Adenosine Receptor Activation Research Articles

Abstract P08: Adenosine-based cancer immunotherapy for COVID-19

Abstract Cancer immunotherapy has shaped the way in which we design cancer treatments, introducing the paradigm of taking advantage of our immune system to fight cancer. We propose that the same concept could be applied to infectious diseases and, especially, to those that hamper the immune system like COVID-19. It is well known that an adaptive immune response is able to eradicate viral infections and CD8+ T-cells are key in such anti-viral response. Furthermore, several studies reported that the number of CD8+ T-cells is reduced in COVID-19 patients since the beginning of SARS-CoV-2 infection and further decreased in severe cases. CD8+ T-cells often show signs of exhaustion, loss of T-cell functions and suppression in COVID-19 patients, suggesting that hampering CD8+ T-cells could be a way by which SARS-CoV-2 infection progresses. Importantly, the number of CD8+ T cells appears to re-increase in patients that are recovering from COVID-19, suggesting that CD8+ T-cells could be a key factor that determines whether our body can recover from the disease. We propose that therapies that boost CD8+ T cells could be effective in clearing SARS-CoV-2 infection in COVID-19. To test this, we will take advantage of the adenosine-mediated immunomodulation. Adenosine, an ATP metabolite that is produced during inflammation, hypoxia and in the tumor microenvironment, was found to suppress the immune response through activation of adenosine receptors present on immune cells. Small molecules antagonists that block one of these receptors, adenosine A2A receptor (A2AR) antagonists, are currently being studied to boost anti-cancer T-cell mediated immune responses. Our data show that treatment with an A2AR antagonist restores and stabilizes Notch1, a key pathway for T-cell functions, along with production of INF-gamma and Granzyme B and proliferation in CD8+ T-cells. As a proof of concept, our data indicates that treatment with an A2AR antagonist increases CD8+ T-cells anti-cancer response in tumor-derived organoids, suggesting that the treatment boosts CD8+ T-cells-mediated immune response. Ongoing work aims to test whether the A2AR antagonist treatment restores several parameters of T-cell function that are specifically modified in COVID-19. This analysis will help to predict the action of the A2AR antagonist on T-cells in vivo and we ultimately aim to test this treatment in a mouse model for COVID-19. Overall, our work could introduce a new paradigm and new therapies for COVID-19 and other infectious diseases. Citation Format: Giulia Monticone, Alistair Ramsay, Lucio Miele. Adenosine-based cancer immunotherapy for COVID-19 [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P08.

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B.

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Seasonal changes in adenosine kinase in tanycytes of the Arctic ground squirrel (Urocitellus parryii)

Hibernation is a seasonal strategy to conserve energy, characterized by modified thermoregulation, an increase in sleep pressure and drastic metabolic changes. Glial cells such as astrocytes and tanycytes are the brain metabolic sensors, but it remains unknown whether they contribute to seasonal expression of hibernation. The onset of hibernation is controlled by an undefined endogenous circannual rhythm in which adenosine plays a role through the activation of the A1 adenosine receptor (A1AR). Seasonal changes in brain levels of adenosine may contribute to an increase in A1AR sensitivity leading to the onset of hibernation. The primary regulator of extracellular adenosine concentration is adenosine kinase, which is located in astrocytes. Using immunohistochemistry to localize and quantify adenosine kinase in Arctic ground squirrels’ brain collected during different seasons, we report lower expression of adenosine kinase in the third ventricle tanycytes in winter compared to summer; a similar change was not seen in astrocytes. Moreover, for the first time, we describe adenosine kinase expression in tanycyte cell bodies in the hypothalamus and in the area postrema, both brain regions involved in energy homeostasis. Next we describe seasonal changes in tanycyte morphology in the hypothalamus. Although still speculative, our findings contribute to a model whereby adenosine kinase in tanycytes regulates seasonal changes in extracellular concentration of adenosine underling the seasonal expression of hibernation.

IFN-γ and CD38 in Hyperprogressive Cancer Development.

