalgesia elicited by CYP-induced cystitis. In addition, the A2a receptor and TRPV1 were coexpressed on DRG neurons. The TRPV1 antagonist capsazepine blocked bladder overactivity induced by the A2a receptor agonist CGS21680. In contrast, ZM241385 significantly inhibited the capsaicin-induced increase in intracellular calcium concentration in DRG neurons. These results suggest that suppression of adenosine A2a receptors in bladder afferents alleviates bladder overactivity and hyperalgesia elicited by CYP-induced cystitis in rats by inhibiting TRPV1, indicating that the adenosine A2a receptor in bladder afferents is a potential therapeutic target for the treatment of IC/BPS.

Effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion injury in diabetic rats: the relationship with autophagy

Objective To evaluate the effect of activating adenosine A2A receptors on myocardial ischemia-reperfusion (I/R) injury in diabetic rats and the relationship with autophagy. Methods Clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 200-250 g, were studied.The diabetic rat model was established by intraperitoneal injection of 1% streptozotocin 60 mg/kg.Forty diabetic rats were divided into 4 groups (n=10 each) using a random number table method: sham operation group (group Sham), I/R group (group I/R), I/R plus adenosine A2A receptor agonist CGS21680 group (group CGS), and I/R plus CGS21680 plus adenosine A2A receptor antagonist ZM241385 group (group CGS+ ZM). Myocardial I/R was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120-min reperfusion.Adenosine A2A receptor agonist CGS21680 10 μg/100g was intravenously injected at 10 min before reperfusion in group CGS.CGS21680 10 ug/100g and ZM241385 0.2 mg/kg were intravenously injected sequentially at 10 min before reperfusion in group CGS+ ZM.Blood samples were obtained at the end of reperfusion for determination of concentrations of creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH) and cardiac troponin I (cTnI) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct volume (by TTC staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3 Ⅱ, p62 and Beclin-1 (by Western blot). LC3 Ⅱ/LC3 Ⅰ ratio was calculated. Results Compared with group Sham, the serum CK-MB, LDH and cTnI concentrations and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was up-regulated, and the LC3 Ⅱ/LC3 Ⅰ ratio was increased in group I/R (P 0.05). Compared with group CGS, the concentrations of serum CK-MB, LDH and cTnI and percentage of myocardial infarct volume were significantly increased, the expression of p62 and Beclin-1 was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in group CGS+ ZM (P<0.05). Conclusion Activating adenosine A2A receptors can mitigate myocardial I/R injury, and the mechanism may be related to enhancing autophagy in diabetic rats. Key words: Receptor, adenosine A2A; Autophagy; Diabetes mellitus; Myocardial reperfusion injury

Effect of cardamonin on acute lung injury induced by hemorrhagic shock and resuscitation in rats

Objective To evaluate the effect of cardamonin on acute lung injury induced by hemorrhagic shock and resuscitation (HSR) in rats. Methods Thirty-two male Sprague-Dawley rats, aged 18-24 weeks, weighing 200-250 g, were divided into 4 groups (n = 8 each) using a random number table: sham operation group (group Sham); group HSR; cardamonin group (group CA); cardamonin + adenosina A2A receptor antagonist ZM241385 group (group CZM). Bilateral common carotid arteries were only cannulated in group Sham.The left common carotid artery was cannulated for blood-letting until mean arterial pressure was reduced to 35-45 mmHg and maintained at this level for 30 min, and the animals were then resuscitated by infusion of shed blood and normal saline two-fold volume of shed blood to establish HSR model in HSR, CA and CZM groups.ZM241385 5 mg/kg was injected intraperitoneally at 30 min before blood-letting in group CZM, and cardamonin 75 mg/kg was injected intraperitoneally immediately after the beginning of resuscitation in CA and CZM groups.The rats were sacrificed at 2 h after completion of resuscitation, bronchoalveolar lavage fluid (BALF) was collected for determination of neutrophil count, and lungs were removed for microscopic examination of the pathological changes and for determination of wet/dry lung weight ratio (W/D ratio), contents of tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1β) and IL-6 (by enzyme-linked immunosorbent assay) and expression of adenosine A2A receptors in lung tissues (by Western blot). Results Compared with group Sham, the neutrophil count in BALF, W/D ratio and contents of TNF-α, IL-1β and IL-6 were significantly increased, the expression of adenosine A2A receptors was significantly down-regulated in group HSR, and the neutrophil count in BALF and contents of TNF-α and IL-6 were significantly increased (P 0.05). Compared with group HSR, the neutrophil count in BALF, W/D ratio and contents of TNF-α, IL-1β and IL-6 were significantly decreased, the expression of adenosine A2A receptors was significantly up-regulated (P 0.05). Compared with group CA, the neutrophil count in BALF, W/D ratio and contents of TNF-α, IL-1β and IL-6 were significantly increased, the expression of adenosine A2A receptors was significantly down-regulated (P<0.05), and the pathological changes were aggravated in group CZM. Conclusion Cardamonin can attenuate acute lung injury induced by HSR in rats, and activated adenosine A2A receptors and inhibited inflammatory responses are involved in the mechanism. Key words: AMOMUM COSTATUM; Shock, hemorrhagic; Respiratory distress syndrome, adult

