Adenosine Receptor Stimulation Research Articles

Further investigations on the influence of protein phosphatases on the signaling of muscarinic receptors in the atria of mouse hearts

The vagal regulation of cardiac function involves acetylcholine (ACh) receptor activation followed by negative chronotropic and negative as well as positive inotropic effects. The resulting signaling pathways may include Gi/o protein-coupled reduction in adenylyl cyclase (AC) activity, direct Gi/o protein-coupled activation of ACh-activated potassium current (IKACh), inhibition of L-type calcium ion channels, and/or the activation of protein phosphatases. Here, we studied the role of the protein phosphatases 1 (PP1) and 2A (PP2A) for muscarinic receptor signaling in isolated atrial preparations of transgenic mice with cardiomyocyte-specific overexpression of either the catalytic subunit of PP2A (PP2A-TG) or the inhibitor-2 (I2) of PP1 (I2-TG) or in double transgenic mice overexpressing both PP2A and I2 (DT). In mouse left atrial preparations, carbachol (CCh), cumulatively applied (1 nM–10 µM), exerted at low concentrations a negative inotropic effect followed by a positive inotropic effect at higher concentrations. This biphasic effect was noted with CCh alone as well as when CCh was added after β-adrenergic pre-stimulation with isoprenaline (1 µM). Whereas the response to stimulation of β-adrenoceptors or adenosine receptors (used as controls) was changed in PP2A-TG, the response to CCh was unaffected in atrial preparations from all transgenic models studied here. Therefore, the present data tentatively indicate that neither PP2A nor PP1, but possibly other protein phosphatases, is involved in the muscarinic receptor-induced inotropic and chronotropic effects in the mouse heart.

A2A adenosine receptor stimulation ameliorated diabetic-induced osteoporosis in rats.

Diabetic osteoporosis is a common health problem that is associated with a disruption in bone metabolism. A2A adenosine receptor (A2AAR) signaling seems to play a critical role in bone homeostasis. This study aimed to evaluate the effect of A2AAR stimulation on the treatment of diabetic-induced osteoporosis versus insulin treatment. Forty adult male rats were allocated into control (C), untreated diabetic-induced osteoporosis (DIO), insulin-treated DIO (I-DIO), and A2AAR agonist-treated DIO (A-DIO) groups. Both insulin and A2AAR agonist treatments significantly increased serum insulin level, glutathione peroxidase (GPx) activity, bone expression of osteoprotegerin (Opg) and β-catenin (Ctnnb1), and cortical and trabecular bone thickness, whereas they decreased serum fasting glucose, malondialdehyde (MDA), tumor necrosis factor α (TNF-α), bone expression of receptor activator of nuclear factor kappa-B ligand (Rankl), runt-related transcription factor-2 (Runx2), and sclerostin (Sost) versus the untreated DIO groups. A2AAR agonist treatment was more effective than insulin in ameliorating diabetic osteoporosis. This might be attributed to the upregulation of β-catenin gene expression, enhancing its anabolic effect on bone, in addition to the A2AAR agonist's anti-oxidative, anti-inflammatory, and anti-diabetic effects.

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.

Adenosine receptor stimulation inhibits methamphetamine-associated cue seeking.

Methamphetamine (METH) is a psychostimulant drug that remains a popular and threatening drug of abuse with high abuse liability. There is no established pharmacotherapy to treat METH dependence, but evidence suggests that stimulation of adenosine receptors reduces the reinforcing properties of METH and could be a potential pharmacological target. This study examines the effects of adenosine receptor subtype stimulation on METH seeking using both a cue-induced reinstatement and cue-craving model of relapse. Male and female rats were trained to self-administer METH during daily 2-h sessions. Cue-induced reinstatement of METH seeking was evaluated after extinction training. A systemic pretreatment of an adenosine A1 receptor (A1R) or A2A receptor (A2AR) agonist was administered prior to an extinction or cue session to evaluate the effects of adenosine receptor subtype stimulation on METH seeking. The effects of a systemic pretreatment of A1R or A2AR agonists were also evaluated in a cue-craving model where the cued-seeking test was conducted after 21 days of forced home-cage abstinence without extinction training. Cue-induced reinstatement was reduced in both male and female rats that received A1R or A2AR agonist pretreatments. Similarly, an A1R or A2AR agonist pretreatment also inhibited cue craving in both male and female rats. Stimulation of either adenosine A1R or A2AR subtypes inhibits METH-seeking behavior elicited by METH-associated cues. These effects may be attributed to the ability of A1R and A2AR stimulation to disrupt cue-induced dopamine and glutamate signaling throughout the brain.