Simple SummaryHyperprogressive disease (HPD) is a pattern of paradoxical tumor progression that has been reported in patients treated with immune checkpoint inhibitors (ICIs). Although a large number of studies have investigated HPD and several associated factors have been reported, the mechanisms that drive the acceleration of tumor growth remain unknown. In this review, we discuss the possible role of IFN-γ and CD38 in the development of HPD, and we report the main findings in the scientific literature on the signaling pathways in which these factors take part, and their involvement in response and resistance to ICI therapy.Immune checkpoint inhibitors (ICIs) improve the survival of patients with multiple types of cancer. However, low response rates and atypical responses limit their success in clinical applications. The paradoxical acceleration of tumor growth after treatment, defined as hyperprogressive disease (HPD), is the most difficult problem facing clinicians and patients alike. The mechanisms that underlie hyperprogression (HP) are still unclear and controversial, although different factors are associated with the phenomenon. In this review, we propose two factors that have not yet been demonstrated to be directly associated with HP, but upon which it is important to focus attention. IFN-γ is a key cytokine in antitumor response and its levels increase during ICI therapy, whereas CD38 is an alternative immune checkpoint that is involved in immunosuppressive responses. As both factors are associated with resistance to ICI therapy, we have discussed their possible involvement in HPD with the conclusion that IFN-γ may contribute to HP onset through the activation of the inflammasome pathway, immunosuppressive enzyme IDO1 and activation-induced cell death (AICD) in effector T cells, while the role of CD38 in HP may be associated with the activation of adenosine receptors, hypoxia pathways and AICD-dependent T-cell depletion.

A Circadian Clock in the Retina Regulates Rod-Cone Gap Junction Coupling and Neuronal Light Responses via Activation of Adenosine A2A Receptors.

Adenosine, a major neuromodulator in the central nervous system (CNS), is involved in a variety of regulatory functions such as the sleep/wake cycle. Because exogenous adenosine displays dark- and night-mimicking effects in the vertebrate retina, we tested the hypothesis that a circadian (24 h) clock in the retina uses adenosine to control neuronal light responses and information processing. Using a variety of techniques in the intact goldfish retina including measurements of adenosine overflow and content, tracer labeling, and electrical recording of the light responses of cone photoreceptor cells and cone horizontal cells (cHCs), which are post-synaptic to cones, we demonstrate that a circadian clock in the retina itself—but not activation of melatonin or dopamine receptors—controls extracellular and intracellular adenosine levels so that they are highest during the subjective night. Moreover, the results show that the clock increases extracellular adenosine at night by enhancing adenosine content so that inward adenosine transport ceases. Also, we report that circadian clock control of endogenous cone adenosine A2A receptor activation increases rod-cone gap junction coupling and rod input to cones and cHCs at night. These results demonstrate that adenosine and A2A receptor activity are controlled by a circadian clock in the retina, and are used by the clock to modulate rod-cone electrical synapses and the sensitivity of cones and cHCs to very dim light stimuli. Moreover, the adenosine system represents a separate circadian-controlled pathway in the retina that is independent of the melatonin/dopamine pathway but which nevertheless acts in concert to enhance the day/night difference in rod-cone coupling.

Ticagrelor alleviates sepsis-induced myocardial injury via an adenosine-dependent pathway in a mouse sepsis model.

The purpose of this study was to determine whether ticagrelor, a classic anti-platelet drug, has a therapeutic effect on sepsis-induced myocardial injury. The C57BL6J mice received oral ticagrelor (10, 25 and 50 mg/kg) for seven days after which cecum ligation and puncture (CLP) were performed. An adenosine-receptor antagonist (CGS15943) was administered two hours before CLP. After 24 h, cardiac function was measured using cardiac echocardiography, then the heart and blood were collected. Hematoxylin and eosin (HE) staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL staining) were used to observe pathological changes and cardiomyocyte apoptosis. Plasma concentration of TNF-α, IL-6 and adenosine and myocardial tissue levels of TNF-α and IL-6 were determined. Survival analysis was performed. Western blot was used to determine the expression of a signalling protein in the myocardial tissue. The HE and TUNEL staining showed less inflammatory cell infiltration and less cardiomyocyte apoptosis in the ticagrelor group. Cardiac echocardiography showed preserved heart function in the ticagrelor group. Plasma TNF-α, IL-6 and relative expression of TNF-α and IL-6 in myocardial tissue were significantly lower in the ticagrelor group. Plasma adenosine levels were significantly higher in the ticagrelor group. Adenosine-receptor antagonists significantly blocked the protective effect of ticagrelor. Ticagrelor reduced the mortality of sepsis mice, and this reduction was blocked by the adenosine-receptor antagonist. Western blot showed that ticagrelor activated the phosphorylation of AKT and mTOR. Adenosine-receptor antagonists inhibited the activation of AKT and mTOR. The protective effect of ticagrelor was dependent on adenosine-receptor activation, with downstream upregulation of phosphorylation of AKT and mTOR.