The Effect of A2A Receptor Antagonist on Microglial Activation in Experimental Glaucoma.

We investigated the effect of A2A receptor (A2AR) antagonist on microglial activation and retinal ganglion cell (RGC) survival under chronic ocular hypertension (COH), and explored the relationship between microglial activation and RGC survival by means of in vitro and in vivo experiments. An animal model of COH was induced in one eye of male Sprague-Dawley (SD) rats by ligation of three episcleral veins. The survival of RGCs and the activation of microglia under COH without or with intravitreous injection of A2AR antagonist ZM241385 were assessed by fluorescent labeling, real time PCR and Western blot. ELISA was used to measure the secretion of inflammatory mediators by microglia when glutamate and/or ZM241385 was added into the culture system. Compared to the baseline, RGC density 2 weeks after COH induction decreased at the central (2436 ± 143 cells/mm2 pre- and 2130 ± 148 cells/mm2 post-COH induction) and peripheral (2219 ± 140 cells/mm2 pre- and 1953 ± 142 cells/mm2 post-COH induction) retina. The microglia changed their ramified morphology to an amoeboid form with increase in TNF-α and IL-1β expression after COH. These changes, however, were ameliorated with intravitreous ZM241385 (RGC density only dropped to 2287 ± 135 cells/mm2). The upregulation of those proinflammatory cytokines secreted by microglia in vitro under high concentration of glutamate was downregulated when ZM241385 was added into the culture system. A2AR antagonist ZM241385 could reduce the activation of microglia and downregulate the proinflammatory cytokines expression under the conditions of COH and high concentration of glutamate, which may be one of the mechanisms that protected RGCs in experimental glaucoma.

PI3K/Akt signaling pathway is involved in the neurotrophic effect of senegenin.

Neurodegenerative diseases are frequently associated with the loss of synapses and neurons. Senegenin, extracted from the Chinese herb Polygala tenuifolia Willd, was previously found to promote neurite outgrowth and neuronal survival in primary cultured rat cortical neurons. The aim of the present study was to investigate the underlying mechanisms of senegenin-induced neurotrophic effects on rat cortical neurons. Primary cortical rat neurons were treated with various pharmacological antagonists and with or without senegenin, and subjected to MTT and western blot analysis to explore the effects of senegenin on cell survival as well as the activation of signaling pathways. Neurite outgrowth and neuronal survival induced by senegenin were significantly inhibited by A2A receptor antagonist ZM241385 and specific phosphoinositide-3 kinase (PI3K) inhibitor LY294002, but not by tropomyosin receptor kinase A receptor inhibitor K252a, mitogen-activated protein kinase kinase inhibitor PD98059 or protein kinase C inhibitor GÖ6976. Furthermore, senegenin enhanced the phosphorylation of Akt, which was blocked by LY294002. The present study revealed that the PI3K/Akt signaling pathway may be involved in the neurotrophic effects of senegenin.

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.

Alkaline phosphatase protects against renal inflammation through dephosphorylation of lipopolysaccharide and adenosine triphosphate.

