Adenosine A1 Receptor Agonist Research Articles

Anticancer activity study of A3 adenosine receptor agonists

AimsA3 adenosine receptor (A3AR) signalling activation seems to mediate anticancer effect, and it has been targeted for drug development. The identification of potent and selective A3AR agonists could be crucial for cancer drug development. Materials and methodsIn the present study was determined the in vitro activity of known 1–3 and newly 4–6 synthesized compounds with high A3AR affinity and selectivity (Ki in the low nanomolar range) in binding studies. Effect of known and novel A3AR agonists on human prostate cancer (PC3), hepatocellular carcinoma (Hep G2), and epithelial colorectal carcinoma (Caco-2) cells were analysed by cytotoxicity assay, dose and time dependent inhibitor assay, migration, apoptosis, autophagy and reactive oxygen species (ROS) assays. Key findingsResults show that the anticancer effect is not due to A3AR activation alone. In fact, the more active and selective agonist versus A3AR, compound 1, results inactive on cancer cells such as compounds 2–4. Moreover, results show that the novel compound 5, at micromolar concentration range (IC50 = 28.0 μM), inhibits the growth of PC3, Hep G2, and Caco-2 cells and their migration in time- and dose- dependent manner. The mechanism involved in cell death is attributable to apoptosis. At the same time compound 5 promotes autophagy, which induce apoptosis producing autophagic cell death. Further investigation revealed that compound 5 elevates the level of ROS in all cancer cells tested, suggesting the involvement of ROS in cell death. SignificanceThese results show that the new compound 5 exerts inhibitory effect on cancer cells through differential effect and may serve as a potential anticancer agent.

Mechanisms of acetylcholinesterase protection against sarin and soman by adenosine A1 receptor agonist N6-cyclopentyladenosine

Organophosphorus nerve agents (NAs) irreversibly inhibit acetylcholinesterase (AChE), the enzyme responsible for breaking down the neurotransmitter acetylcholine (ACh). The over accumulation of ACh after NA exposure leads to cholinergic toxicity, seizure, and death. Current medical countermeasures effectively mitigate peripheral symptoms, however; the brain is often unprotected. Alternative acute treatment with the adenosine A1 receptor agonist N6-cyclopentyladensosine (CPA) has previously been demonstrated to prevent AChE inhibition as well as to suppress neuronal activity. The mechanism of AChE protection is unknown. To elucidate the feasibility of potential CPA-AChE interaction mechanisms, we applied a truncated molecular model approach and density functional theory. The candidate mechanisms studied are reversible enzyme inhibition, enzyme reactivation, and NA blocking prior to enzyme conjugation. Our thermodynamic data suggest that CPA can compete with the NAs sarin and soman for the active site of AChE, but may, in contrast to NAs, undergo back-reaction. We found a strong interaction between CPA and NA conjugated AChE, making enzyme reactivation unlikely but possibly allowing for CPA protection through the prevention of NA aging. The data also indicates that there is an affinity between CPA and unbound NAs. The results from this study support the hypothesis that CPA counters NA toxicity via multiple mechanisms and is a promising therapeutic strategy that warrants further development.

Effects of adenosine receptor agonist on the rocuroniuminduced neuromuscular block and sugammadex-induced recovery.

BackgroundSeveral types of receptors are found at neuromuscular presynaptic membranes. Presynaptic inhibitory A1 and facilitatory A2A receptors mediate different modulatory functions on acetylcholine release. This study investigated whether adenosine A1 receptor agonist contributes to the first twitch tension (T1) of train-of-four (TOF) stimulation depression and TOF fade during rocuronium-induced neuromuscular blockade, and sugammadex-induced recovery.MethodsPhrenic nerve-diaphragm tissues were obtained from 30 adult Sprague-Dawley rats. Each tissue specimen was randomly allocated to either control group or 2-chloroadenosine (CADO, 10 μM) group. One hour of reaction time was allowed before initiating main experimental data collection. Loading and boost doses of rocuronium were sequentially administered until > 95% depression of the T1 was achieved. After confirming that there was no T1 twitch tension response, 15 min of resting time was allowed, after which sugammadex was administered. Recovery profiles (T1, TOF ratio [TOFR], and recovery index) were collected for 1 h and compared between groups.ResultsThere were statistically significant differences on amount of rocuronium (actually used during experiment), TOFR changes during concentration-response of rocuronium (P = 0.04), and recovery profiles (P < 0.01) of CADO group comparing with the control group. However, at the initial phase of this experiment, dose-response of rocuronium in each group demonstrated no statistically significant differences (P = 0.12).ConclusionsThe adenosine A1 receptor agonist (CADO) influenced the TOFR and the recovery profile. After activating adenosine receptor, sugammadex-induced recovery from rocuronium-induced neuromuscular block was delayed.