Activation of adenosine A3 receptor reduces early brain injury by alleviating neuroinflammation after subarachnoid hemorrhage in elderly rats.

The incidence of subarachnoid hemorrhage (SAH) and hazard ratio of death increase with age. Overactivation of microglia contributes to brain damage. This study aimed to investigate the effects of A3 adenosine receptors (A3R) activation on neurofunction and microglial phenotype polarization in the context of SAH in aged rats. The A3R agonist (CI-IB-MECA) and antagonist (MRS1523) were used in the SAH model. Microglia were cultured to mimic SAH in the presence or absence of CI-IB-MECA and/or siRNA for A3R. The neurofunction and status of the microglial phenotype were evaluated. The P38 inhibitor SB202190 and the STAT6 inhibitor AS1517499 were used to explore the signaling pathway. The results showed that SAH induced microglia to polarize to the M(LPS) phenotype both in vivo and in vitro. CI-IB-MECA distinctly skewed microglia towards the M(IL-4) phenotype and ameliorated neurological dysfunction, along with the downregulation of inflammatory cytokines. Knockdown of A3R or inhibition of P38 and/or STAT6 weakened the effects of CI-IB-MECA on microglial phenotypic shifting. Collectively, our findings suggest that activation of A3R exerted anti-inflammatory and neuroprotective effects by regulating microglial phenotype polarization through P38/STAT6 pathway and indicated that A3R agonists may be a promising therapeutic options for the treatment of brain injury after SAH.

Abstract PR17: Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis

Abstract African-American (AA) men have a 60% higher incidence of prostate cancer compared to European-American (EA) men and tend to have a more aggressive clinical outcome. An understanding of the altered biological responses and the factors that lead to health disparity in prostate cancer development and progression is unclear. Metabolism defines the physiological state of the cell and metabolic reprogramming is a hallmark of cancer. Thus, studying the metabolic landscape will be critical for understanding the biological changes that might contribute to this disparity. Analysis of 190 metabolites across prostate cancer/benign adjacent tissue pairs from ancestry typed AA and EA men performed in our laboratory, revealed altered levels of nucleosides adenosine and inosine. High inosine to adenosine ratio is observed in AA men compared to EA men. In line with this finding, the enzyme adenosine deaminase (ADA), which converts adenosine to inosine was found elevated in AA men, potentially explaining the high inosine to adenosine ratio in AA PCa. The consequences of the accumulation of these metabolites on AA PCa progression are unknown. The current study will address the knowledge gap on the consequences of elevated ADA activity in AA PCa and attempt to dissect its role in effecting an aggressive phenotype in PCa. To determine the role of elevated ADA in PCa, it was overexpressed in ancestry-typed AA (MDA-PCa-2A) and EA PCa(LNCaP) cell lines (termed ADA OE). Phenotypic examination of these cells revealed increased ADA enzyme activity decreases the cell-ECM adhesion. This adhesion decrease is facilitated by a decrease in cell adhesion protein integrin β1 (ITGB1). Elevated ADA enzyme activity decreases cAMP which mediates this adhesion decrease by disrupting the Epac-Rap1 pathway. It is postulated that high inosine upon ADA OE activates adenosine receptors A1 and A3, which causes this decrease in cAMP levels. These molecular findings suggest that ADA-mediated adhesion decrease could facilitate metastatic dissemination from the primary tumor. From a translational viewpoint, ADA enzyme activity could serve as a biomarker for metastatic prostate cancer. Therefore, we further look to analyze the inosine to adenosine levels (ADA enzyme activity) in patient samples and correlate with various clinical outcomes in PCa. Our initial analysis reveals high inosine to adenosine ratio in biopsy positive patient samples compared to biopsy negative samples. Our next steps of analysis include correlating the enzyme activity with (i) Biochemical Recurrence (ii) Time to attaining castration resistance in castration sensitive patients (iii) Time to metastasis (iv) Response to Androgen deprivation in patients after BCr. Based on our current findings, we expect inosine to adenosine ratio in plasma or urine to serve as a predictive marker for PCa incidence or progression in AA men. In addition, we expect ADA to serve as a potential therapeutic target for AA PCa. Citation Format: Christy Charles, Jie Gohlke, Stacy Lloyd, Uttam Rasaily, James Henderson, Balasubramanyam Karanam, Brian Simons, Nora Navone, Rick Kittles, Stefan Ambs, George Michailidis, Nagireddy Putluri, Arun Sreekumar. Metabolic re-wiring in African-American prostate cancer: A role of adenosine-inosine axis [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PR17.