Recently, two phase-II trials demonstrated improved renal function in critically ill patients with sepsis-associated acute kidney injury treated with the enzyme alkaline phosphatase. Here, we elucidated the dual active effect on renal protection of alkaline phosphatase. The effect of human recombinant alkaline phosphatase (recAP) on LPS-induced renal injury was studied in Sprague-Dawley rats. Renal function was assessed by transcutaneous measurement of FITC-sinistrin elimination in freely moving, awake rats. The mechanism of action of recAP was further investigated in vitro using conditionally immortalized human proximal tubular epithelial cells (ciPTEC). In vivo, LPS administration significantly prolonged FITC-sinistrin half-life and increased fractional urea excretion, which was prevented by recAP co-administration. Moreover, recAP prevented LPS-induced increase in proximal tubule injury marker, kidney injury molecule-1 expression and excretion. In vitro, LPS-induced production of TNF-α, IL-6 and IL-8 was significantly attenuated by recAP. This effect was linked to dephosphorylation, as enzymatically inactive recAP had no effect on LPS-induced cytokine production. RecAP-mediated protection resulted in increased adenosine levels through dephosphorylation of LPS-induced extracellular ADP and ATP. Also, recAP attenuated LPS-induced increased expression of adenosine A2A receptor. However, the A2A receptor antagonist ZM-241385 did not diminish the effects of recAP. These results indicate that the ability of recAP to reduce renal inflammation may account for the beneficial effect observed in septic acute kidney injury patients, and that dephosphorylation of ATP and LPS are responsible for this protective effect.

Japanese sake yeast supplementation improves the quality of sleep: a double-blind randomised controlled clinical trial.

Activation of adenosine A2a receptors in cerebral neurons induces sleep in various mammals. It was previously found that Japanese sake yeast enriched in adenosine analogues activates A2a receptors in vitro and induces sleep in mice. Here it is reported that sake yeast activated A2a receptors in a cultured human cell line and improved human sleep quality in a clinical trial. Sake yeast activated A2a receptors in HEK cells in a dose-dependent manner with an EC50 of 40 μg mL(-1), and the activation was attenuated almost completely by the A2a receptor antagonist ZM241385 with an IC50 of 73 nm. In a double-blind placebo-controlled crossover clinical study, 68 healthy participants ingested tablets containing either 500 mg of sake yeast powder or a placebo (cellulose) 1 h before sleep for 4 days. Electroencephalograms were recorded during sleep at home with a portable device for 4 week days. Electroencephalogram analyses revealed that sake yeast supplementation significantly (P = 0.03) increased delta power during the first cycle of slow-wave sleep by 110%, without changing other sleep parameters. Sake yeast supplementation also significantly increased growth hormone secretion in the urine on awakening by 137% from 3.17 ± 0.41 (placebo) to 4.33 ± 0.62 (sake yeast) pg mg(-1) creatinine (P = 0.03). Subjective sleepiness (P = 0.02) and fatigue (P = 0.06) in the morning were improved by sake yeast. Given these benefits and the absence of adverse effects during the study period, it was concluded that sake yeast supplementation is an effective and safe way to support daily high-quality, deep sleep.

Brief Report: Methotrexate Prevents Wear Particle-Induced Inflammatory Osteolysis in Mice Via Activation of Adenosine A2A Receptor.

Adenosine, acting at the A2A receptor, mediates the antiinflammatory effects of methotrexate (MTX) in models of inflammation. We previously reported that A2A receptor ligation diminishes wear particle-driven osteolysis. The aim of this study was to investigate whether MTX treatment could prevent bone resorption caused by inflammatory osteolysis. C57BL/6 mice (6-8 weeks old) received intraperitoneal injections of 1 mg/kg MTX (n = 20) or 0.9% saline (n = 10), starting 2 weeks prior to surgical implantation of 3 mg of wear particles (ultrahigh molecular weight polyethylene [UHMWPE] particles). The MTX-treated mice received daily injections of vehicle or ZM241385 at the surgical site until they were killed, 14 days later. XenoLight RediJect Bone Probe 680 was injected intravenously, and fluorescence analysis of the calvaria using an IVIS imaging system was performed to assess bone formation. Micro-computed tomography (micro-CT) and immunostaining for osteoclast and osteoblast markers were performed. Implantation of wear particles induced bone pitting and thinning, as shown by micro-CT. MTX treatment markedly reduced osteolysis, and this effect was abrogated by treatment with the A2A receptor antagonist ZM241385. Implantation of UHMWPE reduced new bone formation, and MTX treatment restored new bone formation, an effect that was completely reversed by treatment with ZM241385. Histologic examination of particle-exposed calvariae demonstrated that MTX prevented accumulation of an inflammatory infiltrate at the site of particle implantation, increased the number of osteoblasts, and reduced the number of osteoclasts at the site of inflammation, an effect that was reversed by treatment with ZM241385. MTX reduces inflammatory osteolysis indirectly via stimulation of A2A receptor and may represent a novel approach to enhance orthopedic implant survival, delaying or eliminating the need for revision arthroplasty surgery.

