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Sample records for a2 receptor tp

  1. Interaction of prostanoid EP3 and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE2

    PubMed Central

    Jones, RL; Woodward, DF

    2011-01-01

    BACKGROUND AND PURPOSE Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E2 (DX-DM PGE2) on pig cerebral artery when used as a selective EP3 receptor agonist. This study investigated the selectivity profile of DX-DM PGE2, focusing on the interaction between its EP3 and TP (thromboxane A2-like) agonist activities. EXPERIMENTAL APPROACH Contraction of guinea-pig trachea (EP1 system) and aorta (EP3 and TP systems) was measured in conventional organ baths. KEY RESULTS Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE2 (EP3 agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE2 induced strong contraction, which on the basis of treatment with (DG)-3ap (EP3 antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP3 and TP receptors. EP3/TP self-synergism also accounted for contraction induced by PGF2α and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE2 also showed significant EP1 agonism on guinea-pig trachea as defined by the EP1 antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM. CONCLUSIONS AND IMPLICATIONS EP3/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP3 agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP3 system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed. PMID:20955363

  2. Molecular docking, molecular modeling, and molecular dynamics studies of azaisoflavone as dual COX-2 inhibitors and TP receptor antagonists.

    PubMed

    Hadianawala, Murtuza; Mahapatra, Amarjyoti Das; Yadav, Jitender K; Datta, Bhaskar

    2018-02-26

    Designed multi-target ligand (DML) is an emerging strategy for the development of new drugs and involves the engagement of multiple targets with the same moiety. In the context of NSAIDs it has been suggested that targeting the thromboxane prostanoid (TP) receptor along with cyclooxygenase-2 (COX-2) may help to overcome cardiovascular (CVS) complications associated with COXIBs. In the present work, azaisoflavones were studied for their COX-2 and TP receptor binding activities using structure based drug design (SBDD) techniques. Flavonoids were selected as a starting point based on their known COX-2 inhibitory and TP receptor antagonist activity. Iterative design and docking studies resulted in the evolution of a new class scaffold replacing the benzopyran-4-one ring of flavonoids with quinolin-4-one. The docking and binding parameters of these new compounds are found to be promising in comparison to those of selective COX-2 inhibitors, such as SC-558 and celecoxib. Owing to the lack of structural information, a model for the TP receptor was generated using a threading base alignment method with loop optimization performed using an ab initio method. The model generated was validated against known antagonists for TP receptor using docking/MMGBSA. Finally, the molecules that were designed for selective COX-2 inhibition were docked into the active site of the TP receptor. Iterative structural modifications and docking on these molecules generated a series which displays optimum docking scores and binding interaction for both targets. Molecular dynamics studies on a known TP receptor antagonist and a designed molecule show that both molecules remain in contact with protein throughout the simulation and interact in similar binding modes. Graphical abstract ᅟ.

  3. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells.

    PubMed

    Petri, Marcelo H; Tellier, Céline; Michiels, Carine; Ellertsen, Ingvill; Dogné, Jean-Michel; Bäck, Magnus

    2013-11-15

    The prothrombotic mediator thromboxane A2 is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels of different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Expression of the TPα and TPβ isoforms of the thromboxane prostanoid receptor (TP) in prostate cancer: clinical significance and diagnostic potential.

    PubMed

    Mulvaney, Eamon P; Shilling, Christine; Eivers, Sarah B; Perry, Antoinette S; Bjartell, Anders; Kay, Elaine W; Watson, R William; Kinsella, B Therese

    2016-11-08

    The prostanoid thromboxane (TX)A2 plays a central role in haemostasis and is increasingly implicated in cancer progression. TXA2 signals through two T Prostanoid receptor (TP) isoforms termed TPα and TPβ, with both encoded by the TBXA2R gene. Despite exhibiting several functional and regulatory differences, the role of the individual TP isoforms in neoplastic diseases is largely unknown.This study evaluated expression of the TPα and TPβ isoforms in tumour microarrays of the benign prostate and different pathological (Gleason) grades of prostate cancer (PCa). Expression of TPβ was significantly increased in PCa relative to benign tissue and strongly correlated with increasing Gleason grade. Furthermore, higher TPβ expression was associated with increased risk of biochemical recurrence (BCR) and significantly shorter disease-free survival time in patients post-surgery. While TPα was more variably expressed than TPβ in PCa, increased/high TPα expression within the tumour also trended toward increased BCR and shorter disease-free survival time. Comparative genomic CpG DNA methylation analysis revealed substantial differences in the extent of methylation of the promoter regions of the TBXA2R that specifically regulate expression of TPα and TPβ, respectively, both in benign prostate and in clinically-derived tissue representative of precursor lesions and progressive stages of PCa. Collectively, TPα and TPβ expression is differentially regulated both in the benign and tumourigenic prostate, and coincides with clinical pathology and altered CpG methylation of the TBXA2R gene. Analysis of TPβ, or a combination of TPα/TPβ, expression levels may have significant clinical potential as a diagnostic biomarker and predictor of PCa disease recurrence.

  5. Adenosine A2A receptors and depression.

    PubMed

    El Yacoubi, Malika; Costentin, Jean; Vaugeois, Jean-Marie

    2003-12-09

    Adenosine and its analogues have been shown to induce "behavioral despair" in animal models believed to be relevant to depression. Recent data have shown that selective adenosine A2A receptor antagonists (e.g., SCH 58261, ZM241385, and KW6002) or genetic inactivation of the receptor was effective in reversing signs of behavioral despair in the tail suspension and forced swim tests, two screening procedures predictive of antidepressant activity. A2A antagonists were active in the tail suspension test using either mice previously screened for having high immobility scores or mice that were selectively bred for their spontaneous "helplessness" in this test. At stimulant doses, caffeine, a nonselective A1/A2A receptor antagonist, was effective in the forced swim test. The authors have hypothesized that the antidepressant-like effect of selective A2A antagonists is linked to an interaction with dopaminergic transmission, possibly in the frontal cortex. In support of this idea, administration of the dopamine D2 receptor antagonist haloperidol prevented antidepressant-like effects elicited by SCH 58261 in the forced swim test (putatively involving cortex), whereas it had no effect on stimulant motor effects of SCH 58261 (putatively linked to ventral striatum). The interaction profile of caffeine with haloperidol differed markedly from that of SCH 58261 in the forced swim and motor activity tests. Therefore, a clear-cut antidepressant-like effect could not be ascribed to caffeine. In conclusion, available data support the proposition that a selective blockade of the adenosine A2A receptor may be an interesting target for the development of effective antidepressant agents.

  6. The association of TP53 mutations with the resistance of colorectal carcinoma to the insulin-like growth factor-1 receptor inhibitor picropodophyllin

    PubMed Central

    2013-01-01

    Background There is growing evidence indicating the insulin-like growth factor 1 receptor (IGF-1R) plays a critical role in the progression of human colorectal carcinomas. IGF-1R is an attractive drug target for the treatment of colon cancer. Picropodophyllin (PPP), of the cyclolignan family, has recently been identified as an IGF-1R inhibitor. The aim of this study is to determine the therapeutic response and mechanism after colorectal carcinoma treatment with PPP. Methods Seven colorectal carcinoma cell lines were treated with PPP. Following treatment, cells were analyzed for growth by a cell viability assay, sub-G1 apoptosis by flow cytometry, caspase cleavage and activation of AKT and extracellular signal-regulated kinase (ERK) by western blot analysis. To examine the in vivo therapeutic efficacy of PPP, mice implanted with human colorectal carcinoma xenografts underwent PPP treatment. Results PPP treatment blocked the phosphorylation of IGF-1R, AKT and ERK and inhibited the growth of TP53 wild-type but not mutated colorectal carcinoma cell lines. The treatment of PPP also induced apoptosis in TP53 wild-type cells as evident by the presence of sub-G1 cells and the cleavage of caspase-9, caspase-3, DNA fragmentation factor-45 (DFF45), poly (ADP-ribose) polymerase (PARP), and X-linked inhibitor of apoptosis protein (XIAP). The loss of BAD phosphorylation in the PPP-treated TP53 wild type cells further suggested that the treatment induced apoptosis through the BAD-mediated mitochondrial pathway. In contrast, PPP treatment failed to induce the phosphorylation of AKT and ERK and caspase cleavage in TP53 mutated colorectal carcinoma cell lines. Finally, PPP treatment suppressed the growth of xenografts derived from TP53 wild type but not mutated colorectal carcinoma cells. Conclusions We report the association of TP53 mutations with the resistance of treatment of colorectal carcinoma cells in culture and in a xenograft mouse model with the IGF-1R inhibitor PPP. TP53

  7. LPA1 receptor-mediated thromboxane A2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction.

    PubMed

    Dancs, Péter Tibor; Ruisanchez, Éva; Balogh, Andrea; Panta, Cecília Rita; Miklós, Zsuzsanna; Nüsing, Rolf M; Aoki, Junken; Chun, Jerold; Offermanns, Stefan; Tigyi, Gábor; Benyó, Zoltán

    2017-04-01

    Lysophosphatidic acid (LPA) has been recognized recently as an endothelium-dependent vasodilator, but several lines of evidence indicate that it may also stimulate vascular smooth muscle cells (VSMCs), thereby contributing to vasoregulation and remodeling. In the present study, mRNA expression of all 6 LPA receptor genes was detected in murine aortic VSMCs, with the highest levels of LPA 1 , LPA 2 , LPA 4 , and LPA 6 In endothelium-denuded thoracic aorta (TA) and abdominal aorta (AA) segments, 1-oleoyl-LPA and the LPA 1-3 agonist VPC31143 induced dose-dependent vasoconstriction. VPC31143-induced AA contraction was sensitive to pertussis toxin (PTX), the LPA 1&3 antagonist Ki16425, and genetic deletion of LPA 1 but not that of LPA 2 or inhibition of LPA 3 , by diacylglycerol pyrophosphate. Surprisingly, vasoconstriction was also diminished in vessels lacking cyclooxygenase-1 [COX1 knockout (KO)] or the thromboxane prostanoid (TP) receptor (TP KO). VPC31143 increased thromboxane A 2 (TXA 2 ) release from TA of wild-type, TP-KO, and LPA 2 -KO mice but not from LPA 1 -KO or COX1-KO mice, and PTX blocked this effect. Our findings indicate that LPA causes vasoconstriction in VSMCs, mediated by LPA 1 -, G i -, and COX1-dependent autocrine/paracrine TXA 2 release and consequent TP activation. We propose that this new-found interaction between the LPA/LPA 1 and TXA 2 /TP pathways plays significant roles in vasoregulation, hemostasis, thrombosis, and vascular remodeling.-Dancs, P. T., Ruisanchez, E., Balogh, A., Panta, C. R., Miklós, Z., Nüsing, R. M., Aoki, J., Chun, J., Offermanns, S., Tigyi, G., Benyó, Z. LPA 1 receptor-mediated thromboxane A 2 release is responsible for lysophosphatidic acid-induced vascular smooth muscle contraction. © FASEB.

  8. Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

    PubMed

    Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P

    2013-03-01

    Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.

  9. Effects of the dual TP receptor antagonist and thromboxane synthase inhibitor EV-077 on human endothelial and vascular smooth muscle cells

    SciTech Connect

    Petri, Marcelo H.; Tellier, Céline; Michiels, Carine

    2013-11-15

    Highlights: •EV-077 reduced TNF-α induced inflammation in endothelial cells. •The thromboxane mimetic U69915 enhanced vascular smooth muscle cell proliferation. •EV-077 inhibited smooth muscle cell proliferation. -- Abstract: The prothrombotic mediator thromboxane A{sub 2} is derived from arachidonic acid metabolism through the cyclooxygenase and thromboxane synthase pathways, and transduces its effect through the thromboxane prostanoid (TP) receptor. The aim of this study was to determine the effect of the TP receptor antagonist and thromboxane synthase inhibitor EV-077 on inflammatory markers in human umbilical vein endothelial cells and on human coronary artery smooth muscle cell proliferation. To this end, mRNA levels ofmore » different proinflammatory mediators were studied by real time quantitative PCR, supernatants were analyzed by enzyme immune assay, and cell proliferation was assessed using WST-1. EV-077 significantly decreased mRNA levels of ICAM-1 and PTX3 after TNFα incubation, whereas concentrations of 6-keto PGF1α in supernatants of endothelial cells incubated with TNFα were significantly increased after EV-077 treatment. Although U46619 did not alter coronary artery smooth muscle cell proliferation, this thromboxane mimetic enhanced the proliferation induced by serum, insulin and growth factors, which was significantly inhibited by EV-077. In conclusion, EV-077 inhibited TNFα-induced endothelial inflammation and reduced the enhancement of smooth muscle cell proliferation induced by a thromboxane mimetic, supporting that the thromboxane pathway may be associated with early atherosclerosis in terms of endothelial dysfunction and vascular hypertrophy.« less

  10. Glutaraldehyde pretreatment blocks phospholipase A2 modulation of adrenergic receptors.

    PubMed

    Cohen, R M; McLellan, C; Dauphin, M; Hirata, F

    1985-01-07

    Treatment of rat cerebral cortical membranes with phospholipase A2 affects, in a parallel fashion, beta-, alpha 1- and alpha 2-adrenergic receptor binding, but not the affinity of these receptors for their respective ligands. Pretreatment of membranes with 0.1 percent glutaraldehyde blocks the effects of phospholipase A2 on adrenergic receptor binding. The results support the hypothesis that desensitization or "masking" of adrenergic receptors may involve changes in membrane lipid composition. Furthermore, glutaraldehyde may prove a useful tool in the investigation of the dynamic roles of lipids in receptor function and more specifically, their regulation and coupling to physiological events.

  11. Low-dose thromboxane A2 receptor stimulation promotes closure of the rat ductus arteriosus with minimal adverse effects.

    PubMed

    Yokota, Tomohiro; Aida, Takashi; Ichikawa, Yasuhiro; Fujita, Takayuki; Yokoyama, Utako; Minamisawa, Susumu

    2012-08-01

    Patent ductus arteriosus (PDA) is a common life-threatening complication among premature infants. Although cyclooxygenase inhibitors are frequently used to treat PDA, as they inhibit the synthesis of prostaglandin E(2), the most potent vasodilator in the ductus arteriosus (DA), their efficacy is often limited. As thromboxane A(2) (TXA(2)) induces vascular contraction via the TXA(2) receptor (TP), we hypothesized that TP stimulation would promote DA closure. To measure the inner diameter of the vessels, a rapid whole-body freezing method was used. Injection of the selective TP agonists U46619 and I-BOP constricted the fetal DA at embryonic day 19 (e19) and e21 in a dose-dependent manner. Of note, U46619 also exerted a vasoconstrictive effect on two different types of postnatal PDA models: premature PDA and hypoxia-induced PDA. We also found that U46619 constricted the ex vivo DA ring to a greater extent than it constricted the ex vivo aorta. Furthermore, we found that U46619 at lower concentrations (up to 0.05 mg/g of body weight) had a minimal vasoconstrictive effect on other vessels and did not induce microthrombosis in the pulmonary capillary arteries. Low-dose TP stimulation constricts the DA with minimal adverse effects at least in rat neonates and our results could point to an alternative potent vasoconstrictor for PDA.