Stimulation of adenosine A1 receptor prevents oxidative injury in H9c2 cardiomyoblasts: Role of Gβγ-mediated Akt and ERK1/2 signaling

Oxidative stress causes cellular injury and damage in the heart primarily through apoptosis resulting in cardiac abnormalities such as heart failure and cardiomyopathy. During oxidative stress, stimulation of adenosine receptor (AR) has been shown to protect against oxidative damage due to their cytoprotective properties. However, the subtype specificity and signal transductions of adenosine A1 receptor (A1R) on cardiac protection during oxidative stress have remained elusive. In this study, we found that stimulation of A1Rs with N6-cyclopentyladenosine (CPA), a specific A1R agonist, attenuated the H2O2-induced intracellular and mitochondrial reactive oxygen species (ROS) production and apoptosis. In addition, A1R stimulation upregulated the synthesis of antioxidant enzymes (catalase and GPx-1), antiapoptotic proteins (Bcl-2 and Bcl-xL), and mitochondria-related markers (UCP2 and UCP3). Blockades of Gβγ subunit of heterotrimeric Gαi protein antagonized A1R-mediated antioxidant and antiapoptotic effects, confirming the potential role of Gβγ subunit-mediated A1R signaling. Additionally, cardioprotective effects of CPA mediated through PI3K/Akt- and ERK1/2-dependent signaling pathways. Thus, we propose that A1R represents a promising therapeutic target for prevention of oxidative injury in the heart.

A3 adenosine receptor agonist IB-MECA reverses chronic cerebral ischemia-induced inhibitory avoidance memory deficit

BackgroundChronic cerebral ischemia (CCI) is a major cause of subcortical ischemic vascular dementia. Here, we examined the neuroprotective action of the A3 adenosine receptor agonist N6-(3-iodobenzyl)-adenosine-5′-N-methylcarboxamide (IB-MECA) on white matter lesions following CCI. MethodsA CCI mouse model was established using unilateral common carotid artery occlusion. IB-MECA and 3-propyl-6-ethyl-5-[(ethylthio)carbonyl]-2 phenyl-4-propyl-3-pyridine carboxylate (MRS1523), an antagonist of the A3 adenosine receptor, were administered by intraperitoneal injection. The inhibitory avoidance test was used to examine changes in memory performance. Microtubule-associated protein 2 (MAP-2) and neurofilament were assessed by immunohistochemistry, while expressions of phosphorylated extracellular signal-regulated kinase (ERK), ERK, interferon-beta (IFN-β), and glial fibrillary acidic protein (GFAP) were assessed by western blot assay. ResultsThe memory retention score was reduced in vehicle-treated mice compared with IB-MECA-treated (p < 0.05) mice. Compared to sham-operated mice, p-ERK, GFAP and IFN-β were increased, while MAP-2 and neurofilament were reduced in vehicle-treated mice (p < 0.01 for each). IB-MECA reduced ERK phosphorylation (p < 0.01) and GFAP expression (p < 0.05), but upregulated MAP-2 and IFN-β (p < 0.01 for both), compared with vehicle. MRS1523 suppressed the effects of IB-MECA on the memory deficit, ERK phosphorylation, and on MAP-2, neurofilament, GFAP and IFN-β levels. ConclusionOur results suggest that A3 adenosine receptor stimulation ameliorates CCI-induced memory deficits, modulates the ERK signaling pathway, preserves MAP-2 and neurofilament expression, regulates GFAP expression, and upregulates the anti-inflammatory cytokine IFN-β. Thus, the A3 adenosine receptor may be a therapeutic target for treatment of cognitive disorders and cerebral inflammatory diseases.