Reduced adenosine A2a receptor–mediated efferent arteriolar vasodilation contributes to diabetes-induced glomerular hyperfiltration

Diabetes is associated with increased risk for development of kidney disease, and an increased glomerular filtration rate is an early indication of altered kidney function. Here we determine whether reduced adenosine A2a receptor-mediated vasodilation of the efferent arteriole contributes to the increased glomerular filtration rate in diabetes. The glomerular filtration rate, renal blood flow, and proximal tubular stop flow pressure were investigated in control and streptozotocin-diabetic rats during baseline and after administration of the adenosine A2a receptor antagonist ZM241385 or the adenosine A2a receptor agonist CGS21680. The diabetes-induced glomerular hyperfiltration was reduced by 24% following A2a receptor stimulation but was unaffected by A2a receptor inhibition. Contrarily, glomerular filtration rate in controls increased by 22% after A2a receptor inhibition and was unaffected by A2a stimulation. The increased glomerular filtration rate after A2a receptor inhibition in controls and decreased glomerular filtration rate after A2a receptor activation in diabetics were caused by increased and decreased stop flow pressure, respectively. None of the interventions affected renal blood flow. Thus, the normal adenosine A2a receptor-mediated tonic vasodilation of efferent arterioles is abolished in the diabetic kidney. This causes increased efferent arteriolar resistance resulting in increased filtration fraction and hyperfiltration.

Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices.

Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X1-5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X1, P2X3, P2X2/3, and P2X7 receptors, respectively. Moreover, adenosine receptors blockade with A1 receptor antagonist DPCPX (100 nM) or with A2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A2A receptor antagonist ZM241385 (50 nM), or A3 receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A1, and A2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.

Acute hyperammonemia and systemic inflammation is associated with increased extracellular brain adenosine in rats: a biosensor study.

Acute liver failure (ALF) can lead to brain edema, cerebral hyperperfusion and intracranial hypertension. These complications are thought to be mediated by hyperammonemia and inflammation leading to altered brain metabolism. As increased levels of adenosine degradation products have been found in brain tissue of patients with ALF we investigated whether hyperammonemia could induce adenosine release in brain tissue. Since adenosine is a potent vasodilator and modulator of cerebral metabolism we furthermore studied the effect of adenosine receptor ligands on intracranial pressure (ICP) and cerebral blood flow (CBF). We measured the adenosine concentration with biosensors in rat brain slices exposed to ammonia and in a rat model with hyperammonemia and systemic inflammation. Exposure to ammonia in concentrations from 0.15-10mM led to increases in the cortical adenosine concentration up to 18µM in brain slices. In vivo recordings showed a tendency towards increased adenosine levels in rats with hyperammonemia and systemic inflammation compared to a control group (3.7±0.7 vs. 0.8±0.2µM, P=0.06). This was associated with a significant increase in ICP and CBF. Intervention with the non-selective adenosine receptor antagonist theophyllamine, the A2A receptor antagonist ZM241385, or the A1 receptor agonist N6-Cyclopentyladenosine did not reduce ICP or CBF. In conclusion, our results show that the adenosine concentration in cortex increases during exposure to ammonia, and is associated with a rise in intracranial pressure and cerebral perfusion. However adenosine receptor antagonism/agonism did not reduce the ICP or CBF which indicates that adenosine may not be of direct importance for these cerebral complications in ALF.

Cellular signalling pathways mediating dilation of porcine pial arterioles to adenosine A2A receptor activation