  12. Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane-mediated thromboxane A2 receptor activation.

    PubMed

    Bauer, Jochen; Ripperger, Anne; Frantz, Stefan; Ergün, Süleyman; Schwedhelm, Edzard; Benndorf, Ralf A

    2014-07-01

    Isoprostanes are free radical-catalysed PG-like products of unsaturated fatty acids, such as arachidonic acid, which are widely recognized as reliable markers of systemic lipid peroxidation and oxidative stress in vivo. Moreover, activation of enzymes, such as COX-2, may contribute to isoprostane formation. Indeed, formation of isoprostanes is considerably increased in various diseases which have been linked to oxidative stress, such as cardiovascular disease (CVD), and may predict the atherosclerotic burden and the risk of cardiovascular complications in the latter patients. In addition, several isoprostanes may directly contribute to the functional consequences of oxidant stress via activation of the TxA2 prostanoid receptor (TP), for example, by affecting endothelial cell function and regeneration, vascular tone, haemostasis and ischaemia/reperfusion injury. In this context, experimental and clinical data suggest that selected isoprostanes may represent important alternative activators of the TP receptor when endogenous TxA2 levels are low, for example, in aspirin-treated individuals with CVD. In this review, we will summarize the current understanding of isoprostane formation, biochemistry and (patho) physiology in the cardiovascular context. © 2014 The British Pharmacological Society.

  13. Pathophysiology of isoprostanes in the cardiovascular system: implications of isoprostane-mediated thromboxane A2 receptor activation

    PubMed Central

    Bauer, Jochen; Ripperger, Anne; Frantz, Stefan; Ergün, Süleyman; Schwedhelm, Edzard; Benndorf, Ralf A

    2014-01-01

    Isoprostanes are free radical-catalysed PG-like products of unsaturated fatty acids, such as arachidonic acid, which are widely recognized as reliable markers of systemic lipid peroxidation and oxidative stress in vivo. Moreover, activation of enzymes, such as COX-2, may contribute to isoprostane formation. Indeed, formation of isoprostanes is considerably increased in various diseases which have been linked to oxidative stress, such as cardiovascular disease (CVD), and may predict the atherosclerotic burden and the risk of cardiovascular complications in the latter patients. In addition, several isoprostanes may directly contribute to the functional consequences of oxidant stress via activation of the TxA2 prostanoid receptor (TP), for example, by affecting endothelial cell function and regeneration, vascular tone, haemostasis and ischaemia/reperfusion injury. In this context, experimental and clinical data suggest that selected isoprostanes may represent important alternative activators of the TP receptor when endogenous TxA2 levels are low, for example, in aspirin-treated individuals with CVD. In this review, we will summarize the current understanding of isoprostane formation, biochemistry and (patho) physiology in the cardiovascular context. PMID:24646155

  14. EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

    PubMed Central

    Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F.; Rudel, Thomas

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and

  15. EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis.

    PubMed

    Subbarayal, Prema; Karunakaran, Karthika; Winkler, Ann-Cathrin; Rother, Marion; Gonzalez, Erik; Meyer, Thomas F; Rudel, Thomas

    2015-04-01

    The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and

  16. Novel approaches for targeting the adenosine A2A receptor.

    PubMed

    Yuan, Gengyang; Gedeon, Nicholas G; Jankins, Tanner C; Jones, Graham B

    2015-01-01

    The adenosine A2A receptor (A2AR) represents a drug target for a wide spectrum of diseases. Approaches for targeting this membrane-bound protein have been greatly advanced by new stabilization techniques. The resulting X-ray crystal structures and subsequent analyses provide deep insight to the A2AR from both static and dynamic perspectives. Application of this, along with other biophysical methods combined with fragment-based drug design (FBDD), has become a standard approach in targeting A2AR. Complementarities of in silico screening based- and biophysical screening assisted- FBDD are likely to feature in future approaches in identifying novel ligands against this key receptor. This review describes evolution of the above approaches for targeting A2AR and highlights key modulators identified. It includes a review of: adenosine receptor structures, homology modeling, X-ray structural analysis, rational drug design, biophysical methods, FBDD and in silico screening. As a drug target, the A2AR is attractive as its function plays a role in a wide spectrum of diseases including oncologic, inflammatory, Parkinson's and cardiovascular diseases. Although traditional approaches such as high-throughput screening and homology model-based virtual screening (VS) have played a role in targeting A2AR, numerous shortcomings have generally restricted their applications to specific ligand families. Using stabilization methods for crystallization, X-ray structures of A2AR have greatly accelerated drug discovery and influenced development of biophysical-in silico hybrid screening methods. Application of these new methods to other ARs and G-protein-coupled receptors is anticipated in the future.

  17. Adenosine A2A receptor agonists with potent antiplatelet activity.

    PubMed

    Fuentes, Eduardo; Fuentes, Manuel; Caballero, Julio; Palomo, Iván; Hinz, Sonja; El-Tayeb, Ali; Müller, Christa E

    2018-05-01

    Selected adenosine A 2A receptor agonists (PSB-15826, PSB-12404, and PSB-16301) have been evaluated as new antiplatelet agents. In addition, radioligand-binding studies and receptor-docking experiments were performed in order to explain their differential biological effects on a molecular level. Among the tested adenosine derivatives, PSB-15826 was the most potent compound to inhibit platelet aggregation (EC 50 0.32 ± 0.05 µmol/L) and platelet P-selectin cell-surface localization (EC 50 0.062 ± 0.2 µmol/L), and to increase intraplatelets cAMP levels (EC 50 0.24 ± 0.01 µmol/L). The compound was more active than CGS21680 (EC 50 0.97±0.07 µmol/L) and equipotent to NECA (EC 50 0.31 ± 0.05 µmol/L) in platelet aggregation induced by ADP. In contrast to the results from cAMP assays, K i values determined in radioligand-binding studies were not predictive of the A 2A agonists' antiplatelet activity. Docking studies revealed the key molecular determinants of this new family of adenosine A 2A receptor agonists: differences in activities are related to π-stacking interactions between the ligands and the residue His264 in the extracellular loop of the adenosine A 2A receptor which may result in increased residence times. In conclusion, these results provide an improved understanding of the requirements of antiplatelet adenosine A 2A receptor agonists.

  18. ST 1535: a preferential A2A adenosine receptor antagonist.

    PubMed

    Stasi, Maria Antonietta; Borsini, Franco; Varani, Katia; Vincenzi, Fabrizio; Di Cesare, Maria Assunta; Minetti, Patrizia; Ghirardi, Orlando; Carminati, Paolo

    2006-10-01

    Antagonism of the A2A adenosine function has proved beneficial in the treatment of Parkinson's disease, in that it increases L-dopa therapeutical effects without concomitant worsening of its side-effects. In this paper we describe a preferential A2A adenosine antagonist, ST 1535, with long-lasting pharmacodynamic effects. It competitively antagonizes the effects of the A2A adenosine agonist NECA on cAMP in cells cloned with the human A2A adenosine receptor (IC50=353+/-30 nM), and the effects of the A1 adenosine agonist CHA on cAMP in cells cloned with the human A1 adenosine receptor (IC50=510+/-38 nM). ST 1535, at oral doses of 5 and 10 mg/kg, antagonizes catalepsy induced by intracerebroventricular administration of the A2A adenosine agonist CGS 21680 (10 microg/5 microl) in mice. At oral doses ranging between 5 and 20 mg/kg, ST 1535 induces hypermotility and antagonizes haloperidol-induced catalepsy in mice up to 7 h. Oral ST 1535, at 1.25 and 2.5 mg/kg, potentiates L-dopa effects in reducing haloperidol-induced catalepsy. ST 1535 represents a potential new compound, with long-lasting activity, for the treatment of Parkinson's disease.

  19. Functional efficacy of adenosine A2A receptor agonists is positively correlated to their receptor residence time

    PubMed Central

    Guo, Dong; Mulder-Krieger, Thea; IJzerman, Adriaan P; Heitman, Laura H

    2012-01-01

    BACKGROUND AND PURPOSE The adenosine A2A receptor belongs to the superfamily of GPCRs and is a promising therapeutic target. Traditionally, the discovery of novel agents for the A2A receptor has been guided by their affinity for the receptor. This parameter is determined under equilibrium conditions, largely ignoring the kinetic aspects of the ligand-receptor interaction. The aim of this study was to assess the binding kinetics of A2A receptor agonists and explore a possible relationship with their functional efficacy. EXPERIMENTAL APPROACH We set up, validated and optimized a kinetic radioligand binding assay (a so-called competition association assay) at the A2A receptor from which the binding kinetics of unlabelled ligands were determined. Subsequently, functional efficacies of A2A receptor agonists were determined in two different assays: a novel label-free impedance-based assay and a more traditional cAMP determination. KEY RESULTS A simplified competition association assay yielded an accurate determination of the association and dissociation rates of unlabelled A2A receptor ligands at their receptor. A correlation was observed between the receptor residence time of A2A receptor agonists and their intrinsic efficacies in both functional assays. The affinity of A2A receptor agonists was not correlated to their functional efficacy. CONCLUSIONS AND IMPLICATIONS This study indicates that the molecular basis of different agonist efficacies at the A2A receptor lies within their different residence times at this receptor. PMID:22324512

  20. EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling*

    PubMed Central

    Singh, Deo R.; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B.; Hristova, Kalina

    2015-01-01

    The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. PMID:26363067

  1. EphA2 Receptor Unliganded Dimers Suppress EphA2 Pro-tumorigenic Signaling.

    PubMed

    Singh, Deo R; Ahmed, Fozia; King, Christopher; Gupta, Nisha; Salotto, Matt; Pasquale, Elena B; Hristova, Kalina

    2015-11-06

    The EphA2 receptor tyrosine kinase promotes cell migration and cancer malignancy through a ligand- and kinase-independent distinctive mechanism that has been linked to high Ser-897 phosphorylation and low tyrosine phosphorylation. Here, we demonstrate that EphA2 forms dimers in the plasma membrane of HEK293T cells in the absence of ephrin ligand binding, suggesting that the current seeding mechanism model of EphA2 activation is incomplete. We also characterize a dimerization-deficient EphA2 mutant that shows enhanced ability to promote cell migration, concomitant with increased Ser-897 phosphorylation and decreased tyrosine phosphorylation compared with EphA2 wild type. Our data reveal a correlation between unliganded dimerization and tumorigenic signaling and suggest that EphA2 pro-tumorigenic activity is mediated by the EphA2 monomer. Thus, a therapeutic strategy that aims at the stabilization of EphA2 dimers may be beneficial for the treatment of cancers linked to EphA2 overexpression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

    PubMed

    Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

    2017-01-01

    Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. © 2016 International Society for Neurochemistry.

  3. Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPβ isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

    PubMed

    O'Sullivan, Aine G; Mulvaney, Eamon P; Kinsella, B Therese

    2017-04-01

    The prostanoid thromboxane (TX) A 2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA 2 /TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA 2 -TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA 2 /TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA 2 /TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA 2 /TP signalling axis in PCa, including potentially in CRPC. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. The importance of the adenosine A(2A) receptor-dopamine D(2) receptor interaction in drug addiction.

    PubMed

    Filip, M; Zaniewska, M; Frankowska, M; Wydra, K; Fuxe, K

    2012-01-01

    Drug addiction is a serious brain disorder with somatic, psychological, psychiatric, socio-economic and legal implications in the developed world. Illegal (e.g., psychostimulants, opioids, cannabinoids) and legal (alcohol, nicotine) drugs of abuse create a complex behavioral pattern composed of drug intake, withdrawal, seeking and relapse. One of the hallmarks of drugs that are abused by humans is that they have different mechanisms of action to increase dopamine (DA) neurotransmission within the mesolimbic circuitry of the brain and indirectly activate DA receptors. Among the DA receptors, D(2) receptors are linked to drug abuse and addiction because their function has been proven to be correlated with drug reinforcement and relapses. The recognition that D(2) receptors exist not only as homomers but also can form heteromers, such as with the adenosine (A)(2A) receptor, that are pharmacologically and functionally distinct from their constituent receptors, has significantly expanded the range of potential drug targets and provided new avenues for drug design in the search for novel drug addiction therapies. The aim of this review is to bring current focus on A(2A) receptors, their physiology and pharmacology in the central nervous system, and to discuss the therapeutic relevance of these receptors to drug addiction. We concentrate on the contribution of A(2A) receptors to the effects of different classes of drugs of abuse examined in preclinical behavioral experiments carried out with pharmacological and genetic tools. The consequences of chronic drug treatment on A(2A) receptor-assigned functions in preclinical studies are also presented. Finally, the neurochemical mechanism of the interaction between A(2A) receptors and drugs of abuse in the context of the heteromeric A(2A)-D(2) receptor complex is discussed. Taken together, a significant amount of experimental analyses provide evidence that targeting A(2A) receptors may offer innovative translational strategies

  5. Adenosine A2B and A3 receptor location at the mouse neuromuscular junction.

    PubMed

    Garcia, Neus; Priego, Mercedes; Hurtado, Erica; Obis, Teresa; Santafe, Manel M; Tomàs, Marta; Lanuza, Maria Angel; Tomàs, Josep

    2014-07-01

    To date, four subtypes of adenosine receptors have been cloned (A(1)R, A(2A)R, A(2B)R, and A(3)R). In a previous study we used confocal immunocytochemistry to identify A(1)R and A(2A)R receptors at mouse neuromuscular junctions (NMJs). The data shows that these receptors are localized differently in the three cells (muscle, nerve and glia) that configure the NMJs. A(1)R localizes in the terminal teloglial Schwann cell and nerve terminal, whereas A(2A)R localizes in the postsynaptic muscle and in the axon and nerve terminal. Here, we use Western blotting to investigate the presence of A(2B)R and A(3)R receptors in striated muscle and immunohistochemistry to localize them in the three cells of the adult neuromuscular synapse. The data show that A(2B)R and A(3)R receptors are present in the nerve terminal and muscle cells at the NMJs. Neither A(2B)R nor A(3)R receptors are localized in the Schwann cells. Thus, the four subtypes of adenosine receptors are present in the motor endings. The presence of these receptors in the neuromuscular synapse allows the receptors to be involved in the modulation of transmitter release. © 2014 Anatomical Society.

  6. EphA2 is a functional receptor for the growth factor progranulin.

    PubMed

    Neill, Thomas; Buraschi, Simone; Goyal, Atul; Sharpe, Catherine; Natkanski, Elizabeth; Schaefer, Liliana; Morrione, Andrea; Iozzo, Renato V

    2016-12-05

    Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. © 2016 Neill et al.