Myelin Defects in Niemann-Pick Type C Disease: Mechanisms and Possible Therapeutic Perspectives.

Niemann–Pick type C (NPC) disease is a wide-spectrum clinical condition classified as a neurovisceral disorder affecting mainly the liver and the brain. It is caused by mutations in one of two genes, NPC1 and NPC2, coding for proteins located in the lysosomes. NPC proteins are deputed to transport cholesterol within lysosomes or between late endosome/lysosome systems and other cellular compartments, such as the endoplasmic reticulum and plasma membrane. The first trait of NPC is the accumulation of unesterified cholesterol and other lipids, like sphingosine and glycosphingolipids, in the late endosomal and lysosomal compartments, which causes the blockade of autophagic flux and the impairment of mitochondrial functions. In the brain, the main consequences of NPC are cerebellar neurodegeneration, neuroinflammation, and myelin defects. This review will focus on myelin defects and the pivotal importance of cholesterol for myelination and will offer an overview of the molecular targets and the pharmacological strategies so far proposed, or an object of clinical trials for NPC. Finally, it will summarize recent data on a new and promising pharmacological perspective involving A2A adenosine receptor stimulation in genetic and pharmacological NPC dysmyelination models.

A2 B Adenosine Receptors and Sphingosine 1-Phosphate Signaling Cross-Talk in Oligodendrogliogenesis.

Oligodendrocyte-formed myelin sheaths allow fast synaptic transmission in the brain. Impairments in the process of myelination, or demyelinating insults, might cause chronic diseases such as multiple sclerosis (MS). Under physiological conditions, remyelination is an ongoing process throughout adult life consisting in the differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes (OLs). During pathological events, this process fails due to unfavorable environment. Adenosine and sphingosine kinase/sphingosine 1-phosphate signaling axes (SphK/S1P) play important roles in remyelination processes. Remarkably, fingolimod (FTY720), a sphingosine analog recently approved for MS treatment, plays important roles in OPC maturation. We recently demonstrated that the selective stimulation of A2B adenosine receptors (A2BRs) inhibit OPC differentiation in vitro and reduce voltage-dependent outward K+ currents (IK) necessary to OPC maturation, whereas specific SphK1 or SphK2 inhibition exerts the opposite effect. During OPC differentiation A2BR expression increases, this effect being prevented by SphK1/2 blockade. Furthermore, selective silencing of A2BR in OPC cultures prompts maturation and, intriguingly, enhances the expression of S1P lyase, the enzyme responsible for irreversible S1P catabolism. Finally, the existence of an interplay between SphK1/S1P pathway and A2BRs in OPCs was confirmed since acute stimulation of A2BRs activates SphK1 by increasing its phosphorylation. Here the role of A2BR and SphK/S1P signaling during oligodendrogenesis is reviewed in detail, with the purpose to shed new light on the interaction between A2BRs and S1P signaling, as eventual innovative targets for the treatment of demyelinating disorders.

Adenosine Metabotropic Receptors in Chronic Pain Management.