Adenosine is a potent vasodilator contributing to cerebral blood flow regulation during metabolic stress. However, the distribution of adenosine receptor subtypes and underlying signalling mechanisms for dilation of pial arterioles remain unclear. The present study aimed at addressing these issues. Isolated porcine pial arterioles were subjected to study of vasomotor function, localization of adenosine receptors, and production of nitric oxide (NO). Concentration-dependent vasodilation to adenosine was inhibited by A₂A receptor antagonist ZM241385 but not by A₁ receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. A₂A receptors were detected in endothelium and smooth muscle of pial arterioles via immunohistochemistry. Adenosine significantly increased arteriolar production of NO, and the induced dilation was insensitive to KATP channel blocker glibenclamide but was attenuated by endothelial denudation, NO synthase inhibitor L-NAME, or guanylyl cyclase inhibitor ODQ in a similar manner. Both inward rectifier potassium (Kir) channel inhibitor barium and cAMP signalling inhibitor Rp-8-Br-cAMPS attenuated adenosine-induced dilation. In the presence of L-NAME or the absence of endothelium, addition of Rp-8-Br-cAMPS but not barium further reduced adenosine-induced responses. Barium diminished endothelium-independent vasodilation to NO donor sodium nitroprusside. Comparable to the adenosine-induced response, vasodilation to A₂A receptor agonist CGS21680 was attenuated by endothelial removal, ZM241385, L-NAME, barium, or Rp-8-Br-cAMPS, but not by glibenclamide. Adenosine evokes dilation of porcine pial arterioles via parallel activation of endothelial and smooth muscle A₂A receptors. Stimulation of endothelial NO production activates smooth muscle guanylyl cyclase for vasodilation by opening Kir channels. Adenosine also activates smooth muscle cAMP signalling leading to vasodilation.

Adenosine A2A and A3 Receptors Are Involved in the Human Endothelial Progenitor Cells Migration

Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A₂A, A₂B, and A₃ in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A₂A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A₂A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A₂A and A₃ but not A₂B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.

Effect of adenosine A2A receptor antagonist ZM241385 on amygdala-kindled seizures and progression of amygdala kindling

The purpose of this study was to evaluate the effect of adenosine A2A receptor antagonist ZM241385 on amygdala-kindled seizures and its roles in epileptogenesis. Electrodes were implanted into the right amygdala of male adult Wistar rats. Kindling was accomplished by using stimulus strength of 500 μA applied daily to the amygdala until 10 consecutive stage 5 seizues were induced. Then effect of ZM241385 was studied in fully kindled rats after intracerebroventricular administration of the drug. In addition, the effect on kindling progression was evaluated through ZM241385 injection before daily stimulation. In all experiments, behavioral changes in the rats in response to ZM241385 were monitored closely. The results showed that, in fully amygdala-kindled rats, ZM241385 (0.001-0.1 nmol/L) decreased afterdischage duration (ADD), motor seizure duration (MSD), stage 5 duration (S5D) and seizure duration (SD), but only the effect on ADD was dose-dependent. The doses of 0.001-0.1 nmol/L had no influence on stage 4 latency (S4L) and seizure stage (SS). The dosages of 0.0001 and 1 nmol/L of ZM241385 did not exert any effect on all seizure parameters. In contrast to the results in fully amygdala-kindled rats, ZM241385 (0.001-0.1 nmol/L) had minimal or no effects on the progression of amygdala-kindled seizures. We are led to the conclusion that although ZM241385 had no influence on the progression of amygdala-kindled seizures, it had potent anti-convulsant profile and little adverse effects at the dosage of 0.001-0.1 nmol/L, suggesting that the agent is effective against the amygdala-kindled seizures.

Suppression of Bladder Overactivity by Adenosine A2A Receptor Antagonist in a Rat Model of Parkinson Disease

Overactive bladder is highly prevalent among patients with Parkinson disease. Adenosine is an important neurotransmitter in the central nervous system but it is not fully clarified how adenosine receptors regulate the micturition reflex. Thus, we examined the effect of an adenosine A2A receptor antagonist on the micturition reflex in a rat model of Parkinson disease. In a rat model of Parkinson disease induced by 6-hydroxydopamine (Tocris Bioscience, Ellisville, Missouri) injection we examined the effects of the adenosine A2A receptor antagonist ZM241385, the dopamine D1 receptor agonist SKF38393 and the dopamine D2 receptor agonist quinpirole on bladder activity. Intravenous administration of ZM241385 increased the intercontraction interval in a dose dependent manner in rats with Parkinson disease and sham operated rats but the inhibitory effect was greater in the Parkinson disease group. Intrathecal and intracerebroventricular administration of ZM241385 increased the intercontraction interval in each group. However, in rats with Parkinson disease the inhibitory effects induced by intracerebroventricular administration of ZM241385 were greater than in sham operated rats. Intravenous administration of SKF38393 increased the intercontraction interval in rats with Parkinson disease and subsequent administration of ZM further increased the intercontraction interval. However, SKF38393 did not increase the intercontraction interval after ZM241385 application. Also, ZM241385 increased the intercontraction interval without being affected by pre-administration or post-administration of quinpirole, which decreased the intercontraction interval. Results indicate that the adenosine A2A receptor mediated excitatory mechanism is enhanced at a supraspinal site to induce bladder overactivity and A2A receptor inhibition effectively suppresses bladder overactivity in rats with Parkinson disease. Thus, adenosine A2A receptor antagonists could be useful for bladder dysfunction in Parkinson disease cases.