  7. EphA2 is a functional receptor for the growth factor progranulin

    PubMed Central

    Neill, Thomas; Goyal, Atul; Sharpe, Catherine

    2016-01-01

    Although the growth factor progranulin was discovered more than two decades ago, the functional receptor remains elusive. Here, we discovered that EphA2, a member of the large family of Ephrin receptor tyrosine kinases, is a functional signaling receptor for progranulin. Recombinant progranulin bound with high affinity to EphA2 in both solid phase and solution. Interaction of progranulin with EphA2 caused prolonged activation of the receptor, downstream stimulation of mitogen-activated protein kinase and Akt, and promotion of capillary morphogenesis. Furthermore, we found an autoregulatory mechanism of progranulin whereby a feed-forward loop occurred in an EphA2-dependent manner that was independent of the endocytic receptor sortilin. The discovery of a functional signaling receptor for progranulin offers a new avenue for understanding the underlying mode of action of progranulin in cancer progression, tumor angiogenesis, and perhaps neurodegenerative diseases. PMID:27903606

  8. Adenosine A2a receptors and O2 sensing in development

    PubMed Central

    2011-01-01

    Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O2 sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5′-nucleotidase and the resulting activation of adenosine A2A receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A2A receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A2A receptors mediate hypoxic inhibition of breathing and rapid eye movements. A2A receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A2A receptors play virtually no role in O2 sensing by the carotid bodies, but brain A2A receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A2A receptors have been implicated in O2 sensing by carotid glomus cells, while central A2A receptors likely blunt hypoxic hyperventilation. In conclusion, A2A receptors are crucially involved in the transduction mechanisms of O2 sensing in fetal carotid bodies and brains. Postnatally, central A2A receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O2 sensing in carotid chemoreceptors, particularly in developing lambs. PMID:21677265

  9. Adenosine A2B receptor: from cell biology to human diseases

    NASA Astrophysics Data System (ADS)

    Sun, Ying; Huang, Pingbo

    2016-08-01

    Extracellular adenosine is a ubiquitous signaling molecule that modulates a wide array of biological processes. Recently, significant advances have been made in our understanding of A2B adenosine receptor (A2BAR). In this review, we first summarize some of the general characteristics of A2BAR, and then we describe the multiple binding partners of the receptor, such as newly identified α-actinin-1 and p105, and discuss how these associated proteins could modulate A2BAR’s functions, including certain seemingly paradoxical functions of the receptor. Growing evidence indicates a critical role of A2BAR in cancer, renal disease, and diabetes, in addition to its importance in the regulation of vascular diseases and lung disease. Here, we also discuss the role of A2BAR in cancer, renal disease, and diabetes and the potential of the receptor as a target for treating these three diseases.

  10. Design and synthesis of small molecule agonists of EphA2 receptor.

    PubMed

    Petty, Aaron; Idippily, Nethrie; Bobba, Viharika; Geldenhuys, Werner J; Zhong, Bo; Su, Bin; Wang, Bingcheng

    2018-01-01

    Ligand-independent activation of EphA2 receptor kinase promotes cancer metastasis and invasion. Activating EphA2 receptor tyrosine kinase with small molecule agonist is a novel strategy to treat EphA2 overexpressing cancer. In this study, we performed a lead optimization of a small molecule Doxazosin that was identified as an EphA2 receptor agonist. 33 new analogs were developed and evaluated; a structure-activity relationship was summarized based on the EphA2 activation of these derivatives. Two new derivative compounds 24 and 27 showed much improved activity compared to Doxazosin. Compound 24 possesses a bulky amide moiety, and compound 27 has a dimeric structure that is very different to the parental compound. Compound 27 with a twelve-carbon linker of the dimer activated the kinase and induced receptor internalization and cell death with the best potency. Another dimer with a six-carbon linker has significantly reduced potency compared to the dimer with a longer linker, suggesting that the length of the linker is critical for the activity of the dimeric agonist. To explore the receptor binding characteristics of the new molecules, we applied a docking study to examine how the small molecule binds to the EphA2 receptor. The results reveal that compounds 24 and 27 form more hydrogen bonds to EphA2 than Doxazosin, suggesting that they may have higher binding affinity to the receptor. Published by Elsevier Masson SAS.

  11. A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

    PubMed

    Kataoka, Shinji; Baquero, Arian; Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C; Finger, Thomas E

    2012-01-01

    In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

  12. A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

    PubMed Central

    Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

    2012-01-01

    In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

  13. Activation of Adenosine A2A Receptors Inhibits Neutrophil Transuroepithelial Migration ▿

    PubMed Central

    Säve, Susanne; Mohlin, Camilla; Vumma, Ravi; Persson, Katarina

    2011-01-01

    Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A2A receptors. In this study, we examined the role of adenosine and A2A receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A1, A2A, A2B, and A3 receptors), but A3 receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A2A receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A2A receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A2A receptor activation, possibly through inhibition of NF-κB signaling pathways. PMID:21646447

  14. Progranulin and the receptor tyrosine kinase EphA2, partners in crime?

    PubMed Central

    Chitramuthu, Babykumari; Bateman, Andrew

    2016-01-01

    Progranulin is a secreted protein with roles in tumorigenesis, inflammation, and neurobiology, but its signaling receptors have remained unclear. In this issue, Neill et al. (2016. J. Cell Biol. https://doi.org/10.1083/jcb.201603079) identify the tyrosine kinase EphA2 as a strong candidate for such a receptor, providing insight into progranulin and EphA2 signaling. PMID:27903608

  15. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    PubMed

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  16. A role of the SAM domain in EphA2 receptor activation.

    PubMed

    Shi, Xiaojun; Hapiak, Vera; Zheng, Ji; Muller-Greven, Jeannine; Bowman, Deanna; Lingerak, Ryan; Buck, Matthias; Wang, Bing-Cheng; Smith, Adam W

    2017-03-24

    Among the 20 subfamilies of protein receptor tyrosine kinases (RTKs), Eph receptors are unique in possessing a sterile alpha motif (SAM domain) at their C-terminal ends. However, the functions of SAM domains in Eph receptors remain elusive. Here we report on a combined cell biology and quantitative fluorescence study to investigate the role of the SAM domain in EphA2 function. We observed elevated tyrosine autophosphorylation levels upon deletion of the EphA2 SAM domain (EphA2ΔS) in DU145 and PC3 prostate cancer cells and a skin tumor cell line derived from EphA1/A2 knockout mice. These results suggest that SAM domain deletion induced constitutive activation of EphA2 kinase activity. In order to explain these effects, we applied fluorescence correlation spectroscopy to investigate the lateral molecular organization of EphA2. Our results indicate that SAM domain deletion (EphA2ΔS-GFP) increases oligomerization compared to the full length receptor (EphA2FL-GFP). Stimulation with ephrinA1, a ligand for EphA2, induced further oligomerization and activation of EphA2FL-GFP. The SAM domain deletion mutant, EphA2ΔS-GFP, also underwent further oligomerization upon ephrinA1 stimulation, but the oligomers were larger than those observed for EphA2FL-GFP. Based on these results, we conclude that the EphA2 SAM domain inhibits kinase activity by reducing receptor oligomerization.

  17. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer

    PubMed Central

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I.; Lluís, Carme; Cortés, Antoni; Volkow, Nora D.; Schiffmann, Serge N.; Ferré, Sergi; Casadó, Vicent

    2015-01-01

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain. PMID:26100888

  18. Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

    PubMed

    Bonaventura, Jordi; Navarro, Gemma; Casadó-Anguera, Verònica; Azdad, Karima; Rea, William; Moreno, Estefanía; Brugarolas, Marc; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Cortés, Antoni; Volkow, Nora D; Schiffmann, Serge N; Ferré, Sergi; Casadó, Vicent

    2015-07-07

    Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

  19. Adenosine A(2A) receptors are necessary and sufficient to trigger memory impairment in adult mice.

    PubMed

    Pagnussat, N; Almeida, A S; Marques, D M; Nunes, F; Chenet, G C; Botton, P H S; Mioranzza, S; Loss, C M; Cunha, R A; Porciúncula, L O

    2015-08-01

    Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer's disease, an effect mimicked by adenosine A2 A receptor, but not A1 receptor, antagonists. Hence, we investigated the effects of adenosine receptor agonists and antagonists on memory performance and scopolamine-induced memory impairment in mice. We determined whether A2 A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2 A receptor activation triggers memory deficits in naïve mice, using three tests to assess short-term memory, namely the object recognition task, inhibitory avoidance and modified Y-maze. Scopolamine (1.0 mg·kg(-1) , i.p.) impaired short-term memory performance in all three tests and this scopolamine-induced amnesia was prevented by the A2 A receptor antagonist (SCH 58261, 0.1-1.0 mg·kg(-1) , i.p.) and by the A1 receptor antagonist (DPCPX, 0.2-5.0 mg·kg(-1) , i.p.), except in the modified Y-maze where only SCH58261 was effective. Both antagonists were devoid of effects on memory or locomotion in naïve rats. Notably, the activation of A2 A receptors with CGS 21680 (0.1-0.5 mg·kg(-1) , i.p.) before the training session was sufficient to trigger memory impairment in the three tests in naïve mice, and this effect was prevented by SCH 58261 (1.0 mg·kg(-1) , i.p.). Furthermore, i.c.v. administration of CGS 21680 (50 nmol) also impaired recognition memory in the object recognition task. These results show that A2 A receptors are necessary and sufficient to trigger memory impairment and further suggest that A1 receptors might also be selectively engaged to control the cholinergic-driven memory impairment. © 2015 The British Pharmacological Society.

  20. a2* Nicotinic Acetylcholine Receptors Influence Hippocampus-Dependent Learning and Memory in Adolescent Mice

    ERIC Educational Resources Information Center

    Lotfipour, Shahrdad; Mojica, Celina; Nakauchi, Sakura; Lipovsek, Marcela; Silverstein, Sarah; Cushman, Jesse; Tirtorahardjo, James; Poulos, Andrew; Elgoyhen, Ana Belén; Sumikawa, Katumi; Fanselow, Michael S.; Boulter, Jim

    2017-01-01

    The absence of a2* nicotinic acetylcholine receptors (nAChRs) in oriens lacunosum moleculare (OLM) GABAergic interneurons ablate the facilitation of nicotine-induced hippocampal CA1 long-term potentiation and impair memory. The current study delineated whether genetic mutations of a2* nAChRs ("Chrna2"[superscript L9'S/L9'S] and…

  1. The A2a adenosine receptor modulates the reinforcement efficacy and neurotoxicity of MDMA.

    PubMed

    Ruiz-Medina, Jessica; Ledent, Catherine; Carretón, Olga; Valverde, Olga

    2011-04-01

    Adenosine is an endogenous purine nucleoside that plays a neuromodulatory role in the central nervous system. A2a adenosine receptors have been involved in reward-related processes, inflammatory phenomena and neurotoxicity reactions. In the present study, we investigated the role of A2a adenosine receptors on the acute pharmacological effects, reinforcement and neuroinflammation induced by MDMA administration. First, the acute effects of MDMA on body temperature, locomotor activity and anxiety-like responses were measured in A2a knockout mice and wild-type littermates. Second, MDMA reinforcing properties were evaluated using the intravenous self-administration paradigm. Finally, we assessed striatal astrogliosis and microgliosis as markers of MDMA neurotoxicity. Our results showed that acute MDMA produced a biphasic effect on body temperature and increased locomotor activity and anxiogenic-like responses in both genotypes. However, MDMA reinforcing properties were dramatically affected by the lack of A2a adenosine receptors. Thus, wild-type mice maintained MDMA self-administration under a fixed ratio 1 reinforcement schedule, whereas the operant response appeared completely abolished in A2a knockout mice. In addition, the MDMA neurotoxic regime produced an enhanced inflammatory response in striatum of wild-type mice, revealed by a significant increase in glial expression, whereas such activation was attenuated in mutant mice. This is the first report indicating that A2a adenosine receptors play a key role in reinforcement and neuroinflammation induced by the widely used psychostimulant.

  2. Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers

    PubMed Central

    Brozmanova, M.; Mazurova, L.; Ru, F.; Tatar, M.; Hu, Y.; Yu, S.

    2015-01-01

    Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10–60 mmHg) in a concentration-dependent fashion (1–100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1. PMID:26564719

  3. Targeting Thromboxane A2 Receptor for Antimetastasis Therapy of Breast Cancer

    DTIC Science & Technology

    2012-09-01

    Figure 1B, right). The predominant localization of TP in the cytosol of MDA-MB-231 cells led us to examine whether a subset of TP is expressed at cell...in the Figure 1C, MDA-MB-231 cells had positive staining at cell surface, suggesting that a subset of TP was localized at plasma membrane. We...34, ISBN 979- 953-307-183-0. 2011. Robbins GT, Nie D. PPAR gamma, bioactive lipids, and cancer progression. Front Biosci. 17:1816-34, 2012. Grants

  4. Behavioral control by striatal adenosine A2A -dopamine D2 receptor heteromers.

    PubMed

    Taura, J; Valle-León, M; Sahlholm, K; Watanabe, M; Van Craenenbroeck, K; Fernández-Dueñas, V; Ferré, S; Ciruela, F

    2018-04-01

    G protein-coupled receptors (GPCR) exhibit the ability to form receptor complexes that include molecularly different GPCR (ie, GPCR heteromers), which endow them with singular functional and pharmacological characteristics. The relative expression of GPCR heteromers remains a matter of intense debate. Recent studies support that adenosine A 2A receptors (A 2A R) and dopamine D 2 receptors (D 2 R) predominantly form A 2A R-D 2 R heteromers in the striatum. The aim of the present study was evaluating the behavioral effects of pharmacological manipulation and genetic blockade of A 2A R and D 2 R within the frame of such a predominant striatal heteromeric population. First, in order to avoid possible strain-related differences, a new D 2 R-deficient mouse with the same genetic background (CD-1) than the A 2A R knock-out mouse was generated. Locomotor activity, pre-pulse inhibition (PPI) and drug-induced catalepsy were then evaluated in wild-type, A 2A R and D 2 R knock-out mice, with and without the concomitant administration of either the D 2 R agonist sumanirole or the A 2A R antagonist SCH442416. SCH442416-mediated locomotor effects were demonstrated to be dependent on D 2 R signaling. Similarly, a significant dependence on A 2A R signaling was observed for PPI and for haloperidol-induced catalepsy. The results could be explained by the existence of one main population of striatal postsynaptic A 2A R-D 2 R heteromers, which may constitute a relevant target for the treatment of Parkinson's disease and other neuropsychiatric disorders. © 2017 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

  5. Tobacco smoke induces epithelial barrier dysfunction via receptor EphA2 signaling.

    PubMed

    Nasreen, Najmunnisa; Khodayari, Nazli; Sriram, Peruvemba S; Patel, Jawaharlal; Mohammed, Kamal A

    2014-06-15

    Erythropoietin-producing human hepatocellular carcinoma (Eph) receptors are the largest family of receptor tyrosine kinases (RTKs) that mediate various cellular and developmental processes. The degrees of expression of these key molecules control the cell-cell interactions. Although the role of Eph receptors and their ligand Ephrins is well studied in developmental processes, their function in tobacco smoke (TS)-induced epithelial barrier dysfunction is unknown. We hypothesized that TS may induce permeability in bronchial airway epithelial cell (BAEpC) monolayer by modulating receptor EphA2 expression, actin cytoskeleton, adherens junction, and focal adhesion proteins. Here we report that in BAEpCs, acute TS exposure significantly upregulated EphA2 and EphrinA1 expression, disrupted the actin filaments, decreased E-cadherin expression, and increased protein permeability, whereas the focal adhesion protein paxillin was unaffected. Silencing the receptor EphA2 expression with silencing interference RNA (siRNA) significantly attenuated TS-induced hyperpermeability in BAEpCs. In addition, when BAEpC monolayer was transfected with EphA2-expressing plasmid and treated with recombinant EphrinA1, the transepithelial electrical resistance decreased significantly. Furthermore, TS downregulated E-cadherin expression and induced hyperpermeability across BAEpC monolayer in a Erk1/Erk2, p38, and JNK MAPK-dependent manner. TS induced hyperpermeability in BAEpC monolayer by targeting cell-cell adhesions, and interestingly cell-matrix adhesions were unaffected. The present data suggest that TS causes significant damage to the BAEpCs via induction of EphA2 and downregulation of E-cadherin. Induction of EphA2 in the BAEpCs exposed to TS may be an important signaling event in the pathogenesis of TS-induced epithelial injury.