Critical roles of TRPV2 channels, histamine H1 and adenosine A1 receptors in the initiation of acupoint signals for acupuncture analgesia

Acupuncture is one of the most promising modalities in complimentary medicine. However, the underlying mechanisms are not well understood yet. We found that in TRPV2 knockout male mice, acupuncture-induced analgesia was suppressed with a decreased activation of mast cells in the acupoints stimulated. The mast cell stabilizer sodium cromolyn could suppress the release of adenosine in the acupoints on male rats. A direct injection of adenosine A1 receptor agonist or histamine H1 receptor agonist increased β-endorphin in the cerebral-spinal fluid in the acute adjuvant arthritis male rats and thus replicated the analgesic effect of acupuncture. These observations suggest that the mast cell is the central structure of acupoints and is activated by acupuncture through TRPV2 channels. The mast cell transduces the mechanical stimuli to acupuncture signal by activating either H1 or A1 receptors, therefore triggering the acupuncture effect in the subject. These findings might open new frontiers for acupuncture research.

Role of A1 and A2B Adenosine receptors in Angiotensin II dependent hypertension in mice.

Our recent finding showed that acute angiotensin (ANG) II stimulation enhanced A1 adenosine receptor (AR) dependent cytochrome P450 (Cyp) 4A mediated contraction in mouse mesenteric arteries (MA). In addition, A2BAR dependent and partly KATP channel mediated relaxation was reduced. These data suggest a possible interaction between ANG II and ARs at a vascular level, which may play a role in ANG II dependent hypertension (Yadav et. al. Br J Pharmacol. 2015).We used ANG II (1000 ng/kg/min, 28 days) infused mouse model of hypertension in WT, A1 and A2B AR KO mice. Non‐invasive tail cuff method was used to measure mean arterial blood pressure (MABP). Aorta as a conduit and MA (second order) as a resistance artery were chosen for muscle tension studies. Protein expression in aortas and MAs were analyzed by western blot.Infusion of ANG II increased MABP in mice. Particularly, in ANG II infused WT mice, MABP (mmHg, mean ± SE) at day 0 was 77.1 ± 1.7 which increased to 119.9 ± 5.6 on day 28 (p&lt;0.05). Importantly, in A1 and A2B KO mice, increase in MABP was significantly attenuated. At day 28, in A1AR KO ANG II mice MABP was 98.5 ± 6 and in A2B KO ANG II mice was 96.3 ± 6.8.Higher expression of A1AR (~155%) and Cyp4A (~50%) was noted in hypertensive MAs compared to control. Expression of A1AR and Cyp4A was significantly increased in WT aorta of hypertensive and control mice.In MAs, CCPA‐induced contraction was reduced in hypertensive mice but NECA induced relaxation was comparable. Pinacidil (KATP channel opener) induced relaxation was significantly lower (−25% at 100 nM) in MAs of hypertensive mice. Importantly, in A1 and A2BAR KO mice MAs, pinacidil induced responses were comparable in control and ANG II groups. CCPA (A1AR agonist) ‐induced concentration (% PE) was reduced (~15% at 100 nM, p&lt;0.01) in aorta of hypertensive mice compared to control. NECA, non‐selective AR agonist, produced higher (~ −15% at 10 μM, p&lt;0.05) relaxation in aorta of hypertensive mice. Our histological analysis of aorta showed significantly reduced elastin to collagen ratio and higher media to lumen ratio suggesting vascular remodeling in ANG II hypertensive mice.In conclusion, our data revealed important roles for A1 and A2B AR dependent signaling in ANG II induced hypertension. Reduced pinacidil induced relaxation of resistance arteries may aid vascular contraction and blood pressure. Further studies involving pharmacological inhibitors and genetic models (ANGII KOs) are required to understand the interaction between ANG II and ARs at vascular level in the development of hypertension.Support or Funding InformationSupported from NIH grant HL 027339.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