“Adenosine an old player with new possibilities in kidney diseases”: Preclinical evidences and clinical perspectives

Renal injury might originate from multiple factors like ischemia reperfusion (I/R), drug toxicity, cystic fibrosis, radio contrast agent etc. The four adenosine receptor subtypes have been identified and found to show diverse physiological and pathological roles in kidney diseases. The activation of A1 adenosine receptor (A1) protects against acute kidney injury by improving renal hemodynamic alterations, decreasing tubular necrosis and its inhibition might facilitate removal of toxin or drug metabolite in chronic kidney disease models. Furthermore, recent findings revealed that A2A receptor subtype activation regulates macrophage phenotype in experimental models of nephritis. Interestingly the emerging role of adenosine kinase inhibitors in kidney diseases has been discussed which act by increasing adenosine availability at target sites and thereby promote A2A receptor stimulation. In addition, the least explored adenosine receptor subtype A3 inhibition was observed to exert anti- oxidant, immunosuppressive and anti-fibrotic effects, but more studies are required to confirm its benefits in other renal injury models. The clinical studies targeting A1 receptor in patients with pre-existing kidney disease have yielded disappointing results, perhaps owing to the origin of unexpected neurological complications during the course of trial. Importantly, conducting well designed clinical trials and testing adenosine modulators with lesser brain penetrability could clear the way for clinical approval of these agents for patients with renal functional impairments.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNβ expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.

Advantages in Wound Healing Process in Female Mice Require Upregulation A2A-Mediated Angiogenesis under the Stimulation of 17β-Estradiol.

Estrogenic steroids and adenosine A2A receptors promote the wound healing and angiogenesis processes. However, so far, it is unclear whether estrogen may regulate the expression and pro-angiogenic activity of A2A receptors. Using in vivo analyses, we showed that female wild type (WT) mice have a more rapid wound healing process than female or male A2A-deficient mice (A2AKO) mice. We also found that pulmonary endothelial cells (mPEC) isolated from female WT mice showed higher expression of A2A receptor than mPEC from male WT mice. mPEC from female WT mice were more sensitive to A2A-mediated pro-angiogenic response, suggesting an ER and A2A crosstalk, which was confirmed using cells isolated from A2AKO. In those female cells, 17β-estradiol potentiated A2A-mediated cell proliferation, an effect that was inhibited by selective antagonists of estrogen receptors (ER), ERα, and ERβ. Therefore, estrogen regulates the expression and/or pro-angiogenic activity of A2A adenosine receptors, likely involving activation of ERα and ERβ receptors. Sexual dimorphism in wound healing observed in the A2AKO mice process reinforces the functional crosstalk between ER and A2A receptors.

Effects of Adenosine A2A Receptor Agonist on Histopathology of Some Oral Tissues in Rabbits

Adenosine is a protective regulator that act endogenously to restore equilibrium of cellular energy in response to tissue trauma. It can perform such function of different systems in the body by activation of adenosine receptors. Study the effects of systemic administration of the adenosine on tongue and salivary glands tissues in the rabbit model. Thirty male rabbits of body weight of 1.5 ± 0.25kg were included in the study. In control group (15 animals), one ml of distilled water was injected intraperitoneally while in treatment group (15 animals) were injected by adenosine intraperitoneally at a dose of one mg/ml, All animals were sacrificed after 30 days. Serum samples were separated and used for analysis of adenosine deaminase (ADA)and glutathione(GSH). Tissue samples sections from tongue and salivary glands were stained with hematoxylin-eosin (H&E) and examined under a light microscope for histological changes by a blinded pathologist. Histological sections in treatment group showed congestion of blood vessels and infiltration of inflammatory cells with mild hemorrhage among acini of salivary glands. Increased level of adenosine in the body microenvironment may affect tongue and salivary glands tissues by modulating some processes including inflammation and blood vessels.