  6. Silencing Receptor EphA2 Enhanced Sensitivity to Lipoplatin™ in Lung Tumor and MPM Cells.

    PubMed

    Lee, Hung-Yen; Mohammed, Kamal A; Goldberg, Eugene P; Kaye, Frederic; Najmunnisa, Nasreen

    2016-08-08

    Receptor EphA2 is overexpressed in lung cancer and malignant pleural mesothelioma (MPM) which promote tumorogenesis. Lipoplatin™, a new liposomal cisplatin formulation, is used against resistant tumors. Use of cisplatin-based drugs leads to unacceptable toxicities. To improve the effectiveness of Lipoplatin, enhancing the cellular sensitivity of lung tumor and MPM cells is critical. Therefore, we targeted receptor EphA2 by silencing interference RNA (siRNA) and treated tumor cells with Lipoplatin. The combined effects of siRNA-EphA2 and Lipoplatin were determined. We report that silencing EphA2 significantly enhanced the cellular sensitivity of lung tumor and MPM cells to Lipoplatin and maybe a potential therapy for lung cancer.

  7. Emerging strategies for EphA2 receptor targeting for cancer therapeutics.

    PubMed

    Tandon, Manish; Vemula, Sai Vikram; Mittal, Suresh K

    2011-01-01

    High mortality rates with cancers warrant further development of earlier diagnostics and better treatment strategies. Membrane-bound erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is overexpressed in breast, prostate, urinary bladder, skin, lung, ovary and brain cancers. EphA2 overexpression in cancers, its signaling mechanisms and strategies to target its deregulation. High EphA2 expression in cancer cells is correlated with a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., ephrinA1) triggers events that are deregulated and implicated in carcinogenesis. EphrinA1-independent oncogenic activity and ephrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches. EphA2 has been tested as a drug target using multiple approaches such as agonist antibodies, RNA interference, immunotherapy, virus vector-mediated gene transfer, small-molecule inhibitors and nanoparticles. With over a decade of research, encouraging results with targeting of EphA2 expression in various pre-clinical cancer models necessitate further studies.

  8. Overexpression of Adenosine A2A Receptors in Rats: Effects on Depression, Locomotion, and Anxiety.

    PubMed

    Coelho, Joana E; Alves, Pedro; Canas, Paula M; Valadas, Jorge S; Shmidt, Tatiana; Batalha, Vânia L; Ferreira, Diana G; Ribeiro, Joaquim A; Bader, Michael; Cunha, Rodrigo A; do Couto, Frederico Simões; Lopes, Luísa V

    2014-01-01

    Adenosine A2A receptors (A2AR) are a sub-type of receptors enriched in basal ganglia, activated by the neuromodulator adenosine, which interact with dopamine D2 receptors. Although this reciprocal antagonistic interaction is well-established in motor function, the outcome in dopamine-related behaviors remains uncertain, in particular in depression and anxiety. We have demonstrated an upsurge of A2AR associated to aging and chronic stress. Furthermore, Alzheimer's disease patients present A2AR accumulation in cortical areas together with depressive signs. We now tested the impact of overexpressing A2AR in forebrain neurons on dopamine-related behavior, namely depression. Adult male rats overexpressing human A2AR under the control of CaMKII promoter [Tg(CaMKII-hA2AR)] and aged-matched wild-types (WT) of the same strain (Sprague-Dawley) were studied. The forced swimming test (FST), sucrose preference test (SPT), and the open-field test (OFT) were performed to evaluate behavioral despair, anhedonia, locomotion, and anxiety. Tg(CaMKII-hA2AR) animals spent more time floating and less time swimming in the FST and presented a decreased sucrose preference at 48 h in the SPT. They also covered higher distances in the OFT and spent more time in the central zone than the WT. The results indicate that Tg(CaMKII-hA2AR) rats exhibit depressive-like behavior, hyperlocomotion, and altered exploratory behavior. This A2AR overexpression may explain the depressive signs found in aging, chronic stress, and Alzheimer's disease.

  9. A2A adenosine receptor agonists and their potential therapeutic applications. An update.

    PubMed

    Guerrero, Angel

    2018-03-12

    In the last 20 years, an increasing interest of medicinal chemists on the development of potent and selective agonists and antagonists of adenosine receptors has been noticed due to the large impact they have shown in a variety of important biological processes and diseases. Among these, it should be mentioned vasodilation, inflammation, cancer, wound healing, ischemia reperfusion injury, Parkinson disease, infectious diseases, and other CNS disorders. In this review, I will provide an update of the structures of the A2A agonists known, their selectivity versus other adenosine receptors, and their latest therapeutic applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  10. Restriction of Receptor Movement Alters Cellular Response: Physical Force Sensing by EphA2

    SciTech Connect

    Salaita, Khalid; Nair, Pradeep M; Petit, Rebecca S

    2009-09-09

    Activation of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands presented on apposed cell surfaces plays important roles in development and exhibits poorly understood functional alterations in cancer. We reconstituted this intermembrane signaling geometry between live EphA2-expressing human breast cancer cells and supported membranes displaying laterally mobile ephrin-A1. Receptor-ligand binding, clustering, and subsequent lateral transport within this junction were observed. EphA2 transport can be blocked by physical barriers nanofabricated onto the underlying substrate. This physical reorganization of EphA2 alters the cellular response to ephrin-A1, as observed by changes in cytoskeleton morphology and recruitment of a disintegrin and metalloprotease 10. Quantitativemore » analysis of receptor-ligand spatial organization across a library of 26 mammary epithelial cell lines reveals characteristic differences that strongly correlate with invasion potential. These observations reveal a mechanism for spatio-mechanical regulation of EphA2 signaling pathways.« less

  11. A2A Adenosine Receptor Antagonists as Therapeutic Candidates: are they still an interesting challenge?

    PubMed

    Cacciari, Barbara; Federico, Stephanie; Spalluto, Giampiero

    2018-04-22

    In the past decades, many efforts were done to develope ligands for the adenosine receptors, with the purpose to individuate agonists and antagonists affine and selective for each subtypes , named A1, A2A, A2B, and A3. These intense studies allowed a deeper and deeper knowledge of the nature and, moreover, of the pathophysiological roles of all the adenosine receptor subtypes. In particular, the involvment of the A2A adenosine receptor subtype in some physiological mechanisms in the brain, that could be related to important diseases such as the Parkinson's disease, encouraged the research in this field. Particular attention was given to the antagonists endowed with high affinity and selectivity since they could have a real employment in the treatment of Parkinson's disease, and some compounds, such as istradefylline, preladenant and tozadenant, are already studied in clinical trials. Actually, the role of A2A antagonists in Parkinson's disease is becoming contradictory due to contrasting results in the last studies, but, at the same time, new possible employments are emerging for this class of antagonists in cancer pathologies as much interesting to legitimate further efforts in the research of A2A ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  12. Adenosine A2B Receptor Deficiency Promotes Host Defenses against Gram-Negative Bacterial Pneumonia

    PubMed Central

    Barletta, Kathryn E.; Cagnina, R. Elaine; Burdick, Marie D.; Linden, Joel

    2012-01-01

    Rationale: Activation of the adenosine A2B receptor (A2BR) promotes antiinflammatory effects in diverse biological settings, but the role of this receptor in antimicrobial host defense in the lung has not been established. Gram-negative bacillary pneumonia is a common and serious illness associated with high morbidity and mortality, the treatment of which is complicated by increasing rates of antibiotic resistance. Objectives: To test the hypothesis that absence of adenosine A2B receptor signaling promotes host defense against bacterial pneumonia. Methods: We used a model of Klebsiella pneumoniae pneumonia in wild-type mice and mice with targeted deletion of the A2BR. Host responses were compared in vivo and leukocyte responses to the bacteria were examined in vitro. Measurements and Main Results: A2BR–/– mice demonstrated enhanced bacterial clearance from the lung and improved survival after infection with K. pneumoniae compared with wild-type controls, an effect that was mediated by bone marrow–derived cells. Leukocyte recruitment to the lungs and expression of inflammatory cytokines did not differ between A2BR–/– and wild-type mice, but A2BR–/– neutrophils exhibited sixfold greater bactericidal activity and enhanced production of neutrophil extracellular traps compared with wild-type neutrophils when incubated with K. pneumoniae. Consistent with this finding, bronchoalveolar lavage fluid from A2BR–/– mice with Klebsiella pneumonia contained more extracellular DNA compared with wild-type mice with pneumonia. Conclusions: These data suggest that the absence of A2BR signaling enhances antimicrobial activity in gram-negative bacterial pneumonia. PMID:22997203

  13. Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes

    PubMed Central

    Baranovic, Jelena; Ramanujan, Chandra S.; Kasai, Nahoko; Midgett, Charles R.; Madden, Dean R.; Torimitsu, Keiichi; Ryan, John F.

    2013-01-01

    AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. PMID:23382380

  14. The A2 Adenosine Receptor: Guanine Nucleotide Modulation of Agonist Binding Is Enhanced by Proteolysis

    PubMed Central

    NANOFF, CHRISTIAN; JACOBSON, KENNETH A.; STILES, GARY L.

    2012-01-01

    SUMMARY Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125l-2-[4-(2-{2-[(4-ammnophenyl)methylcarbonylamino]ethylaminnocarbonyl}ethyl)phenyl]ethylamino-5′-N-ethylcarboxamidoadenosine (1251-PAPA-APEC) and its photoaffinity analog 125l-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is Iabeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125l-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr ~47,O00 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory Gs-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit stnatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5′-(β,γ-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by

  15. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  16. Adenosine A2A Receptor Activation and Macrophage-mediated Experimental Glomerulonephritis

    PubMed Central

    Garcia, Gabriela E.; Truong, Luan D.; Li, Ping; Zhang, Ping; Du, Jie; Chen, Jiang-Fan; Feng, Lili

    2010-01-01

    In immune-induced inflammation, leukocytes are key mediators of tissue damage. Since A2A adenosine receptors (A2AR) are endogenous suppressors of inflammation, we examined cellular and molecular mechanisms of kidney damage to determine whether selective activation of A2AR will suppress inflammation in a rat model of glomerulonephritis. Activation of A2AR reduced the degree of kidney injury in both the acute inflammatory phase and the progressive phase of glomerulonephritis. This protection against acute and chronic inflammation was associated with suppression of the glomerular expression of the MDC/CCL22 chemokine and down-regulation of MIP-1α/CCL3, RANTES/CCL5, MIP-1β/CCL4, and MCP-1/CCL2 chemokines. The expression of anti-inflammatory cytokines, IL-4 and IL-10, also increased. The mechanism for these anti-inflammatory responses to the A2AR agonist was suppression of macrophages function. A2AR expression was increased in macrophages, macrophage-derived chemokines were reduced in response to the A2AR agonist, and chemokines not expressed in macrophages did not respond to A2AR activation. Thus, activation of the A2AR on macrophages inhibits immune-associated inflammation. In glomerulonephritis, A2AR activation modulates inflammation and tissue damage even in the progressive phase of glomerulonephritis. Accordingly, pharmacological activation of A2AR could be developed into a novel treatment for glomerulonephritis and other macrophage-related inflammatory diseases. PMID:17898087

  17. Malaria parasites target the hepatocyte receptor EphA2 for successful host infection.

    PubMed

    Kaushansky, Alexis; Douglass, Alyse N; Arang, Nadia; Vigdorovich, Vladimir; Dambrauskas, Nicholas; Kain, Heather S; Austin, Laura S; Sather, D Noah; Kappe, Stefan H I

    2015-11-27

    The invasion of a suitable host hepatocyte by mosquito-transmitted Plasmodium sporozoites is an essential early step in successful malaria parasite infection. Yet precisely how sporozoites target their host cell and facilitate productive infection remains largely unknown. We found that the hepatocyte EphA2 receptor was critical for establishing a permissive intracellular replication compartment, the parasitophorous vacuole. Sporozoites productively infected hepatocytes with high EphA2 expression, and the deletion of EphA2 protected mice from liver infection. Lack of host EphA2 phenocopied the lack of the sporozoite proteins P52 and P36. Our data suggest that P36 engages EphA2, which is likely to be a key step in establishing the permissive replication compartment. Copyright © 2015, American Association for the Advancement of Science.

  18. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies

    DTIC Science & Technology

    2012-12-01

    in Traumatic Optic Neuropathies ” PRINCIPAL INVESTIGATOR: Gregory I. Liou, PhD CONTRACTING ORGANIZATION: Georgia Health Sciences...Adenosine Receptor A2A in Traumatic Optic Neuropathies 5b. GRANT NUMBER W81XWH-11-2-0046 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT...ABSTRACT Our goal is to develop an early therapeutic intervention before the progression of traumatic optic neuropathy (TON), a vision-threatening

  19. Emerging strategies for EphA2 receptor targeting for cancer therapeutics

    PubMed Central

    Tandon, Manish; Vemula, Sai Vikram; Mittal, Suresh K.