REVERSAL OF PERIPHERAL AND CNS MEDIATED A1 ADENOSINE RECEPTOR HYPOTENSION

IntroductionA1 adenosine receptor (A1AR) agonists such as N6‐cyclohexyladenosine (CHA) are known for their neuroprotective and anti‐convulsant properties and for modulation of thermogenesis. We have demonstrated a neuroprotective, thermolytic effect following systemic administration of CHA combined with the peripherally acting adenosine receptor antagonist, 8‐(p‐sulfophenyl)theophylline (8‐SPT). However, A1AR‐mediated cardiovascular side effects limit the translation of this drug class, and clear understanding of the effects of this drug combination on hypotension and bradycardia are lacking. CHA delivered into the CNS increases para‐sympathetic influence on the heart to produce bradycardia similar to what is seen during onset of hibernation. Systemic administration of A1AR agonist produces bradycardia via direct negative chronotropic and inotropic effects. Evidence also supports other mechanisms of A1AR agonist modulation of blood pressure including indirect activation of peripheral β2 adrenergic receptors and central H1 receptors.HypothesisWe test the hypothesis that CHA lowers blood pressure via increased para‐sympathetic tone to the heart and direct effects on the heart.MethodsRats were implanted with telemetry transmitters to monitor Tb, heart rate (HR), mean arterial pressure (MAP) and administered drugs IP as follows, CHA (1.0mg/kg), 8‐SPT (25.0mg/kg), Atropine (1.0mg/kg), diphenhydramine (4.0mg/kg), or pro‐pranolol (4.0mg/kg), 15min prior to CHA. Cage surface temperature was initially cooled to 4°C and adjusted to maintain Tb at 32°C.Results8‐SPT with atropine reversed hypotension. 8‐SPT alone increased rate of resolution in MAP to baseline value. Pre‐treatment with propranolol, atropine, or diphenhydramine alone did not influence MAP. Diphenhydramine with 8‐SPT reduced the magnitude of hypotension (− 24% average one hour baseline change after CHA) compared to pre‐treatment with 8‐SPT (− 36%) alone.ConclusionsOur results suggest that A1AR induced hypotension is mediated through increased parasympathetic tone to the heart in addition to direct effect on the heart. Histamine receptor activation may also play a role, although the resolution of MAP seen may have been due to the anticholinergic effect of diphenhydramine instead of H1 receptor antagonism. Reversing hypotension necessitates addressing both mechanism with atropine and 8‐SPTSupport or Funding InformationNIH U54GM115371, NIH R15 NS070779, NIH: RL5GM118900, TL4 GM 18992, 1UL1GN118991This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Precise Control of Target Temperature Using N6-Cyclohexyladenosine and Real-Time Control of Surface Temperature.

Targeted temperature management is standard of care for cardiac arrest and is in clinical trials for stroke. N6-cyclohexyladenosine (CHA), an A1 adenosine receptor (A1AR) agonist, inhibits thermogenesis and induces onset of hibernation in hibernating species. Despite promising thermolytic efficacy of CHA, prior work has failed to achieve and maintain a prescribed target core body temperature (Tb) between 32°C and 34°C for 24 hours. We instrumented Sprague-Dawley rats (n = 19) with indwelling arterial and venous cannulae and a transmitter for monitoring Tb and ECG, then administered CHA via continuous IV infusion or intraperitoneal (IP) injection. In the first experiment (n = 11), we modulated ambient temperature and increased the dose of CHA in an attempt to manage Tb. In the second experiment (n = 8), we administered CHA (0.25 mg/[kg·h]) via continuous IV infusion and modulated cage surface temperature to control Tb. We rewarmed animals by increasing surface temperature at 1°C h-1 and discontinued CHA after Tb reached 36.5°C. Tb, brain temperature (Tbrain), heart rate, blood gas, and electrolytes were also monitored. Results show that titrating dose to adjust for individual variation in response to CHA led to tolerance and failed to manage a prescribed Tb. Starting with a dose (0.25 mg/[kg·h]) and modulating surface temperature to prevent overcooling proved to be an effective means to achieve and maintain Tb between 32°C and 34°C for 24 hours. Increasing surface temperature to 37°C during CHA administration brought Tb back to normothermic levels. All animals treated in this way rewarmed without incident. During the initiation of cooling, we observed bradycardia within 30 minutes of the start of IV infusion, transient hyperglycemia, and a mild hypercapnia; the latter normalized via metabolic compensation. In conclusion, we describe an intravenous delivery protocol for CHA at 0.25 mg/(kg·h) that, when coupled with conductive cooling, achieves and maintains a prescribed and consistent target Tb between 32°C and 34°C for 24 hours.