The adenosine A2A receptor antagonist ZM241385 enhances neuronal survival after oxygen-glucose deprivation in rat CA1 hippocampal slices.

Activation of adenosine A(2A) receptors in the CA1 region of rat hippocampal slices during oxygen-glucose deprivation (OGD), a model of cerebral ischaemia, was investigated. We made extracellular recordings of CA1 field excitatory postsynaptic potentials (fepsps) followed by histochemical and immunohistochemical techniques coupled to Western blots. OGD (7 or 30 min duration) elicited an irreversible loss of fepsps invariably followed by the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. The application of the selective adenosine A(2A) receptor antagonist, ZM241385 (4-(2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-ylamino]ethyl)phenol; 100-500 nmolxL(-1)) prevented or delayed AD appearance induced by 7 or 30 min OGD and protected from the irreversible fepsp depression elicited by 7 min OGD. Two different selective adenosine A(2A) receptor antagonists, SCH58261 and SCH442416, were less effective than ZM241385 during 7 min OGD. The extent of CA1 cell injury was assessed 3 h after the end of 7 min OGD by propidium iodide. Substantial CA1 pyramidal neuronal damage occurred in untreated slices, exposed to OGD, whereas injury was significantly prevented by 100 nmolxL(-1) ZM241385. Glial fibrillary acid protein (GFAP) immunostaining showed that 3 h after 7 min OGD, astrogliosis was appreciable. Western blot analysis indicated an increase in GFAP 30 kDa fragment which was significantly reduced by treatment with 100 nmolxL(-1) ZM241385. In the CA1 hippocampus, antagonism of A(2A) adenosine receptors by ZM241385 was protective during OGD (a model of cerebral ischaemia) by delaying AD appearance, decreasing astrocyte activation and improving neuronal survival.

Adenosine Promotion of Cellular Migration in Bronchial Epithelial Cells Is Mediated by the Activation of Cyclic Adenosine Monophosphate–Dependent Protein Kinase A

Migration of neighboring cells into the injury is important for rapid repair of damaged airway epithelium. We previously reported that activation of the A2A receptors (A2AARs) mediates adenosine-stimulated epithelial wound...

Adenosine closes the K+ channel KCa3.1 in human lung mast cells and inhibits their migration via the adenosine A2A receptor.

Human lung mast cells (HLMC) express the Ca2+-activated K+ channel KCa3.1, which opens following IgE-dependent activation. This hyperpolarises the cell membrane and potentiates both Ca2+ influx and degranulation. In addition, blockade of KCa3.1 profoundly inhibits HLMC migration to a variety of diverse chemotactic stimuli. KCa3.1 activation is attenuated by the β2adrenoceptor through a Gαs-coupled mechanism independent of cyclic AMP. Adenosine is an important mediator that both attenuates and enhances HLMC mediator release through the Gαs-coupled A2A and A2B adenosine receptors, respectively. We show that at concentrations that inhibit HLMC degranulation (10–5–10–3 M), adenosine closes KCa3.1 both dose-dependently and reversibly. KCa3.1 suppression by adenosine was reversed partially by the selective adenosine A2A receptor antagonist ZM241385 but not by the A2B receptor antagonist MRS1754, and the effects of adenosine were mimicked by the selective A2A receptor agonist CGS21680. Adenosine also opened a depolarising current carried by non-selective cations. As predicted from the role of KCa3.1 in HLMC migration, adenosine abolished HLMC chemotaxis to asthmatic airway smooth muscle-conditioned medium. In summary, the Gαs-coupled adenosine A2A receptor closes KCa3.1, providing a clearly defined mechanism by which adenosine inhibits HLMC migration and degranulation. A2A receptor agonists with channel-modulating function may be useful for the treatment of mast cell-mediated disease.