    2010-01-01

    Importance of the field High mortality rates with cancers warrant further development of earlier diagnostics and better treatment strategies. Membrane-bound hepatocellular receptor tyrosine kinase class A2 (EphA2) is overexpressed in breast, prostate, urinary bladder, skin, lung, ovary and brain cancers. Areas covered in this review This review describes EphA2 overexpression in cancers, its signaling mechanisms and strategies to target its deregulation. What will the reader will gain High EphA2 expression in cancer cells is correlated to a poor prognosis associated with recurrence due to enhanced metastasis. Interaction of the EphA2 receptor with its ligand (e.g., EphrinA1) triggers events that are deregulated and implicated in carcinogenesis. Both EphrinA1-independent oncogenic activity and EphrinA1-dependent tumor suppressor roles for EphA2 are described. Molecular interactions of EphA2 with signaling proteins are associated with the modulation of cytoskeleton dynamics, cell adhesion, proliferation, differentiation and metastasis. The deregulated signaling by EphA2 and its involvement in oncogenesis provide multiple avenues for the rational design of intervention approaches. Take home message EphA2 has been tested as a drug target using multiple approaches such as agonist antibodies, RNA interference, immunotherapy, virus vectors-mediated gene transfer, small molecule inhibitors and nanoparticles. With over a decade of research, encouraging results with successful targeting of EphA2 expression in various pre-clinical cancer models necessitate further studies. PMID:21142802

  20. Adenosine A2A receptors in ventral striatum, hypothalamus and nociceptive circuitry. Implications for drug addiction, sleep and pain

    PubMed Central

    Ferré, S.; Diamond, I.; Goldberg, S.R.; Yao, L.; Hourani, S.M.O.; Huang, Z.L.; Urade, Y.; Kitchen, I.

    2007-01-01

    Adenosine A2A receptors localized in the dorsal striatum are considered as a new target for the development of antiparkinsonian drugs. Co-administration of A2A receptor antagonists has shown a significant improvement of the effects of L-DOPA. The present review emphasizes the possible application of A2A receptor antagonists in pathological conditions other than parkinsonism, including drug addiction, sleep disorders and pain. In addition to the dorsal striatum, the ventral striatum (nucleus accumbens) contains a high density of A2A receptors, which presynaptically and postsynaptically regulate glutamatergic transmission in the cortical glutamatergic projections to the nucleus accumbens. It is currently believed that molecular adaptations of the cortico-accumbens glutamatergic synapses are involved in compulsive drug seeking and relapse. Here we review recent experimental evidence suggesting that A2A antagonists could become new therapeutic agents for drug addiction. Morphological and functional studies have identified lower levels of A2A receptors in brain areas other than the striatum, such as the ventrolateral preoptic area of the hypothalamus, where adenosine plays an important role in sleep regulation. Although initially believed to be mostly dependent on A1 receptors, here we review recent studies that demonstrate that the somnogenic effects of adenosine are largely mediated by hypothalamic A2A receptors. A2A receptor antagonists could therefore be considered as a possible treatment for narcolepsy and other sleep-related disorders. Finally, nociception is another adenosine-regulated neural function previously thought to mostly involve A1 receptors. Although there is some conflicting literature on the effects of agonists and antagonists, which may partly be due to the lack of selectivity of available drugs, the studies in A2A receptor knockout mice suggest that A2A receptor antagonists might have some therapeutic potential in pain states, in particular where

  1. A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction.

    PubMed

    Nisar, Shaista P; Lordkipanidzé, Marie; Jones, Matthew L; Dawood, Ban; Murden, Sherina; Cunningham, Margaret R; Mumford, Andrew D; Wilde, Jonathan T; Watson, Steve P; Mundell, Stuart J; Lowe, Gillian C

    2014-05-05

    A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

  2. Targeting Thromboxane A2 Receptor for Anti-Metastasis Therapy of Breast Cancer

    DTIC Science & Technology

    2011-09-01

    of cell function by Rho GTPases." Drug News Perspect 14(7): 389-95. Erickson, J. W., R. A. Cerione, et al. (1997). "Identification of an actin...Focusing Tumor Microenvironment, Stem Cells and Metastasis 570 (MTOC) and Golgi apparatus to the front of the nucleus, oriented toward the direction of...define the function of TP in tumor cell motility and to validate TP as a target for anti-metastasis therapy of breast cancer. In the first aim, the

  3. The adenosine A2A receptor — Myocardial protectant and coronary target in endotoxemia

    PubMed Central

    Reichelt, Melissa E.; Ashton, Kevin J.; Tan, Xing Lin; Mustafa, S. Jamal; Ledent, Catherine; Delbridge, Lea M.D.; Hofmann, Polly A.; Headrick, John P.; Morrison, R. Ray

    2013-01-01

    Background Cardiac injury and dysfunction are contributors to disease progression and mortality in sepsis. This study evaluated the cardiovascular role of intrinsic A2A adenosine receptor (A2AAR) activity during lipopolysaccharide (LPS)-induced inflammation. Methods We assessed the impact of 24 h of LPS challenge (20 mg/kg, IP) on cardiac injury, coronary function and inflammatory mediator levels in Wild-Type (WT) mice and mice lacking functional A2AARs (A2AAR KO). Results Cardiac injury was evident in LPS-treated WTs, with ∼7-fold elevation in serum cardiac troponin I (cTnI), and significant ventricular and coronary dysfunction. Absence of A2AARs increased LPS-provoked cTnI release at 24 h by 3-fold without additional demise of contraction function. Importantly, A2AAR deletion per se emulated detrimental effects of LPS on coronary function, and LPS was without effect in coronary vessels lacking A2AARs. Effects of A2AAR KO were independent of major shifts in circulating C-reactive protein (CRP) and haptoglobin. Cytokine responses were largely insensitive to A2AAR deletion; substantial LPS-induced elevations (up to 100-fold) in IFN-γ and IL-10 were unaltered in A2AAR KO mice, as were levels of IL-4 and TNF-α. However, late elevations in IL-2 and IL-5 were differentially modulated by A2AAR KO (IL-2 reduced, IL-5 increased). Data demonstrate that in the context of LPS-triggered cardiac and coronary injury, A2AAR activity protects myocardial viability without modifying contractile dysfunction, and selectively modulates cytokine (IL-2, IL-5) release. A2AARs also appear to be targeted by LPS in the coronary vasculature. Conclusions These experimental data suggest that preservation of A2AAR functionality might provide therapeutic benefit in human sepsis. PMID:22192288

  4. Adenosine A2A Receptor Activation Prevents Wear Particle-Induced Osteolysis

    PubMed Central

    Mediero, Aránzazu; Frenkel, Sally R.; Wilder, Tuere; He, Wenjie; Mazumder, Amitabha; Cronstein, Bruce N.

    2012-01-01

    Prosthesis loosening, associated with wear-particle–induced inflammation and osteoclast-mediated bone destruction, is a common cause for joint implant failure, leading to revision surgery. Adenosine A2A receptors (A2AR) mediate potent anti-inflammatory effects in many tissues and prevent osteoclast differentiation. We tested the hypothesis that an A2AR agonist could reduce osteoclast-mediated bone resorption in a murine calvaria model of wear-particle–induced bone resorption. C57Bl/6 and A2A knockout (A2ARKO) mice received ultrahigh-molecular weight polyethylene particles (UHMWPE) and were treated daily with either saline or the A2AR agonist CGS21680. After 2 weeks, micro-computed tomography of calvaria demonstrated that CGS21680 reduced particle-induced bone pitting and porosity in a dose-dependent manner, increasing cortical bone and bone volume compared to control mice. Histological examination demonstrated diminished inflammation after treatment with CGS21680. In A2AKO mice, CGS21680 did not affect osteoclast-mediated bone resorption or inflammation. Levels of bone-resorption markers receptor activator of nuclear factor-kB (RANK), RANK ligand (RANKL), cathepsin K, CD163, and osteopontin were reduced following CGS21680 treatment, together with a reduction in osteoclasts. Secretion of interleukin 1β (IL-1β) and TNFα was significantly decreased, whereas IL-10 was markedly increased in bone by CGS21680. These results in mice suggest that site-specific delivery of an adenosine A2AR agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery. PMID:22623741

  5. Adenosine A2A Receptors in the Amygdala Control Synaptic Plasticity and Contextual Fear Memory.

    PubMed

    Simões, Ana Patrícia; Machado, Nuno J; Gonçalves, Nélio; Kaster, Manuella P; Simões, Ana T; Nunes, Ana; Pereira de Almeida, Luís; Goosens, Ki Ann; Rial, Daniel; Cunha, Rodrigo A

    2016-11-01

    The consumption of caffeine modulates working and reference memory through the antagonism of adenosine A 2A receptors (A 2A Rs) controlling synaptic plasticity processes in hippocampal excitatory synapses. Fear memory essentially involves plastic changes in amygdala circuits. However, it is unknown if A 2A Rs in the amygdala regulate synaptic plasticity and fear memory. We report that A 2A Rs in the amygdala are enriched in synapses and located to glutamatergic synapses, where they selectively control synaptic plasticity rather than synaptic transmission at a major afferent pathway to the amygdala. Notably, the downregulation of A 2A Rs selectively in the basolateral complex of the amygdala, using a lentivirus with a silencing shRNA (small hairpin RNA targeting A 2A R (shA 2A R)), impaired fear acquisition as well as Pavlovian fear retrieval. This is probably associated with the upregulation and gain of function of A 2A Rs in the amygdala after fear acquisition. The importance of A 2A Rs to control fear memory was further confirmed by the ability of SCH58261 (0.1 mg/kg; A 2A R antagonist), caffeine (5 mg/kg), but not DPCPX (0.5 mg/kg; A 1 R antagonist), treatment for 7 days before fear conditioning onwards, to attenuate the retrieval of context fear after 24-48 h and after 7-8 days. These results demonstrate that amygdala A 2A Rs control fear memory and the underlying process of synaptic plasticity in this brain region. This provides a neurophysiological basis for the association between A 2A R polymorphisms and phobia or panic attacks in humans and prompts a therapeutic interest in A 2A Rs to manage fear-related pathologies.

  6. TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

    PubMed Central

    Haricharan, Svasti; Brown, Powel

    2015-01-01

    Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30–50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types. PMID:26063617

  7. TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth.

    PubMed

    Haricharan, Svasti; Brown, Powel

    2015-06-23

    Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30-50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types.

  8. EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans

    PubMed Central

    Swidergall, Marc; Solis, Norma V.; Lionakis, Michail S.; Filler, Scott G.

    2017-01-01

    Oral epithelial cells discriminate between pathogenic and non-pathogenic stimuli, and only induce an inflammatory response when they are exposed to high levels of a potentially harmful microorganism. The pattern recognition receptors (PRRs) in epithelial cells that mediate this differential response are poorly understood. Here, we demonstrate that the ephrin type-A receptor 2 (EphA2) is an oral epithelial cell PRR that binds to exposed β-glucans on the surface of the fungal pathogen Candida albicans. Binding of C. albicans to EphA2 on oral epithelial cells activates signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase signaling in an inoculum-dependent manner, and is required for induction of a pro-inflammatory and antifungal response. EphA2−/− mice have impaired inflammatory responses and reduced IL-17 signaling during oropharyngeal candidiasis, resulting in more severe disease. Our study reveals that EphA2 functions as PRR for β-glucans that senses epithelial cell fungal burden and is required for the maximal mucosal inflammatory response to C. albicans. PMID:29133884

  9. EphA2 receptor is a key player in the metastatic onset of Ewing sarcoma.

    PubMed

    Garcia-Monclús, Silvia; López-Alemany, Roser; Almacellas-Rabaiget, Olga; Herrero-Martín, David; Huertas-Martinez, Juan; Lagares-Tena, Laura; Alba-Pavón, Piedad; Hontecillas-Prieto, Lourdes; Mora, Jaume; de Álava, Enrique; Rello-Varona, Santi; Giangrande, Paloma H; Tirado, Oscar M

    2018-03-26

    Ewing sarcoma (ES) is the second most common bone malignancy affecting children and young adults with poor prognosis due to high metastasis incidence. Our group previously described that EphA2, a tyrosine kinase receptor, promotes angiogenesis in Ewing sarcoma (ES) cells via ligand-dependent signaling. Now we wanted to explore EphA2 ligand-independent activity, controlled upon phosphorylation at S897 (p-EphA2 S897 ), as it has been linked to metastasis in several malignancies. By reverse genetic engineering we explored the phenotypic changes after EphA2 removal or reintroduction. Gene expression microarray was used to identify key players in EphA2 signaling. Mice were employed to reproduce metastatic processes from orthotopically implanted engineered cells. We established a correlation between ES cells aggressiveness and p-EphA2 S897 . Moreover, stable overexpression of EphA2 in low EphA2 expression ES cells enhanced proliferation and migration, but not a non-phosphorylable mutant (S987A). Consistently, silencing of EphA2 reduced tumorigenicity, migration and invasion in vitro, and lung metastasis incidence in experimental and spontaneous metastasis assays in vivo. A gene expression microarray revealed the implication of EphA2 in cell signaling, cellular movement and survival. ADAM19 knockdown by siRNA technology strongly reproduced the negative effects on cell migration observed after EphA2 silencing. Altogether, our results suggest that p-EphA2 S897 correlates with aggressiveness in ES, so blocking its function may be a promising treatment. © 2018 UICC.

  10. EphA2 Receptor Signaling Mediates Inflammatory Responses in Lipopolysaccharide-Induced Lung Injury.

    PubMed

    Hong, Ji Young; Shin, Mi Hwa; Chung, Kyung Soo; Kim, Eun Young; Jung, Ji Ye; Kang, Young Ae; Kim, Young Sam; Kim, Se Kyu; Chang, Joon; Park, Moo Suk

    2015-07-01

    Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30±2.93 vs. 11.45±1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33×10(4)±8.84×10(4) vs. IgG+LPS: 208.0×10(4)±122.6×10(4); p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52±0.41 mg/mL vs. IgG+LPS: 1.38±1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110γ, phospho-Akt, nuclear factor κB, and proinflammatory cytokines. This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation.