Activation of A1 and A2a adenosine receptors promotes neural progenitor cell proliferation

Neural progenitor cells (NPCs) play a key role not only in the maintenance of the adult central nervous system (CNS) but also in the ability to recover from injury and disease. In this study, we established a 96-well-based screening system to screen small molecules modulating the proliferation of NPCs. A compound library composed of 1280 compounds was screened. We found that the A1 adenosine receptor agonist cyclopentyladenosine (CPA) and the A2a adenosine receptor agonist CGS-21680 increased proliferation of NPCs. The A1 adenosine receptor agonist-induced cell proliferation was attenuated by A1 adenosine receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). Accordingly, the A2a adenosine receptor agonist-induced cell proliferation was attenuated by A2a adenosine receptor antagonist SCH-58261. Further study indicated that CPA and CGS-21680 treatment induced phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, and CPA-induced or CGS-21680-induced cell proliferation was inhibited by ERK and Akt inhibitors. These results suggested that the activation of A1 and A2a adenosine receptor stimulated the proliferation of NPCs via the ERK and Akt signaling pathways.

Involvement of orexin neurons in fasting- and central adenosine-induced hypothermia

We examined whether orexin neurons might play a protective role against fasting- and adenosine-induced hypothermia. We first measured body temperature (BT) in orexin neuron-ablated (ORX-AB) mice and wild-type (WT) controls during 24 hours of fasting. As expected, the magnitude of BT drop and the length of time suffering from hypothermia were greater in ORX-AB mice than in WT mice. Orexin neurons were active just before onset of hypothermia and during the recovery period as revealed by calcium imaging in vivo using G-CaMP. We next examined adenosine-induced hypothermia via an intracerebroventricular administration of an adenosine A1 receptor agonist, N6-cyclohexyladenosine (CHA), which induced hypothermia in both ORX-AB and WT mice. The dose of CHA required to initiate a hypothermic response in ORX-AB mice was more than 10 times larger than the dose for WT mice. Once hypothermia was established, the recovery was seemingly slower in ORX-AB mice. Activation of orexin neurons during the recovery phase was confirmed by immunohistochemistry for c-Fos. We propose that orexin neurons play dual roles (enhancer in the induction phase and compensator during the recovery phase) in adenosine-induced hypothermia and a protective/compensatory role in fasting-induced hypothermia.

Adenosine Receptor Ligands on Cancer Therapy: A Review of Patent Literature.

Adenosine is a purine, with an adenine group and a ribose sugar, formed endogenously by ATP catabolism both intracellularly and extracellularly. Among the medicinal features of adenosine and its receptors (A1, A2A, A2B and A3), anticancer activity has been an intense field of research. The anticancer potential of adenosine receptor ligands has been brought to the forefront of research and evidenced in innumerous research articles and patents. The present review focuses on the patent literature from 2002 onwards (2002-May 2017). Patents were searched and downloaded from the open access patent data bases and are available online. A significant number of patents (65) have been published on adenosine receptor ligands claiming anticancer activity, or presenting new methods of preparation or treatment thereof, from 2002-2017 (May). From these, 35 were published highlighting the promising attributes of compounds/ methods to fight cancer. Most of the compounds act as adenosine A3 receptor agonists, while others act as antagonists for the other adenosine receptor subtypes. The signaling events triggered by activation of adenosine A3 receptor or by blockade of adenosine A1, A2A and A2B receptors can reverse an environment from being pro-cancer to an anti-cancer in the body. The promising anticancer effects mediated by adenosine receptor ligands put them in the forefront as new drug candidates. The present compilation can be worthy to medicinal chemists, pharmacologists, biochemists and other researchers focusing on the putative anticancer activity of adenosine receptor ligands.