The pharmacological treatment of chronic pain is still unsatisfactory. The cellular and molecular mechanisms at the basis of pain chronification are still poorly understood. The commercially available drugs for treating chronic pain, including neuropathic pain, are effective in few patients and own several side effects that often limit the compliance of the patients. In addition, the opioids, which are the most potent analgesics, often fail in chronic pain conditions, especially in neuropathic pain syndromes. Moreover, it is well known that opiates can lead to addiction, tolerance and hyperalgesia. Therefore, new targets for treating neuropathic pain are needed. Purines, including ATP, ADP and adenosine and their receptors are deeply involved in the pathophysiology of neuropathic pain, particularly in the immune-mediated reactions that are responsible for the induction of the tactile allodynia, that represents the main symptom of neuropathic pain. The first response to the insult is mediated by the production of ATP and the activation of P2X receptors (Jacobson et al., 2020). In particular, the P2X4 receptors on microglia have been identified to be essential for the involvement of microglia cells in the pain pathophysiology (Trang et al., 2012). Beside the P2X, the P2Y receptors tend to boost the immune cells in response to nucleotides, with their ligands acting as immediate danger signals (Cekic and Linden, 2016). The subsequent stimulation of the metabotropic P1 adenosine receptors (ARs), of which are known four subtypes (A1, A2A, A2B, and A3), is overall associated with the reduction of both immunoinflammatory response and pain (Vincenzi et al., 2020). In the present opinion paper we will focus on the role of the P1 adenosine receptors in the pathophysiology of chronic neuropathic pain. The involvement of P2X and P2Y receptors in chronic pain has been discussed elsewhere (Jacobson et al., 2020).

Evaluation of adenosine A1 receptor agonists as neuroprotective countermeasures against Soman intoxication in rats

Soman, an organophosphorus (OP) compound, disrupts nervous system function through inactivation of acetylcholinesterase (AChE), the enzyme that breaks down acetylcholine at synapses. Left untreated, a state of prolonged seizure activity (status epilepticus, SE) is induced, causing widespread neuronal damage and associated cognitive and behavioral impairments. Previous research demonstrated that therapeutic stimulation of A1 adenosine receptors (A1ARs) can prevent or terminate soman-induced seizure. This study examined the ability of three potent A1AR agonists to provide neuroprotection and, ultimately, prevent observable cognitive and behavioral deficits following exposure to soman. Sprague Dawley rats were challenged with a seizure-inducing dose of soman (1.2 x LD50) and treated 1 min later with one of the following A1AR agonists: (6)-Cyclopentyladenosine (CPA), 2-Chloro-N6-cyclopentyladenosine (CCPA) or N-bicyclo(2.2.1)hept-2-yl-5'-chloro-5'-deoxyadenosine (cdENBA). An active avoidance shuttle box task was used to evaluate locomotor responses to aversive stimuli at 3, 7 and 14 days post-exposure. Animals treated with CPA, CCPA or cdENBA demonstrated a higher number of avoidance responses and a faster reaction to the aversive stimulus than the soman/saline control group across all three sessions. Findings suggest that A1AR agonism is a promising neuroprotective countermeasure, capable of preventing the long-term deficits in learning and memory that are characteristic of soman intoxication.

Dipyridamole Potentiates the Endothelium-Dependent and -Independent Vasomotion in Isolated Human Small Arteries.

BACKGROUND: To investigate the effects of dipyridamole, a drug with phosphodiesterase-, adenosine reuptake-inhibiting, and prostacyclin-stimulating activity on the biological actions of nitric oxide, 30 norepinephrine-precontracted subcutaneous arterioles were prepared from specimens removed during surgery. METHODS AND RESULTS: Specimens were mounted on a myograph and relaxes through either acetylcholine, a muscarinic agonist that stimulates endothelial nitric oxide production, or sodium nitroprusside, an endothelium-independent vasodilator. Studies were performed under control conditions and after dipyridamole which potentiated in a concentration-dependent manner the vasorelaxation induced both by acetylcholine and sodium nitroprusside, indicating an endothelium-independent mechanism of action. The contribution of nitric oxide to the relaxation produced by acetylcholine was confirmed by N-monomethyl-L-arginine, a nitric oxide synthase inhibitor. In contrast, indomethacin, a cyclo-oxygenase inhibitor, was ineffective, indicating that prostacyclin stimulation could not explain the effect of dipyridamole. CGS 21680 C, an A(2)-selective adenosine receptor agonist insensitive to tissue deaminase, did not influence the relaxations induced by acetylcholine, suggesting that interference with adenosine metabolism was not implicated in the potentiating action of dipyridamole. CONCLUSIONS: Dipyridamole potentiated the vasorelaxing effect of acetylcholine and sodium nitroprusside in human subcutaneous arterioles; neither prostacyclin stimulation nor A(2) adenosine receptor stimulation could explain this effect. The data are consistent with an increase in intracellular cyclic 3' 5'-guanosine monophosphate levels secondary to the phosphodiesterase-inhibiting properties of the drug.