  11. Past, present and future of A2A adenosine receptor antagonists in the therapy of Parkinson’s disease

    PubMed Central

    Armentero, Marie Therese; Pinna, Annalisa; Ferré, Sergi; Lanciego, José Luis; Müller, Christa E.; Franco, Rafael

    2011-01-01

    Several selective antagonists for adenosine A2A receptors (A2AR) are currently under evaluation in clinical trials (phases I to III) to treat Parkinson’s disease, and they will probably soon reach the market. The usefulness of these antagonists has been deduced from studies demonstrating functional interactions between dopamine D2 and adenosine A2A receptors in the basal ganglia. At present it is believed that A2AR antagonists can be used in combination with the dopamine precursor L-DOPA to minimize the motor symptoms of Parkinson’s patients. However, a considerable body of data indicates that in addition to ameliorating motor symptoms, adenosine A2AR antagonists may also prevent neurodegeneration. Despite these promising indications, one further issue must be considered in order to develop fully optimized anti-parkinsonian drug therapy, namely the existence of receptor (hetero)dimers/oligomers of G protein-coupled receptors, a topic currently the focus of intense debate within the scientific community. Dopamine D2 receptors (D2Rs) expressed in the striatum are known to form heteromers with A2A adenosine receptors. Thus, the development of heteromer-specific A2A receptor antagonists represents a promising strategy for the identification of more selective and safer drugs. PMID:21810444

  12. Involvement of Cot/Tp12 in bone loss during periodontitis.

    PubMed

    Ohnishi, T; Okamoto, A; Kakimoto, K; Bandow, K; Chiba, N; Matsuguchi, T

    2010-02-01

    Periodontitis causes resorption of alveolar bone, in which RANKL induces osteoclastogenesis. The binding of lipopolysaccharide to Toll-like receptors causes phosphorylation of Cot/Tp12 to activate the MAPK cascade. Previous in vitro studies showed that Cot/Tp12 was essential for the induction of RANKL expression by lipopolysaccharide. In this study, we examined whether Cot/Tp12 deficiency reduced the progression of alveolar bone loss and osteoclastogenesis during experimental periodontitis. We found that the extent of alveolar bone loss and osteoclastogenesis induced by ligature-induced periodontitis was decreased in Cot/Tp12-deficient mice. In addition, reduction of RANKL expression was observed in periodontal tissues of Cot/Tp12-deficient mice with experimental periodontitis. Furthermore, we found that Cot/Tp12 was involved in the induction of TNF-alpha mRNA expression in gingiva of mice with experimental periodontitis. Our observations suggested that Cot/Tp12 is essential for the progression of alveolar bone loss and osteoclastogenesis in periodontal tissue during experimental periodontitis mediated through increased RANKL expression.

  13. Adenosine A2A receptor deficiency attenuates the somnogenic effect of prostaglandin D2 in mice

    PubMed Central

    Zhang, Bin-jia; Huang, Zhi-li; Chen, Jiang-fan; Urade, Yoshihiro; Qu, Wei-min

    2017-01-01

    Prostaglandin D2 (PGD2) is one of the most potent endogenous sleep promoting substances. PGD2 activates the PGD2 receptor (DPR) and increases the extracellular level of adenosine in wild-type (WT) mice but not DPR knockout (KO) mice, suggesting that PGD2-induced sleep is DPR-dependent, and adenosine may be the signaling molecule that mediates the somnogenic effect of PGD2. The aim of this study was to determine the involvement of the adenosine A2A receptor (A2AR) in PGD2-induced sleep. We infused PGD2 into the lateral ventricle of WT and A2AR KO mice between 20:00 and 2:00 for 6 h, and electroencephalograms and electromyograms were simultaneously recorded. In WT mice, PGD2 infusion dose-dependently increased non-rapid eye movement (non-REM, NREM) sleep, which was 139.1%, 145.0% and 202.7% as large as that of vehicle-treated mice at doses of 10, 20 and 50 pmol/min, respectively. PGD2 infusion at doses of 20 and 50 pmol/min also increased REM sleep during the 6-h PGD2 infusion and 4-h post-dosing periods in WT mice to 148.9% and 166.7%, respectively. In A2AR KO mice, however, PGD2 infusion at 10 pmol/min did not change the sleep profile, whereas higher doses at 20 and 50 pmol/min increased the NREM sleep during the 6-h PGD2 infusion to 117.5% and 155.6%, respectively, but did not change the sleep in the post-dosing period. Moreover, PGD2 infusion at 50 pmol/min significantly increased the episode number in both genotypes but only enhanced the episode duration in WT mice. The results demonstrate that PGD2-induced sleep in mice is mediated by both adenosine A2AR-dependent and -independent systems. PMID:28112177

  14. Tp-e interval and Tp-e/QT ratio in patients with celiac disease.

    PubMed

    Demirtaş, K; Yayla, Ç; Yüksel, M; Açar, B; Ünal, S; Ertem, A G; Kaplan, M; Akpinar, M Y; Kiliç, Z M Y; Kayaçetin, E

    2017-11-01

    Celiac disease is a chronic immune-mediated disease of the small intestine. It has been known that dilated cardiomyopathy and ischemic coronary artery disease have become more frequent in patients with celiac disease. The aim of the study was to assess Tp-e interval and Tp-e/QT ratio in patients with celiac disease. This study was conducted at a single center in collaboration with gastroenterology and cardiology clinics. Between January 2014 and June 2015, a total of 76 consecutive patients were enrolled (38 patients with celiac disease and 38 control subjects). Tp-e interval, Tp-e/QT and Tp-e/QTc ratio were measured from the 12-lead electrocardiogram. Tp-e interval (64.2±11.0 vs. 44.5±6.0; p<0.001), Tp-e/QT ratio (0.18±0.02 vs. 0.13±0.02; p<0.001) and Tp-e/QTc ratio (0.16±0.02 vs. 0.11±0.01; p<0.001) were significantly higher in patients with celiac disease than control subjects. There was a significant positive correlation between Tp-e/QTc ratio and disease duration in patients with celiac disease (r=0.480, p=0.003) and also there was a significant positive correlation between Tp-e/QTc ratio and erythrocyte sedimentation rate (r=0.434, p<0.001). Our study showed that Tp-e interval, Tp-e/QT and Tp-e/QTc ratios were increased in patients with celiac disease. Whether these changes increase the risk of ventricular arrhythmia deserve further studies. Copyright © 2017 Elsevier España, S.L.U. and Sociedad Española de Medicina Interna (SEMI). All rights reserved.

  15. Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes.

    PubMed

    Santhosh, K T; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, A J; Dakshinamurti, S

    2011-07-01

    Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor-mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca(2+) response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. TP receptor sensitivity and EC(50) for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca(2+) mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  16. Evidence for an A2/Ra adenosine receptor in the guinea-pig trachea

    PubMed Central

    Brown, C.M.; Collis, M.G.

    1982-01-01

    1 An attempt was made to determine whether the extracellular adenosine receptor that mediates relaxation in the guinea-pig trachea is of the A1/Ri or A2/Ra subtype. 2 Dose-response curves to adenosine and a number of 5′- and N6-substituted analogues were constructed for the isolated guinea-pig trachea, contracted with carbachol. 3 The 5′-substituted analogues of adenosine were the most potent compounds tested, the order of potency being 5′-N-cyclopropylcarboxamide adenosine (NCPCA) > 5′-N-ethylcarboxamide adenosine (NECA) > 2-chloroadenosine > L-N6-phenylisopropyladenosine (L-PIA) > adenosine > D-N6-phenylisopropyladenosine (D-PIA). 4 The difference in potency between the stereoisomers D- and L-PIA on the isolated trachea was at the most five fold. 5 Responses to low doses of adenosine and its analogues were attenuated after treatment with either theophylline or 8-phenyltheophylline. The responses to 2-chloroadenosine were affected to a lesser extent than were those to the other purines. 6 Adenosine transport inhibitors, dipyridamole and dilazep, potentiated responses to adenosine, did not affect those to NCPCA, NECA, L-PIA and D-PIA but significantly reduced the responses to high doses of 2-chloroadenosine. 7 Relaxations evoked by 9-β-D-xylofuranosyladenosine which can activate intracellular but not extracellular adenosine receptors, were attenuated by dipyridamole but unaffected by 8-phenyltheophylline. 8 The results support the existence of an extracellular A2/Ra subtype of adenosine receptor and an intracellular purine-sensitive site, both of which mediate relaxation. PMID:6286021

  17. Elevated Adenosine Induces Placental DNA Hypomethylation Independent of A2B Receptor Signaling in Preeclampsia.

    PubMed

    Huang, Aji; Wu, Hongyu; Iriyama, Takayuki; Zhang, Yujin; Sun, Kaiqi; Song, Anren; Liu, Hong; Peng, Zhangzhe; Tang, Lili; Lee, Minjung; Huang, Yun; Ni, Xin; Kellems, Rodney E; Xia, Yang

    2017-07-01

    Preeclampsia is a prevalent pregnancy hypertensive disease with both maternal and fetal morbidity and mortality. Emerging evidence indicates that global placental DNA hypomethylation is observed in patients with preeclampsia and is linked to altered gene expression and disease development. However, the molecular basis underlying placental epigenetic changes in preeclampsia remains unclear. Using 2 independent experimental models of preeclampsia, adenosine deaminase-deficient mice and a pathogenic autoantibody-induced mouse model of preeclampsia, we demonstrate that elevated placental adenosine not only induces hallmark features of preeclampsia but also causes placental DNA hypomethylation. The use of genetic approaches to express an adenosine deaminase minigene specifically in placentas, or adenosine deaminase enzyme replacement therapy, restored placental adenosine to normal levels, attenuated preeclampsia features, and abolished placental DNA hypomethylation in adenosine deaminase-deficient mice. Genetic deletion of CD73 (an ectonucleotidase that converts AMP to adenosine) prevented the elevation of placental adenosine in the autoantibody-induced preeclampsia mouse model and ameliorated preeclampsia features and placental DNA hypomethylation. Immunohistochemical studies revealed that elevated placental adenosine-mediated DNA hypomethylation predominantly occurs in spongiotrophoblasts and labyrinthine trophoblasts and that this effect is independent of A2B adenosine receptor activation in both preeclampsia models. Extending our mouse findings to humans, we used cultured human trophoblasts to demonstrate that adenosine functions intracellularly and induces DNA hypomethylation without A2B adenosine receptor activation. Altogether, both mouse and human studies reveal novel mechanisms underlying placental DNA hypomethylation and potential therapeutic approaches for preeclampsia. © 2017 American Heart Association, Inc.

  18. Adenosine A2B receptor modulates intestinal barrier function under hypoxic and ischemia/reperfusion conditions.

    PubMed

    Yang, Yang; Qiu, Yuan; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Zhang, Chaojun; Yang, Hanwenbo; Teitelbaum, Daniel H; Sun, Li-Hua; Yang, Hua

    2014-01-01

    Intestinal barrier function failure from ischemia/reperfusion (I/R) and acute hypoxia has been implicated as a critical determinant in the predisposition to intestinal inflammation and a number of inflammatory disorders. Here, we identified the role of Adenosine A2B receptor (A2BAR) in the regulation of intestinal barrier function under I/R and acute hypoxic conditions. C57BL/6J mice were used, and were randomized into three groups: Sham, I/R, IR+PSB1115 (a specific A2BAR antagonist) groups. After surgery, the small bowel was harvested for immunohistochemical staining, RNA and protein content, and intestinal permeability analyses. Using an epithelial cell culture model, we investigated the influence of hypoxia on the epithelial function, and the role of A2BAR in the expressions of tight junction and epithelial permeability. The expressions of Claudin-1, occludin and ZO-1 were detected by RT-PCR and Western-Blot. Epithelial barrier function was assessed with transepithelial resistance (TER). The A2BAR antagonist, PSB1115, significantly increased tight junction protein expression after intestinal I/R or acute hypoxia conditions. PSB1115 also attenuated the disrupted distribution of TJ proteins. Furthermore, inhibition of A2BAR attenuated the decrease in TER induced by I/R or acute hypoxic conditions, and maintained intestinal barrier function. Antagonism of A2BAR activity improves intestinal epithelial structure and barrier function in a mouse model of intestinal I/R and a cell model of acute hypoxia. These findings support a potentially destructive role for A2BAR under intestinal I/R and acute hypoxic conditions.

  19. Adenosine A2A Receptors in the Nucleus Accumbens Bi-Directionally Alter Cocaine Seeking in Rats

    PubMed Central

    O'Neill, Casey E; LeTendre, Mckenzie L; Bachtell, Ryan K

    2012-01-01

    Repeated cocaine administration enhances dopamine D2 receptor sensitivity in the mesolimbic dopamine system, which contributes to drug relapse. Adenosine A2A receptors are colocalized with D2 receptors on nucleus accumbens (NAc) medium spiny neurons where they antagonize D2 receptor activity. Thus, A2A receptors represent a target for reducing enhanced D2 receptor sensitivity that contributes to cocaine relapse. The aim of these studies were to determine the effects of adenosine A2A receptor modulation in the NAc on cocaine seeking in rats that were trained to lever press for cocaine. Following at least 15 daily self-administration sessions and 1 week of abstinence, lever pressing was extinguished in daily extinction sessions. We subsequently assessed the effects of intra-NAc core microinjections of the A2A receptor agonist, CGS 21680 (4-[2-[[6-amino-9-(N-ethyl-b--ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride), and the A2A receptor antagonist, MSX-3 (3,7-dihydro-8-[(1E)-2-(3-methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)propyl-1-(2-propynyl)-1H-purine-2,6-dione disodium salt hydrate), in modulating cocaine- and quinpirole-induced reinstatement to cocaine seeking. Intra-NAc pretreatment of CGS 21680 reduced both cocaine- and quinpirole-induced reinstatement. These effects were specific to cocaine reinstatement as intra-NAc CGS 21680 had no effect on sucrose seeking in rats trained to self-administer sucrose pellets. Intra-NAc treatment with MSX-3 modestly reinstated cocaine seeking when given alone, and exacerbated both cocaine- and quinpirole-induced reinstatement. Interestingly, the exacerbation of cocaine seeking produced by MSX-3 was only observed at sub-threshold doses of cocaine and quinpirole, suggesting that removing tonic A2A receptor activity enables behaviors mediated by dopamine receptors. Taken together, these findings suggest that A2A receptor stimulation reduces, while A2A blockade amplifies, D2 receptor

  20. Adenosine A2A receptors in the nucleus accumbens bi-directionally alter cocaine seeking in rats.

    PubMed

    O'Neill, Casey E; LeTendre, McKenzie L; Bachtell, Ryan K

    2012-04-01

    Repeated cocaine administration enhances dopamine D(2) receptor sensitivity in the mesolimbic dopamine system, which contributes to drug relapse. Adenosine A(2A) receptors are colocalized with D(2) receptors on nucleus accumbens (NAc) medium spiny neurons where they antagonize D(2) receptor activity. Thus, A(2A) receptors represent a target for reducing enhanced D(2) receptor sensitivity that contributes to cocaine relapse. The aim of these studies were to determine the effects of adenosine A(2A) receptor modulation in the NAc on cocaine seeking in rats that were trained to lever press for cocaine. Following at least 15 daily self-administration sessions and 1 week of abstinence, lever pressing was extinguished in daily extinction sessions. We subsequently assessed the effects of intra-NAc core microinjections of the A(2A) receptor agonist, CGS 21680 (4-[2-[[6-amino-9-(N-ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride), and the A(2A) receptor antagonist, MSX-3 (3,7-dihydro-8-[(1E)-2-(3-methoxyphenyl)ethenyl]-7-methyl-3-[3-(phosphonooxy)propyl-1-(2-propynyl)-1H-purine-2,6-dione disodium salt hydrate), in modulating cocaine- and quinpirole-induced reinstatement to cocaine seeking. Intra-NAc pretreatment of CGS 21680 reduced both cocaine- and quinpirole-induced reinstatement. These effects were specific to cocaine reinstatement as intra-NAc CGS 21680 had no effect on sucrose seeking in rats trained to self-administer sucrose pellets. Intra-NAc treatment with MSX-3 modestly reinstated cocaine seeking when given alone, and exacerbated both cocaine- and quinpirole-induced reinstatement. Interestingly, the exacerbation of cocaine seeking produced by MSX-3 was only observed at sub-threshold doses of cocaine and quinpirole, suggesting that removing tonic A(2A) receptor activity enables behaviors mediated by dopamine receptors. Taken together, these findings suggest that A(2A) receptor stimulation reduces, while A(2A) blockade