Pivotal Role of Adenosine Neurotransmission in Restless Legs Syndrome.

The symptomatology of Restless Legs Syndrome (RLS) includes periodic leg movements during sleep (PLMS), dysesthesias, and hyperarousal. Alterations in the dopaminergic system, a presynaptic hyperdopaminergic state, seem to be involved in PLMS, while alterations in glutamatergic neurotransmission, a presynaptic hyperglutamatergic state, seem to be involved in hyperarousal and also PLMS. Brain iron deficiency (BID) is well-recognized as a main initial pathophysiological mechanism of RLS. BID in rodents have provided a pathogenetic model of RLS that recapitulates the biochemical alterations of the dopaminergic system of RLS, although without PLMS-like motor abnormalities. On the other hand, BID in rodents reproduces the circadian sleep architecture of RLS, indicating the model could provide clues for the hyperglutamatergic state in RLS. We recently showed that BID in rodents is associated with changes in adenosinergic transmission, with downregulation of adenosine A1 receptors (A1R) as the most sensitive biochemical finding. It was hypothesized that A1R downregulation leads to hypersensitive striatal glutamatergic terminals and facilitation of striatal dopamine release. Hypersensitivity of striatal glutamatergic terminals was demonstrated by an optogenetic-microdialysis approach in the rodent with BID, indicating that it could represent a main pathogenetic factor that leads to PLMS in RLS. In fact, the dopaminergic agonists pramipexole and ropinirole and the α2δ ligand gabapentin, used in the initial symptomatic treatment of RLS, completely counteracted optogenetically-induced glutamate release from both normal and BID-induced hypersensitive corticostriatal glutamatergic terminals. It is a main tenet of this essay that, in RLS, a single alteration in the adenosinergic system, downregulation of A1R, disrupts the adenosine-dopamine-glutamate balance uniquely controlled by adenosine and dopamine receptor heteromers in the striatum and also the A1R-mediated inhibitory control of glutamatergic neurotransmission in the cortex and other non-striatal brain areas, which altogether determine both PLMS and hyperarousal. Since A1R agonists would be associated with severe cardiovascular effects, it was hypothesized that inhibitors of nucleoside equilibrative transporters, such as dipyridamole, by increasing the tonic A1R activation mediated by endogenous adenosine, could represent a new alternative therapeutic strategy for RLS. In fact, preliminary clinical data indicate that dipyridamole can significantly improve the symptomatology of RLS.

A binding kinetics study of human adenosine A3 receptor agonists

The human adenosine A3 (hA3) receptor has been suggested as a viable drug target in inflammatory diseases and in cancer. So far, a number of selective hA3 receptor agonists (e.g. IB-MECA and 2-Cl-IB-MECA) inducing anti-inflammatory or anticancer effects are under clinical investigation. Drug-target binding kinetics is increasingly recognized as another pharmacological parameter, next to affinity, for compound triage in the early phases of drug discovery. However, such a kinetics-driven analysis has not yet been performed for the hA3 receptor. In this study, we first validated a competition association assay for adenosine A3 receptor agonists to determine the target interaction kinetics. Affinities and Kinetic Rate Index (KRI) values of 11 ribofurano and 10 methanocarba nucleosides were determined in radioligand binding assays. Afterwards, 15 analogues were further selected (KRI <0.70 or KRI >1.35) for full kinetics characterization. The structure-kinetics relationships (SKR) were derived and longer residence times were associated with methanocarba and enlarged adenine N6 and C2 substitutions. In addition, from a kon-koff-KD kinetic map we divided the agonists into three subgroups. A residence time “cliff” was observed, which might be relevant to (N)-methanocarba derivatives’ rigid C2-arylalkynyl substitutions. Our findings provide substantial evidence that, next to affinity, additional knowledge of binding kinetics is useful for developing and selecting new hA3R agonists in the early phase of the drug discovery process.