Targeting adenosinergic pathway and adenosine A2A receptor signaling for the treatment of COVID-19: A hypothesis

The most serious health issue today is the rapid outbreak of Coronavirus Disease 2019 (COVID-19). More than 6,973,427 confirmed cases were diagnosed in nearly 213 countries and territories around the world and two international conveyances, causing globally over 400,000 deaths. Epidemiology, risk factors, and clinical characteristics of COVID-19 patients have been identified, but the factors influencing the immune system against COVID-19 have not been well established. Upon infection or cell damage, high amounts of adenosine triphosphate (ATP) are released from damaged cells, which serve as mediators of inflammation through purinergic cell surface receptor signaling. As a protective mechanism to prevent excessive damage to host tissue, adenosine counteracts ATP's effects by adenosine receptor stimulation to suppress the pro-inflammatory response. Adenosine is seen as a major obstacle to the efficacy of immune therapies, and the adenosinergic axis components are critical therapeutic targets for cancer and microbial infections. Pharmacologic inhibitors or antibodies specific to adenosinergic pathway components or adenosine receptors in microbial and tumor therapy have shown efficacy in pre-clinical studies and are entering the clinical arena. In this review, we provide a novel hypothesis explaining the potential for improving the efficiency of innate and adaptive immune systems by targeting adenosinergic pathway components and adenosine A2A receptor signaling for the treatment of COVID-19.

Plasma levels of the cardiovascular protective endogenous nucleoside adenosine are reduced in patients with primary aldosteronism without affecting ischaemia-reperfusion injury: A prospective case-control study.

BackgroundPatients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animals, mineralocorticoid receptor antagonists limit ischaemia‐reperfusion (IR) injury by increasing extracellular adenosine formation and adenosine receptor stimulation. Adenosine is an endogenous compound with profound cardiovascular protective effects. Firstly, we hypothesized that patients with PA have lower circulating adenosine levels which might contribute to the observed increased cardiovascular risk. Secondly, we hypothesized that by this mechanism, patients with PA are more susceptible to IR compared to patients with EHT.DesignIn our prospective study in 20 patients with PA and 20 patients with EHT, circulating adenosine was measured using a pharmacological blocker solution that halts adenosine metabolism after blood drawing. Brachial artery flow‐mediated dilation (FMD) before and after forearm IR was used as a well‐established method to study IR injury.ResultsPatients with PA had a 33% lower adenosine level compared to patients with EHT (15.3 [13.3‐20.4] vs 22.7 [19.4‐36.8] nmol/L, respectively, P < .01). The reduction in FMD after IR, however, did not differ between patients with PA and patients with EHT (−1.0 ± 2.9% vs −1.6 ± 1.6%, respectively, P = .52).ConclusionsAs adenosine receptor stimulation induces various powerful protective cardiovascular effects, its lower concentration in patients with PA might be an important novel mechanism that contributes to their increased cardiovascular risk. We suggest that modulation of the adenosine metabolism is an exciting novel pharmacological opportunity to limit cardiovascular risk in patients with PA that needs further exploration.

Agomelatine and tianeptine antidepressant activity in mice behavioral despair tests is enhanced by DMPX, a selective adenosine A2A receptor antagonist, but not DPCPX, a selective adenosine A1 receptor antagonist.