A New Series of 1,3-Dimethylxanthine Based Adenosine A2A Receptor Antagonists as a Non-Dopaminergic Treatment of Parkinson's Disease.

Adenosine receptors (AR) have emerged as competent and innovative nondopaminergic targets for the development of potential drug candidates and thus constitute an effective and safer treatment approach for Parkinson's disease (PD). Xanthine derivatives are considered as potential candidates for the treatment Parkinson's disease due to their potent A2A AR antagonistic properties. The objectives of the work are to study the impact of substituting N7-position of 8-m/pchloropropoxyphenylxanthine structure on in vitro binding affinity of compounds with various AR subtypes, in vivo antiparkinsonian activity and binding modes of newly synthesized xanthines with A2A AR in molecular docking studies. Several new 7-substituted 8-m/p-chloropropoxyphenylxanthine analogues have been prepared. Adenosine receptor binding assays were performed to study the binding interactions with various subtypes and perphenazine induced rat catatonia model was used for antiparkinsonian activity. Molecular docking studies were performed using Schrödinger molecular modeling interface. 8-para-substituted xanthine 9b bearing an N7-propyl substituent displayed the highest affinity towards A2A AR (Ki = 0.75 μM) with moderate selectivity versus other AR subtypes. 7-Propargyl analogue 9d produced significantly long-lasting antiparkinsonian effects and also produced potent and selective binding affinity towards A2A AR. In silico docking studies further highlighted the crucial structural components required to develop xanthine derived potential A2A AR ligands as antiparkinsonian agents. A new series of 7-substituted 8-m/p-chloropropoxyphenylxanthines having good affinity for A2A AR and potent antiparkinsonian activity has been developed.

Limonene-induced activation of A2A adenosine receptors reduces airway inflammation and reactivity in a mouse model of asthma.

Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A2A and A2B receptors. The pharmacological studies were carried out with A2A adenosine receptor knock-out (A2AKO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A2AKO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A2AKO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A2AKO. However, limonene reduced neutrophils in sensitized A2AKO mice, suggesting that it may activate A2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A2AAR but A2B receptors may also play a supporting role.

Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner.

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.

Purinergic signalling in liver diseases: Pathological functions and therapeutic opportunities.

SummaryExtracellular nucleotides, including ATP, are essential regulators of liver function and serve as danger signals that trigger inflammation upon injury. Ectonucleotidases, which are expressed by liver-resident cells and recruited immune cells sequentially hydrolyse nucleotides to adenosine. The nucleotide/nucleoside balance orchestrates liver homeostasis, tissue repair, and functional restoration by regulating the crosstalk between liver-resident cells and recruited immune cells. In this review, we discuss our current knowledge on the role of purinergic signals in liver homeostasis, restriction of inflammation, stimulation of liver regeneration, modulation of fibrogenesis, and regulation of carcinogenesis. Moreover, we discuss potential targeted therapeutic strategies for liver diseases based on purinergic signals involving blockade of nucleotide receptors, enhancement of ectonucleoside triphosphate diphosphohydrolase activity, and activation of adenosine receptors.

Heme Synthesis Inhibition Blocks Angiogenesis via Mitochondrial Dysfunction.

SummaryThe relationship between heme metabolism and angiogenesis is poorly understood. The final synthesis of heme occurs in mitochondria, where ferrochelatase (FECH) inserts Fe2+ into protoporphyrin IX to produce proto-heme IX. We previously showed that FECH inhibition is antiangiogenic in human retinal microvascular endothelial cells (HRECs) and in animal models of ocular neovascularization. In the present study, we sought to understand the mechanism of how FECH and thus heme is involved in endothelial cell function. Mitochondria in endothelial cells had several defects in function after heme inhibition. FECH loss changed the shape and mass of mitochondria and led to significant oxidative stress. Oxidative phosphorylation and mitochondrial Complex IV were decreased in HRECs and in murine retina ex vivo after heme depletion. Supplementation with heme partially rescued phenotypes of FECH blockade. These findings provide an unexpected link between mitochondrial heme metabolism and angiogenesis.