Structure-Based Design, Synthesis, and In Vivo Antinociceptive Effects of Selective A1 Adenosine Receptor Agonists.

Our previous work discovered that combining the appropriate 5'- and N6-substitution in adenosine derivatives leads to the highly selective human A1 adenosine receptor (hA1AR) agonists or highly potent dual hA1AR agonists and hA3AR antagonists. In order to explore novel dual adenosine receptor ligands, a series of N6-substituted-5'-pyrazolyl-adenosine and 2-chloro-adenosine derivatives were synthesized and assayed in vitro at all ARs. The N6-(±)-endo-norbornyl derivative 12 was the most potent and selective at A1AR and effective as an analgesic in formalin test in mice, but none of the 5'-pyrazolyl series compounds showed a dual behavior at hA1 and hA3AR. Molecular modeling studies rationalized the structure-activity relationships and the selectivity profiles of the new series of A1AR agonists. Interestingly, an unexpected inverted binding mode of the N6-tetrahydrofuranyl derivative 14 was hypothesized to explain its low affinity at A1AR.

Inhibition of IL-17 and IL-23 in Human Keratinocytes by the A3 Adenosine Receptor Agonist Piclidenoson.

Interleukin-17 and interleukin-23 play major roles in the inflammatory process in psoriasis. The Gi protein-associated A3 adenosine receptor (A3AR) is known to be overexpressed in inflammatory cells and in peripheral blood mononuclear cells (PBMCs) of patients with autoimmune inflammatory conditions. Piclidenoson, a selective agonist at the A3AR, induces robust anti-inflammatory effect in psoriasis patients. In this study, we aimed to explore A3AR expression levels in psoriasis patients and its role in mediating the anti-inflammatory effect of piclidenoson in human keratinocyte cells. A3AR expression levels were evaluated in skin tissue and PBMCs derived from psoriasis patients and healthy subjects. Proliferation assay and the expression of signaling proteins were used to evaluate piclidenoson effect on human keratinocytes (HaCat). High A3AR expression levels were found in a skin biopsy and in PBMCs from psoriasis patients in comparison to healthy subjects. Piclidenoson inhibited the proliferation of HaCat cells through deregulation of the NF-κB signaling pathway, leading to a decrease in interleukin-17 and interleukin-23 expression levels. This effect was counteracted by the specific antagonist MRS 1523. A3AR overexpression in skin and PBMCs of psoriasis patients may be used as a target to inhibit pathological cell proliferation and the production of interleukin-17 and interleukin-23.

Role of transglutaminase 2 in A1 adenosine receptor- and β2-adrenoceptor-mediated pharmacological pre- and post-conditioning against hypoxia-reoxygenation-induced cell death in H9c2 cells

Pharmacologically-induced pre- and post-conditioning represent attractive therapeutic strategies to reduce ischaemia/reperfusion injury during cardiac surgery and following myocardial infarction. We have previously reported that transglutaminase 2 (TG2) activity is modulated by the A1 adenosine receptor and β2-adrenoceptor in H9c2 cardiomyoblasts. The primary aim of this study was to determine the role of TG2 in A1 adenosine receptor and β2-adrenoceptor-induced pharmacological pre- and post-conditioning in the H9c2 cells. H9c2 cells were exposed to 8h hypoxia (1% O2) followed by 18h reoxygenation, after which cell viability was assessed by monitoring mitochondrial reduction of MTT, lactate dehydrogenase release and caspase-3 activation. N6-cyclopentyladenosine (CPA; A1 adenosine receptor agonist), formoterol (β2-adrenoceptor agonist) or isoprenaline (non-selective β-adrenoceptor agonist) were added before hypoxia/reoxygenation (pre-conditioning) or at the start of reoxygenation following hypoxia (post-conditioning). Pharmacological pre- and post-conditioning with CPA and isoprenaline significantly reduced hypoxia/reoxygenation-induced cell death. In contrast, formoterol did not elicit protection. Pre-treatment with pertussis toxin (Gi/o-protein inhibitor), DPCPX (A1 adenosine receptor antagonist) or TG2 inhibitors (Z-DON and R283) attenuated the A1 adenosine receptor-induced pharmacological pre- and post-conditioning. Similarly, pertussis toxin, ICI 118,551 (β2-adrenoceptor antagonist) or TG2 inhibition attenuated the isoprenaline-induced cell survival. Knockdown of TG2 using small interfering RNA (siRNA) attenuated CPA and isoprenaline-induced pharmacological pre- and post-conditioning. Finally, proteomic analysis following isoprenaline treatment identified known (e.g. protein S100-A6) and novel (e.g. adenine phosphoribosyltransferase) protein substrates for TG2. These results have shown that A1 adenosine receptor and β2-adrenoceptor-induced protection against simulated hypoxia/reoxygenation occurs in a TG2 and Gi/o-protein dependent manner in H9c2 cardiomyoblasts.