Adenosine, an endogenous nucleoside, modulates the release of monoamines, e.g., noradrenaline, serotonin, and dopamine in the brain. Both nonselective and selective stimulation of adenosine receptors produce symptoms of depression in some animal models. Therefore, the main objective of our study was to assess the influence of a selective adenosine A1 receptor antagonist (DPCPX) and a selective adenosine A2A receptor antagonist (DMPX) on the activity of agomelatine and tianeptine. The forced swim test (FST) and tail suspension test (TST) were performed to assess the effects of DPCPX and DMPX on the antidepressant-like activity of agomelatine and tianeptine. Drug serum and brain levels were analyzed using HPLC. Co-administration of agomelatine (20 mg/kg) or tianeptine (15 mg/kg) with DMPX (3 mg/kg), but not with DPCPX (1 mg/kg), significantly reduced the immobility time both in the FST and TST in mice. These effects were not associated with an enhancement in animals' spontaneous locomotor activity. The observed changes in the mouse behavior after concomitant injection of DMPX and the tested antidepressant agents were associated with elevated brain concentration of agomelatine and tianeptine. Our study shows a synergistic action of the selective A2A receptor antagonist and the studied antidepressant drugs, and a lack of such interaction in the case of the selective A1 receptor antagonist. The interaction between DMPX and agomelatine/tianeptine at least partly occurs in the pharmacokinetic phase. A combination of a selective A2A receptor antagonist and an antidepressant may be a new strategy for treating depression.

Enzyme activity of circulating CD73 in human serum.

CD73 is an ectonucleotidase able to catabolize 5'-adenosine monophosphate (AMP) into adenosine at the extracellular level. Extracellular adenosine plays a critical role in regulating many processes under physiological and pathological conditions. In the context of cancer, the expression and activity of CD73, either in tissue and in biological fluids, is increased leading to high levels of adenosine that potently suppress T-cell mediated responses, promoting tumor progression through stimulation of adenosine receptors. Compelling evidence indicates that elevated levels of CD73-generating adenosine limit the efficacy of cancer immunotherapy. Inhibitors of ectonucleotidases and antagonists of adenosine receptors have emerged as new therapeutic tools to improve anti-tumor immune response and potentially synergize with currently used immunotherapeutic agents. Measurement of CD73 levels in serum of cancer patients is a promising approach that, although it needs to be validated, may help to select patients who will benefit from adenosine-targeting agents and predict response to immunotherapy. Here, we describe a simple and fast method to evaluate the AMPase activity of CD73 in peripheral blood that may also be applied to other biological fluids.

Role of dopamine D2-like receptors and their modulation by adenosine receptor stimulation in the reinstatement of methamphetamine seeking.

Previous work has demonstrated that dopamine and adenosine receptors are involved in drug-seeking behaviors, yet the pharmacological interactions between these receptors in methamphetamine (MA) seeking are not well characterized. The present studies examined the role of the dopamine D2-like receptors in MA seeking and identified the interactive effects of adenosine receptor stimulation. Adult male Sprague-Dawley rats were trained to lever press for MA in daily 2-h self-administration sessions on a fixed-ratio 1 schedule for 10 consecutive days. After 1day of abstinence, lever pressing was extinguished in six daily extinction sessions. Treatments were administered systemically prior to a 2-h reinstatement test session. An increase in MA seeking was observed following the administration of the dopamine D2-like agonist, quinpirole, or the D3 receptor agonist, 7-OH-DPAT. Stimulation of D2 or D4 receptors was ineffective at inducing MA seeking. Quinpirole-induced MA seeking was inhibited by D3 receptor antagonism (SB-77011A or PG01037), an adenosine A1 agonist, CPA, and an adenosine A2A agonist, CGS 21680. MA seeking induced by a MA priming injection or D3 receptor stimulation was inhibited by a pretreatment with the adenosine A1 agonist, CPA, but not the adenosine A2A agonist, CGS 21680. These results demonstrate the sufficiency of dopamine D3 receptors to reinstate MA seeking that is inhibited when combined with adenosine A1 receptor stimulation.

Patients with primary aldosteronism have lower circulating adenosine levels but similar susceptibility to ischemia-reperfusion compared to patients with essential hypertension

Aim: Patients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animal models, aldosterone increases myocardial ischemia-reperfusion (IR) injury. Mineralocorticoid receptor antagonists limit IR-injury by increasing extracellular adenosine formation and adenosine receptor stimulation.