  1. Milrinone attenuates thromboxane receptor-mediated hyperresponsiveness in hypoxic pulmonary arterial myocytes

    PubMed Central

    Santhosh, KT; Elkhateeb, O; Nolette, N; Outbih, O; Halayko, AJ; Dakshinamurti, S

    2011-01-01

    BACKGROUND AND PURPOSE Neonatal pulmonary hypertension (PPHN) is characterized by pulmonary vasoconstriction, due in part to dysregulation of the thromboxane prostanoid (TP) receptor. Hypoxia induces TP receptor–mediated hyperresponsiveness, whereas serine phosphorylation mediates desensitization of TP receptors. We hypothesized that prostacyclin (IP) receptor activity induces TP receptor phosphorylation and decreases ligand affinity; that TP receptor sensitization in hypoxic myocytes is due to IP receptor inactivation; and that this would be reversible by the cAMP-specific phosphodiesterase inhibitor milrinone. EXPERIMENTAL APPROACH We examined functional regulation of TP receptors by serine phosphorylation and effects of IP receptor stimulation and protein kinase A (PKA) activity on TP receptor sensitivity in myocytes from neonatal porcine resistance pulmonary arteries after 72 h hypoxia in vitro. Ca2+ response curves to U46619 (TP receptor agonist) were determined in hypoxic and normoxic myocytes incubated with or without iloprost (IP receptor agonist), forskolin (adenylyl cyclase activator), H8 (PKA inhibitor) or milrinone. TP and IP receptor saturation binding kinetics were measured in presence of iloprost or 8-bromo-cAMP. KEY RESULTS Ligand affinity for TP receptors was normalized in vitro by IP receptor signalling intermediates. However, IP receptor affinity was compromised in hypoxic myocytes, decreasing cAMP production. Milrinone normalized TP receptor sensitivity in hypoxic myocytes by restoring PKA-mediated regulatory TP receptor phosphorylation. CONCLUSIONS AND IMPLICATIONS TP receptor sensitivity and EC50 for TP receptor agonists was regulated by PKA, as TP receptor serine phosphorylation by PKA down-regulated Ca2+ mobilization. Hypoxia decreased IP receptor activity and cAMP generation, inducing TP receptor hyperresponsiveness, which was reversed by milrinone. PMID:21385177

  2. Multi-Inhibitory Effects of A2A Adenosine Receptor Signaling on Neutrophil Adhesion Under Flow.

    PubMed

    Yago, Tadayuki; Tsukamoto, Hiroki; Liu, Zhenghui; Wang, Ying; Thompson, Linda F; McEver, Rodger P

    2015-10-15

    A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain "swing-out," which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin Ab. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1, and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A and its substrate C-terminal Src kinase, an inhibitor of SFKs. Treating neutrophils with a protein kinase A inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases. Copyright © 2015 by The American Association of Immunologists, Inc.

  3. Multi-inhibitory effects of A2A adenosine receptor signaling on neutrophil adhesion under flow**

    PubMed Central

    Yago, Tadayuki; Tsukamoto, Hiroki; Liu, Zhenghui; Wang, Ying; Thompson, Linda F.; McEver, Rodger P.

    2015-01-01

    A2A adenosine receptor (A2AAR) signaling negatively regulates inflammatory responses in many disease models, but the detailed mechanisms remain unclear. We used the selective A2AAR agonist, ATL313, to examine how A2AAR signaling affects human and murine neutrophil adhesion under flow. Treating neutrophils with ATL313 inhibited selectin-induced, β2 integrin-dependent slow rolling and chemokine-induced, β2 integrin-dependent arrest on ICAM-1. ATL313 inhibited selectin-induced β2 integrin extension, which supports slow rolling, and chemokine-induced hybrid domain “swing-out”, which supports arrest. Furthermore, ATL313 inhibited integrin outside-in signaling as revealed by reduced neutrophil superoxide production and spreading on immobilized anti-β2 integrin antibody. ATL313 suppressed selectin-triggered activation of Src family kinases (SFKs) and p38 MAPK, chemokine-triggered activation of Ras-related protein 1 (Rap1), and β2 integrin-triggered activation of SFKs and Vav cytoskeletal regulatory proteins. ATL313 activated protein kinase A (PKA) and its substrate C-terminal Src kinase (Csk), an inhibitor of SFKs. Treating neutrophils with a PKA inhibitor blocked the actions of ATL313. In vivo, ATL313-treated neutrophils rolled faster and arrested much less frequently in postcapillary venules of the murine cremaster muscle after TNF-α challenge. Furthermore, ATL313 markedly suppressed neutrophil migration into the peritoneum challenged with thioglycollate. ATL313 did not affect A2AAR-deficient neutrophils, confirming its specificity. Our findings provide new insights into the anti-inflammatory mechanisms of A2AAR signaling and the potential utility of A2AAR agonists in inflammatory diseases. PMID:26355151

  4. Reinforcing and neurochemical effects of cannabinoid CB1 receptor agonists, but not cocaine, are altered by an adenosine A2A receptor antagonist.

    PubMed

    Justinová, Zuzana; Ferré, Sergi; Redhi, Godfrey H; Mascia, Paola; Stroik, Jessica; Quarta, Davide; Yasar, Sevil; Müller, Christa E; Franco, Rafael; Goldberg, Steven R

    2011-07-01

    Several recent studies suggest functional and molecular interactions between striatal adenosine A(2A) and cannabinoid CB(1) receptors. Here, we demonstrate that A(2A) receptors selectively modulate reinforcing effects of cannabinoids. We studied effects of A(2A) receptor blockade on the reinforcing effects of delta-9-tetrahydrocannabinol (THC) and the endogenous CB(1) receptor ligand anandamide under a fixed-ratio schedule of intravenous drug injection in squirrel monkeys. A low dose of the selective adenosine A(2A) receptor antagonist MSX-3 (1 mg/kg) caused downward shifts of THC and anandamide dose-response curves. In contrast, a higher dose of MSX-3 (3 mg/kg) shifted THC and anandamide dose-response curves to the left. MSX-3 did not modify cocaine or food pellet self-administration. Also, MSX-3 neither promoted reinstatement of extinguished drug-seeking behavior nor altered reinstatement of drug-seeking behavior by non-contingent priming injections of THC. Finally, using in vivo microdialysis in freely-moving rats, a behaviorally active dose of MSX-3 significantly counteracted THC-induced, but not cocaine-induced, increases in extracellular dopamine levels in the nucleus accumbens shell. The significant and selective results obtained with the lower dose of MSX-3 suggest that adenosine A(2A) antagonists acting preferentially at presynaptic A(2A) receptors might selectively reduce reinforcing effects of cannabinoids that lead to their abuse. However, the appearance of potentiating rather than suppressing effects on cannabinoid reinforcement at the higher dose of MSX-3 would likely preclude the use of such a compound as a medication for cannabis abuse. Adenosine A(2A) antagonists with more selectivity for presynaptic versus postsynaptic receptors could be potential medications for treatment of cannabis abuse. Addiction Biology © 2010 Society for the Study of Addiction. No claim to original US government works.

  5. Ligand-Dependent Activation and Deactivation of the Human Adenosine A2A Receptor

    PubMed Central

    Li, Jianing; Jonsson, Amanda L.; Beuming, Thijs; Shelley, John C.; Voth, Gregory A.

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR’s activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp2466.48). Our findings agree with a previously proposed view, that during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G protein-binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in crystal

  6. Ligand-dependent activation and deactivation of the human adenosine A(2A) receptor.

    PubMed

    Li, Jianing; Jonsson, Amanda L; Beuming, Thijs; Shelley, John C; Voth, Gregory A

    2013-06-12

    G-protein-coupled receptors (GPCRs) are membrane proteins with critical functions in cellular signal transduction, representing a primary class of drug targets. Acting by direct binding, many drugs modulate GPCR activity and influence the signaling pathways associated with numerous diseases. However, complete details of ligand-dependent GPCR activation/deactivation are difficult to obtain from experiments. Therefore, it remains unclear how ligands modulate a GPCR's activity. To elucidate the ligand-dependent activation/deactivation mechanism of the human adenosine A2A receptor (AA2AR), a member of the class A GPCRs, we performed large-scale unbiased molecular dynamics and metadynamics simulations of the receptor embedded in a membrane. At the atomic level, we have observed distinct structural states that resemble the active and inactive states. In particular, we noted key structural elements changing in a highly concerted fashion during the conformational transitions, including six conformational states of a tryptophan (Trp246(6.48)). Our findings agree with a previously proposed view that, during activation, this tryptophan residue undergoes a rotameric transition that may be coupled to a series of coherent conformational changes, resulting in the opening of the G-protein binding site. Further, metadynamics simulations provide quantitative evidence for this mechanism, suggesting how ligand binding shifts the equilibrium between the active and inactive states. Our analysis also proposes that a few specific residues are associated with agonism/antagonism, affinity, and selectivity, and suggests that the ligand-binding pocket can be thought of as having three distinct regions, providing dynamic features for structure-based design. Additional simulations with AA2AR bound to a novel ligand are consistent with our proposed mechanism. Generally, our study provides insights into the ligand-dependent AA2AR activation/deactivation in addition to what has been found in

  7. BM-520, an original TXA2 modulator, inhibits the action of thromboxane A2 and 8-iso-prostaglandin F2alphain vitro and in vivo on human and rodent platelets, and aortic vascular smooth muscles from rodents.

    PubMed

    Rolin, S; Hanson, J; Vastersaegher, C; Cherdon, C; Pratico, D; Masereel, B; Dogne, J M

    2007-08-01

    Thromboxane A(2) (TXA(2)) and 8-iso-PGF(2alpha) are two prostanoid agonists of the thromboxane A(2) receptor (TP), whose activation has been involved in platelet aggregation and atherosclerosis. Agents able to counteract the actions of these agonists are of great interest in the treatment and prevention of cardiovascular events. Here, we investigated in vitro and in vivo the pharmacological profile of BM-520, a new TP antagonist. In our experiments, this compound showed a great binding affinity for human washed platelets TP receptors, and prevented human platelet activation and aggregation induced by U-46619, arachidonic acid and 8-iso-PGF(2alpha). The TP receptor antagonist property of BM-520 was confirmed by its relaxing effect on rat aorta smooth muscle preparations precontracted with U-46619 and 8-iso-PGF(2alpha). Further, its TP antagonism was also demonstrated in vivo in guinea pig after a single intravenous injection (10 mg kg(-1)). We conclude that this novel TP antagonist could be a promising therapeutic tool in pathologies such as atherosclerosis where an increased production of TXA(2) and 8-iso-PGF(2alpha), as well as TP activation are well-established pathogenic events.

  8. Novel therapy in Parkinson's disease: adenosine A(2A) receptor antagonists.

    PubMed

    Szabó, Nikoletta; Kincses, Zsigmond Tamás; Vécsei, László

    2011-04-01

    Parkinson's disease (PD) is a progressive neurodegenerative disorder. To date, most of the currently available therapies in PD target the dopaminergic system and none of these therapeutic approaches have been proven to modify the course of the disease. To various extents, these drugs can also cause motor and non-motor complications. A novel target, the adenosine A(2A) receptor (AA2AR), was recently identified, blockade of which may alleviate Parkinsonian symptoms, reduce motor fluctuations and potentially afford neuroprotection. This review is based on a PubMed search covering the relationship of the adenosine receptors and PD. The role of the AA2AR is reviewed and the results of preclinical investigations of antagonists are assessed. A synopsis of current drug development is provided, with a special focus on the pharmacokinetics and relevant clinical trials. The localization of the AA2AR in the central nervous system, the ultra structural localization and the molecular mechanism of its action reveal the potential importance of the AA2AR in movement disorders. The theoretical background and experimental data indicate that AA2AR antagonists may have a potential therapeutic effect in Parkinson's disease. More importantly, the putative neuroprotective effect needs further investigation.

  9. Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists.

    PubMed

    Schindler, Charles W; Karcz-Kubicha, Marzena; Thorndike, Eric B; Müller, Christa E; Tella, Srihari R; Ferré, Sergi; Goldberg, Steven R

    2005-03-01

    1. The cardiovascular effects of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) and the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) were investigated in rats implanted with telemetry transmitters for the measurement of blood pressure and heart rate. 2. Intraperitoneal (i.p.) injections of the adenosine A1 receptor agonist CPA led to dose-dependent decreases in both blood pressure and heart rate. These effects of 0.3 mg kg(-1) CPA were antagonized by i.p. injections of the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dimethyl-xanthine (CPT), but not by i.p. injections of the adenosine A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3). Injections (i.p.) of the peripherally acting nonselective adenosine antagonist 8-sulfophenyltheophylline (8-SPT) and the purported nonselective adenosine antagonist caffeine also antagonized the cardiovascular effects of CPA. 3. The adenosine A2A agonist CGS 21680 given i.p. produced a dose-dependent decrease in blood pressure and an increase in heart rate. These effects of 0.5 mg kg(-1) CGS 21680 were antagonized by i.p. injections of the adenosine A2A receptor antagonist MSX-3, but not by i.p. injections of the antagonists CPT, 8-SPT or caffeine. 4. Central administration (intracerebral ventricular) of CGS 21680 produced an increase in heart rate, but no change in blood pressure. MSX-3 given i.p. antagonized the effects of the central injection of CGS 21680. 5. These results suggest that adenosine A1 receptor agonists produce decreases in blood pressure and heart rate that are mediated by A1 receptors in the periphery, with little or no contribution of central adenosine A1 receptors to those effects. 6. The heart rate increasing effect of adenosine A2A agonists appears to be mediated by adenosine A2A receptors in the central nervous system. The blood pressure decreasing

  10. Antiphospholipase A2 Receptor Autoantibodies: A Step Forward in the Management of Primary Membranous Nephropathy

    PubMed Central

    Obrisca, Bogdan; Ismail, Gener; Jurubita, Roxana; Baston, Catalin; Andronesi, Andreea; Mircescu, Gabriel

    2015-01-01

    Since the identification of PLA2R (M-type phospholipase A2 receptor) as the first human antigenic target in primary membranous nephropathy (MN), perpetual progress has been made in understanding the pathogenesis of this disease. Accumulating clinical data support a pathogenic role for the anti-PLA2R antibodies (PLA2R ABs), but confirmation in an animal model is still lacking. However, PLA2R ABs were related to disease activity and outcome, as well as to response therapy. Accordingly, PLA2R ABs assay seems to be promising tool not only to diagnose MN but also to predict the course of the disease and could open the way to personalize therapy. Nevertheless, validation of a universal assay with high precision and definition of cut-off levels, followed by larger studies with a prolonged follow-up period, are needed to confirm these prospects. PMID:26576418

  11. Antiphospholipase A2 Receptor Autoantibodies: A Step Forward in the Management of Primary Membranous Nephropathy.

    PubMed

    Obrisca, Bogdan; Ismail, Gener; Jurubita, Roxana; Baston, Catalin; Andronesi, Andreea; Mircescu, Gabriel

    2015-01-01

    Since the identification of PLA2R (M-type phospholipase A2 receptor) as the first human antigenic target in primary membranous nephropathy (MN), perpetual progress has been made in understanding the pathogenesis of this disease. Accumulating clinical data support a pathogenic role for the anti-PLA2R antibodies (PLA2R ABs), but confirmation in an animal model is still lacking. However, PLA2R ABs were related to disease activity and outcome, as well as to response therapy. Accordingly, PLA2R ABs assay seems to be promising tool not only to diagnose MN but also to predict the course of the disease and could open the way to personalize therapy. Nevertheless, validation of a universal assay with high precision and definition of cut-off levels, followed by larger studies with a prolonged follow-up period, are needed to confirm these prospects.