Pharmacologically induced reversible hypometabolic state mitigates radiation induced lethality in mice

Therapeutic hypothermia has proven benefits in critical care of a number of diseased states, where inflammation and oxidative stress are the key players. Here, we report that adenosine monophosphate (AMP) triggered hypometabolic state (HMS), 1–3 hours after lethal total body irradiation (TBI) for a duration of 6 hours, rescue mice from radiation-induced lethality and this effect is mediated by the persistent hypothermia. Studies with caffeine and 6N-cyclohexyladenosine, a non-selective antagonist and a selective agonist of adenosine A1 receptor (A1AR) respectively, indicated the involvement of adenosine receptor (AR) signaling. Intracerebroventricular injection of AMP also suggested possible involvement of central activation of AR signaling. AMP, induced HMS in a strain and age independent fashion and did not affect the behavioural and reproductive capacities. AMP induced HMS, mitigated radiation-induced oxidative DNA damage and loss of HSPCs. The increase in IL-6 and IL-10 levels and a shift towards anti-inflammatory milieu during the first 3–4 hours seems to be responsible for the augmented survival of HSPCs. The syngeneic bone marrow transplantation (BMT) studies further supported the role of radiation-induced inflammation in loss of bone marrow cellularity after TBI. We also showed that the clinically plausible mild hypothermia effectively mitigates TBI induced lethality in mice.

Extracellular 5'-nucleotidase (CD73) promotes human breast cancer cells growth through AKT/GSK-3β/β-catenin/cyclinD1 signaling pathway.

To identify the role and to explore the mechanism of extracellular 5'-nucleotidase (CD73) in human breast cancer growth, CD73 expression was measured firstly in breast cancer tissues and cell lines, and then interfered with or over-expressed by recombinant lentivirus in cell lines. Impacts of CD73 on breast cancer cell proliferation and cell cycle were investigated with colony formation assay, CCK-8 and flow cytometry. The relationship between CD73 and AKT/GSK-3β/β-catenin pathway was assessed with adenosine, adenosine 2A receptor antagonist (SCH-58261), adenosine 2A receptor agonist (NECA), CD73 enzyme inhibitor (APCP) and Akt inhibitor (MK-2206). Moreover, the effect of CD73 on breast cancer growth in vivo was examined with human breast cancer transplanting model of nude mice. The results showed that the expression of CD73 was high in breast cancer tissues and increased with advanced tumor grades and lympho-node status. CD73 expression was higher in more malignant cells, and CD73 overexpression promoted breast cancer cell proliferation in both in vivo and in vitro. It activated AKT/GSK-3β/β-catenin/cyclinD1 signaling pathway through CD73 enzyme activity and other mechanism.