8-Benzylaminoxanthine scaffold variations for selective ligands acting on adenosine A2A receptors. Design, synthesis and biological evaluation

A library of 34 novel compounds based on a xanthine scaffold was explored in biological studies for interaction with adenosine receptors (ARs). Structural modifications of the xanthine core were introduced in the 8-position (benzylamino and benzyloxy substitution) as well as at N1, N3, and N7 (small alkyl residues), thereby improving affinity and selectivity for the A2A AR. The compounds were characterized by radioligand binding assays, and our study resulted in the development of the potent A2A AR ligands including 8-((6-chloro-2-fluoro-3-methoxybenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12d; Ki human A2AAR: 68.5 nM) and 8-((2-chlorobenzyl)amino)-1-ethyl-3,7-dimethyl-3,7-dihydro-1H-purine-2,6-dione (12h; Ki human A2AAR: 71.1 nM). Moreover, dual A1/A2AAR ligands were identified in the group of 1,3-diethyl-7-methylxanthine derivatives. Compound 14b displayed Ki values of 52.2 nM for the A1AR and 167 nM for the A2AAR. Selected A2AAR ligands were further evaluated as inactive for inhibition of monoamine oxidase A, B and isoforms of phosphodiesterase-4B1, -10A, which represent classical targets for xanthine derivatives. Therefore, the developed 8-benzylaminoxanthine scaffold seems to be highly selective for AR activity and relevant for potent and selective A2A ligands. Compound 12d with high selectivity for ARs, especially for the A2AAR subtype, evaluated in animal models of inflammation has shown anti-inflammatory activity. Investigated compounds were found to display high selectivity and may therefore be of high interest for further development as drugs for treating cancer or neurodegenerative diseases.

GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

P0521TICAGRELOR PROTECTS KIDNEY FROM SEPSIS INDUCED ACUTE KIDNEY INJURY THROUGH ADENOSINE DEPENDENT WAY

Abstract Background and Aims Sepsis induced acute kidney injury (AKI) is common in intensive care unit in multiple organ failure patients. Ticagrelor is an anti-platelet drug that widely applied in coronary artery disease. In addition, ticagrelor can increase the level of adenosine. Recent study had indicated that ticagrelor can protect renal function in sepsis induced AKI. However, the detailed mechanism was still unknown. Accordingly, we designed this trial to investigate if the ticagrelor alleviated sepsis induced AKI and demonstrated the potential mechanism that how ticagrelor works. Method C57BL6J mouse received oral ticagrelor (20mg/kg and 50mg/kg) for 7 days and caecum ligation and puncture (CLP) were performed. Adenosine-receptor antagonist was administered (10mg/kg, intraperitoneal injection) to block the adenosine pathway 2h before CLP. After 24h, serum creatinine was measured. PAS staining was used and TUNEL staining was applied to determine the pathological changes and cell apoptosis. Plasma concentrations of TNF-α and IL-1β were detected. Kidney tissue level of TNF-α and IL-1β were determined via qRT-PCR. Western blot was used to determine the expression of signal molecular in kidney. Results In ticagrelor group, PAS staining (fig. 1) showed that less swelling of renal tubules and TUNEL staining (fig. 2) showed the less cell apoptosis compared to CLP. Serum creatinine was significantly lower in ticagrelor group. Plasma TNF-α and IL-1β and kidney expression of TNF-α and IL-1β were significantly lower in ticagrelor group. Adenosine-receptor antagonist significantly blocked the effect of ticagrelor (fig. 3 ang 4). Western blot showed that ticagrelor activate the phosphorylation of AKT and mTOR in kidney. Adenosine-receptor antagonist inhibited the activation of AKT and mTOR (fig. 5). Conclusion The protective effect of ticagrelor was dependent on adenosine-receptor activation with downstream upregulation of phosphorylation of AKT and mTOR in sepsis induced AKI.