Activation of A2A Receptor by PDRN Reduces Neuronal Damage and Stimulates WNT/β-CATENIN Driven Neurogenesis in Spinal Cord Injury.

Spinal cord injury (SCI) is a complex clinical and progressive condition characterized by neuronal loss, axonal destruction and demyelination. In the last few years, adenosine receptors have been studied as a target for many diseases, including neurodegenerative conditions. The aim of this study was to investigate the effects of an adenosine receptor agonist, PDRN, in an experimental model of SCI. Moreover, since adenosine receptors stimulation may also activate the Wnt pathway, we wanted to study PDRN effects on Wnt signaling following SCI. Spinal trauma was induced by extradural compression of spinal cord at T5-T8 level in C57BL6/J mice. Animals were randomly assigned to the following groups: Sham (n = 10), SCI (n = 14), SCI+PDRN (8 mg/kg/i.p.; n = 14), SCI+PDRN+DMPX (8 and 10 mg/kg/i.p., respectively; n = 14). DMPX was used as an adenosine receptor antagonist to evaluate whether adenosine receptor block might prevent PDRN effects. PDRN systemically administered 1 h following SCI, protected from tissue damage, demyelination, and reduced motor deficits evaluated after 10 days. PDRN also reduced the release of the pro-inflammatory cytokines TNF-α and IL-1β, reduced BAX expression and preserved Bcl-2. Furthermore, PDRN stimulated Wnt/β-catenin pathway and decreased apoptotic process 24 h following SCI, whereas DMPX administration prevented PDRN effects on Wnt/β-catenin signaling. These results confirm PDRN anti-inflammatory activity and demonstrate that a crosstalk between Wnt/β-catenin signaling is possible by adenosine receptors activation. Moreover, these data let us hypothesize that PDRN might promote neural repair through axonal regeneration and/or neurogenesis.

Activation of Adenylyl Cyclase Causes Stimulation of Adenosine Receptors

Background/Aims: Signaling of Gs protein-coupled receptors (GsPCRs) is accomplished by stimulation of adenylyl cyclase, causing an increase of the intracellular cAMP concentration, activation of the intracellular cAMP effectors protein kinase A (PKA) and Epac, and an efflux of cAMP, the function of which is still unclear. Methods: Activation of adenylyl cyclase by GsPCR agonists or cholera toxin was monitored by measurement of the intracellular cAMP concentration by ELISA, anti-phospho-PKA substrate motif phosphorylation by immunoblotting, and an Epac-FRET assay in the presence and absence of adenosine receptor antagonists or ecto-nucleotide phosphodiesterase/pyrophosphatase2 (eNPP2) inhibitors. The production of AMP from cAMP by recombinant eNPP2 was measured by HPLC. Extracellular adenosine was determined by LC-MS/MS, extracellular ATP by luciferase and LC-MS/MS. The expression of eNPP isoenzymes 1-3 was examined by RT-PCR. The expression of multidrug resistance protein 4 was suppressed by siRNA. Results: Here we show that the activation of GsPCRs and the GsPCRs-independent activation of Gs proteins and adenylyl cyclase by cholera toxin induce stimulation of cell surface adenosine receptors (A2A or A2B adenosine receptors). In PC12 cells stimulation of adenylyl cyclase by GsPCR or cholera toxin caused activation of A2A adenosine receptors by an autocrine signaling pathway involving cAMP efflux through multidrug resistance protein 4 and hydrolysis of released cAMP to AMP by eNPP2. In contrast, in PC3 cells cholera toxin- and GsPCR-induced stimulation of adenylyl cyclase resulted in the activation of A2B adenosine receptors. Conclusion: Our findings show that stimulation of adenylyl cyclase causes a remarkable activation of cell surface adenosine receptors.

The Regulation of p27Kip-1and Bcl2 Expression Is Involved in the Decrease of Osteoclast Proliferation by A2B Adenosine Receptor Stimulation

The Regulation of p27^<i>Kip-1</i>and Bcl2 Expression Is Involved in the Decrease of Osteoclast Proliferation by A2B Adenosine Receptor Stimulation