Cardiac arrest induced by muscarinic or adenosine receptors agonists is reversed by DPCPX through double mechanism

In the right atrium (RA), adenosine and acetylcholine inhibit the pacemaker function of the sinoatrial node and induce cardiac arrest. Pre-incubation of receptor antagonists is known to inhibit the cardiac arrest induced by these agonists; however, the effect of antagonist administration after established cardiac arrest has not been described. Therefore, we assessed whether specific receptor antagonists could revert cardiac arrest induced by adenosine and muscarinic receptors activation. RA isolated from adults Wistar rats were mounted in an organ bath containing Krebs solution. Cardiac arrest was induced by adenosine or ATP (1mM), the A1 adenosine receptor agonist CPA (0.1–1µM), and muscarinic receptor agonists, carbachol (0.3–1µM) and acetylcholine (1mM). After establishing the cardiac arrest, the A1 adenosine receptor antagonist DPCPX (0.3–30µM), the muscarinic receptor antagonist atropine (10nM to 100µM) or the phosphodiesterase inhibitor IBMX (10–300µM) were incubated in order to check for the return of spontaneous contractions. DPCPX reversed the cardiac arrest induced by adenosine, ATP and CPA. In addition, atropine reversed the cardiac arrest induced by carbachol. Unexpectedly, DPCPX also reversed the cardiac arrest induced by carbachol. Similarly to DPCPX, the phosphodiesterase inhibitor IBMX reversed the cardiac arrest induced by adenosine, CPA and carbachol. The antagonism of adenosine and acetylcholine receptors activation, as well as phosphodiesterase inhibition, are able to revert cardiac arrest. DPCPX restore spontaneous contractions via the selective antagonism of A1 adenosine receptor and through a secondary mechanism likely related to phosphodiesterase inhibition.

Adenosine Receptor Stimulation Improves Glucocorticoid-Induced Osteoporosis in a Rat Model.

Glucocorticoid-induced osteoporosis (GIO) is a secondary cause of bone loss. Bisphosphonates approved for GIO, might induce jaw osteonecrosis; thus additional therapeutics are required. Adenosine receptor agonists are positive regulators of bone remodeling, thus the efficacy of adenosine receptor stimulation for treating GIO was tested. In a preventive study GIO was induced in Sprague-Dawley rats by methylprednisolone (MP) for 60 days. Animals were randomly assigned to receive polydeoxyribonucleotide (PDRN), an adenosine A2 receptor agonist, or PDRN and DMPX (3,7-dimethyl-1-propargylxanthine, an A2 antagonist), or vehicle (0.9% NaCl). Another set of animals was used for a treatment study, following the 60 days of MP-induction rats were randomized to receive (for additional 60 days) PDRN, or PDRN and DMPX (an adenosine A2 receptor antagonist), or zoledronate (as control for gold standard treatment), or vehicle. Control animals were administered with vehicle for either 60 or 120 days. Femurs were analyzed after treatments for histology, imaging, and breaking strength analysis. MP treatment induced severe bone loss, the concomitant use of PDRN prevented the developing of osteoporosis. In rats treated for 120 days, PDRN restored bone architecture and bone strength; increased b-ALP, osteocalcin, osteoprotegerin and stimulated the Wnt canonical and non-canonical pathway. Zoledronate reduced bone resorption and ameliorated the histological features, without significant effects on bone formation. Our results suggest that adenosine receptor stimulation might be useful for preventing and treating GIO.

Polypharmacology of N6-(3-Iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and Related A3 Adenosine Receptor Ligands: Peroxisome Proliferator Activated Receptor (PPAR) γ Partial Agonist and PPARδ Antagonist Activity Suggests Their Antidiabetic Potential.

A3 adenosine receptor (AR) ligands including A3 AR agonist, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (1a, IB-MECA) were examined for adiponectin production in human bone marrow mesenchymal stem cells (hBM-MSCs). In this model, 1a significantly increased adiponectin production, which is associated with improved insulin sensitivity. However, A3 AR antagonists also promoted adiponectin production in hBM-MSCs, indicating that the A3 AR pathway may not be directly involved in the adiponectin promoting activity. In a target deconvolution study, their adiponectin-promoting activity was significantly correlated to their binding activity to both peroxisome proliferator activated receptor (PPAR) γ and PPARδ. They functioned as both PPARγ partial agonists and PPARδ antagonists. In the diabetic mouse model, 1a and its structural analogues A3 AR antagonists significantly decreased the serum levels of glucose and triglyceride, supporting their antidiabetic potential. These findings indicate that the polypharmacophore of these compounds may provide therapeutic insight into their multipotent efficacy against various human diseases.