  12. M-type phospholipase A2 receptor autoantibodies and renal function in patients with primary membranous nephropathy.

    PubMed

    Hoxha, Elion; Harendza, Sigrid; Pinnschmidt, Hans; Panzer, Ulf; Stahl, Rolf A K

    2014-11-07

    Loss of renal function in patients with primary membranous nephropathy cannot be reliably predicted by laboratory or clinical markers at the time of diagnosis. M-type phospholipase A2 receptor autoantibodies have been shown to be associated with changes in proteinuria. Their eventual effect on renal function, however, is unclear. In this prospective, open, multicenter study, the potential role of M-type phospholipase A2 receptor autoantibodies levels on the increase of serum creatinine in 118 consecutive patients with membranous nephropathy and positivity for serum M-type phospholipase A2 receptor autoantibodies was analyzed. Patients were included in the study between April of 2010 and December of 2012 and observed until December of 2013. The clinical end point was defined as an increase of serum creatinine by ≥ 25% and serum creatinine reaching ≥ 1.3 mg/dl. Patients were divided into tertiles according to their M-type phospholipase A2 receptor autoantibody levels at the time of inclusion in the study: tertile 1 levels=20-86 units/ml (low), tertile 2 levels=87-201 units/ml (medium), and tertile 3 levels ≥ 202 units/ml (high). The median follow-up time of all patients in the study was 27 months (interquartile range=18-33 months). The clinical end point was reached in 69% of patients with high M-type phospholipase A2 receptor autoantibodies levels (tertile 3) but only 25% of patients with low M-type phospholipase A2 receptor autoantibodies levels. The average time to reach the study end point was 17.7 months in patients with high M-type phospholipase A2 receptor autoantibodies levels and 30.9 months in patients with low M-type phospholipase A2 receptor autoantibodies levels. A multivariate Cox regression analysis showed that high M-type phospholipase A2 receptor autoantibodies levels-in addition to men and older age-are an independent predictor for progressive loss of renal function. High M-type phospholipase A2 receptor autoantibodies levels were associated

  13. Mechanism and Site of Inhibition of AMPA Receptors: Pairing a Thiadiazole with a 2,3-Benzodiazepine Scaffold

    PubMed Central

    2013-01-01

    2,3-Benzodiazepine compounds are synthesized as drug candidates for treatment of various neurological disorders involving excessive activity of AMPA receptors. Here we report that pairing a thiadiazole moiety with a 2,3-benzodiazepine scaffold via the N-3 position yields an inhibitor type with >28-fold better potency and selectivity on AMPA receptors than the 2,3-benzodiazepine scaffold alone. Using whole-cell recording, we characterized two thiadiazolyl compounds, that is, one contains a 1,3,4-thiadiazole moiety and the other contains a 1,2,4-thiadiazole-3-one moiety. These compounds exhibit potent, equal inhibition of both the closed-channel and the open-channel conformations of all four homomeric AMPA receptor channels and two GluA2R-containing complex AMPA receptor channels. Furthermore, these compounds bind to the same receptor site as GYKI 52466 does, a site we previously termed as the “M” site. A thiadiazole moiety is thought to occupy more fully the side pocket of the receptor site or the “M” site, thereby generating a stronger, multivalent interaction between the inhibitor and the receptor binding site. We suggest that, as a heterocycle, a thiadiazole can be further modified chemically to produce a new class of even more potent, noncompetitive inhibitors of AMPA receptors. PMID:24313227

  14. Therapy of pancreatic cancer via an EphA2 receptor-targeted delivery of gemcitabine

    PubMed Central

    Barile, Elisa; Das, Swadesh K.; Emdad, Luni; Sarkar, Devanand; De, Surya K.; Kharagh, Susan Morvaridi; Stebbins, John L.; Pandol, Stephen J.; Fisher, Paul B.; Pellecchia, Maurizio

    2016-01-01

    First line treatment for pancreatic cancer consists of surgical resection, if possible, and a subsequent course of chemotherapy using the nucleoside analogue gemcitabine. In some patients, an active transport mechanism allows gemcitabine to enter efficiently into the tumor cells, resulting in a significant clinical benefit. However, in most patients, low expression of gemcitabine transporters limits the efficacy of the drug to marginal levels, and patients need frequent administration of the drug at high doses, significantly increasing systemic drug toxicity. In this article we focus on a novel targeted delivery approach for gemcitabine consisting of conjugating the drug with an EphA2 targeting agent. We show that the EphA2 receptor is highly expressed in pancreatic cancers, and accordingly, the drug-conjugate is more effective than gemcitabine alone in targeting pancreatic tumors. Our preliminary observations suggest that this approach may provide a general benefit to pancreatic cancer patients and offers a comprehensive strategy for enhancing delivery of diverse therapeutic agents to a wide range of cancers overexpressing EphA2, thereby potentially reducing toxicity while enhancing therapeutic efficacy. PMID:26959746

  15. The neuronal Ca(2+) -binding protein 2 (NECAB2) interacts with the adenosine A(2A) receptor and modulates the cell surface expression and function of the receptor.

    PubMed

    Canela, Laia; Luján, Rafael; Lluís, Carme; Burgueño, Javier; Mallol, Josefa; Canela, Enric I; Franco, Rafael; Ciruela, Francisco

    2007-09-01

    Heptaspanning membrane also known as G protein-coupled receptors (GPCR) do interact with a variety of intracellular proteins whose function is regulate receptor traffic and/or signaling. Using a yeast two-hybrid screen, NECAB2, a neuronal calcium binding protein, was identified as a binding partner for the adenosine A(2A) receptor (A(2A)R) interacting with its C-terminal domain. Co-localization, co-immunoprecipitation and pull-down experiments showed a close and specific interaction between A(2A)R and NECAB2 in both transfected HEK-293 cells and also in rat striatum. Immunoelectron microscopy detection of NECAB2 and A(2A)R in the rat striatopallidal structures indicated that both proteins are co-distributed in the same glutamatergic nerve terminals. The interaction of NECAB2 with A(2A)R modulated the cell surface expression, the ligand-dependent internalization and the receptor-mediated activation of the MAPK pathway. Overall, these results show that A(2A)R interacts with NECAB2 in striatal neurones co-expressing the two proteins and that the interaction is relevant for A(2A)R function.

  16. TP53 mutation-correlated genes predict the risk of tumor relapse and identify MPS1 as a potential therapeutic kinase in TP53-mutated breast cancers.

    PubMed

    Győrffy, Balázs; Bottai, Giulia; Lehmann-Che, Jacqueline; Kéri, György; Orfi, László; Iwamoto, Takayuki; Desmedt, Christine; Bianchini, Giampaolo; Turner, Nicholas C; de Thè, Hugues; André, Fabrice; Sotiriou, Christos; Hortobagyi, Gabriel N; Di Leo, Angelo; Pusztai, Lajos; Santarpia, Libero

    2014-05-01

    Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)-positive and ER-negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER-positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53-mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53-correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  17. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

    PubMed Central

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I.; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits. PMID:29497379

  18. Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins.

    PubMed

    Moreno, Estefanía; Canet, Júlia; Gracia, Eduard; Lluís, Carme; Mallol, Josefa; Canela, Enric I; Cortés, Antoni; Casadó, Vicent

    2018-01-01

    Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A 2A R present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A 2A R and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A 2A R involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A 2A R-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A 2A R). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

  19. Crystal structure of the adenosine A 2A receptor bound to an antagonist reveals a potential allosteric pocket

    SciTech Connect

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon

    The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phasemore » diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.« less

  20. SCPS-TP: A Satellite-Enhanced TCP

    NASA Technical Reports Server (NTRS)

    Scott, Keith; Torgerson, Leigh

    2004-01-01

    This viewgraph presentation reviews the Space Communications Protocol Standard Transport Protocol (SCPS-TP) which is a satellite enhanced Transport Control Protocol (TCP). The contents include: 1) Purpose; 2) Background; 3) Stressed Communication Environments; 4) SCPS-TP Features; 5) SCPS-TP Performance; 6) Performance Enhancing Proxies (PEPs); and 7) Ongoing and Future SCPS-TP Work.

  1. (99m)Tc-labeled SWL specific peptide for targeting EphA2 receptor.

    PubMed

    Liu, Yu; Lan, Xiaoli; Wu, Tao; Lang, Juntao; Jin, Xueyan; Sun, Xun; Wen, Qiong; An, Rui

    2014-07-01

    EphA2, one member of the Eph receptor family, is widely expressed in multiple aggressive cancers. SWL, a small peptide identified by phage display, has high binding affinity to EphA2, suggesting that it could be exploited for targeted molecular imaging. Therefore, a novel peptide-based probe, (99m)Tc-HYNIC-SWL, was developed and its potential to specifically target EphA2-positive tumors was investigated. The SWL peptide was labeled with hydrazinonicotinic acid (HYNIC), followed by (99m)Tc labeling. Immunofluorescence staining was carried out to detect the expression of EphA2 in A549 lung cancer cells and OCM-1 melanoma cells. Saturation binding experiments were performed by incubating A549 cells with increasing concentrations of radiolabeled peptide in vitro. To test the probe in vivo, nude mice bearing either A549 or OCM-1 derived tumors were established, injected with (99m)Tc-HYNIC-SWL, and subjected to SPECT imaging. Mice injected with excess unlabeled SWL were used as a specific control. Ex vivo γ-counting of dissected tissues from the mice was also performed to evaluate biodistribution. Immunofluorescence staining showed that A549 cells intensively expressed EphA2, while OCM-1 cells had little expression. (99m)Tc-HYNIC-SWL displayed high binding affinity with A549 cells (KD=2.6±0.7nM). From the SPECT images and the results of the biodistribution study, significantly higher uptake of the tracer was seen in A549 tumors (1.44±0.12 %ID/g) than in OCM-1 tumors (0.43±0.20 %ID/g) at 1h after injection. Pre-injection with excess unlabeled peptide in A549-bearing nude mice, significantly reduced tumor uptake of the radiolabeled probe (0.58±0.20 %ID/g) was seen. These data suggest that (99m)Tc-HYNIC-SWL specifically targets EphA2 in tumors. The expression of EphA2 can be noninvasively investigated using (99m)Tc-HYNIC-SWL by SPECT imaging. The in vitro and in vivo characteristics of (99m)Tc-HYNIC-SWL make it a promising probe for EphA2-positive tumor imaging

  2. Genetic polymorphism of the adenosine A2A receptor is associated with habitual caffeine consumption.

    PubMed

    Cornelis, Marilyn C; El-Sohemy, Ahmed; Campos, Hannia

    2007-07-01

    Caffeine is the most widely consumed stimulant in the world, and individual differences in response to its stimulating effects may explain some of the variability in caffeine consumption within a population. We examined whether genetic variability in caffeine metabolism [cytochrome P450 1A2 (CYP1A2) -163A-->C] or the main target of caffeine action in the nervous system [adenosine A(2A) receptor (ADORA2A) 1083C-->T] is associated with habitual caffeine consumption. Subjects (n=2735) were participants from a study of gene-diet interactions and risk of myocardial infarction who did not have a history of hypertension. Genotype frequencies were examined among persons who were categorized according to their self-reported daily caffeine intake, as assessed with a validated food-frequency questionnaire. The ADORA2A, but not the CYP1A2, genotype was associated with different amounts of caffeine intake. Compared with persons consuming <100 mg caffeine/d, the odds ratios for having the ADORA2A TT genotype were 0.74 (95% CI: 0.53, 1.03), 0.63 (95% CI: 0.48, 0.83), and 0.57 (95% CI: 0.42, 0.77) for those consuming 100-200, >200-400, and >400 mg caffeine/d, respectively. The association was more pronounced among current smokers than among nonsmokers (P for interaction = 0.07). Persons with the ADORA2A TT genotype also were significantly more likely to consume less caffeine (ie, <100 mg/d) than were carriers of the C allele [P=0.011 (nonsmokers), P=0.008 (smokers)]. Our findings show that the probability of having the ADORA2A 1083TT genotype decreases as habitual caffeine consumption increases. This observation provides a biologic basis for caffeine consumption behavior and suggests that persons with this genotype may be less vulnerable to caffeine dependence.

  3. Pharmacologic antagonism of thromboxane A2 receptors by trimetoquinol analogs in vitro and in vivo

    SciTech Connect

    Shin, Y.; Romstedt, K.J.; Doyle, K.

    1991-01-01

    Although (-)-(S)-trimetoquinol (1-(3,4,5-trimethoxy-benzyl)- 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; TMQ) is recognized as a potent bronchodilator, (+)-(R)-TMQ is a selective antagonist of human platelet aggregation and serotonin secretion induced by thromboxane A2 (TXA2) agonists. To confirm the pharmacological actions of TMQ analogs, the interaction of the drugs with TXA2 receptors was examined in human platelets and in a mouse sudden death model. The inhibitory potencies of TMQ analogs (pIC50 values) for displacement of (3H)SQ 29,548 binding to platelets showed excellent correlation with the respective pIC50 (-log IC50) values for U46619-induced aggregation (r = 0.99, P less than 0.01) and serotonin secretion (r = 0.99, Pmore » less than 0.01) in human platelet-rich plasma and for whole blood aggregation (r = 0.99, P less than 0.01). In each system, the rank order of inhibitory potencies was rac-iodoTMQ greater than or equal to (+)-(R)-TMQ greater than rac-TMQ much greater than (-)-(S)-TMQ. Antithrombotic effects of TMQ analogs were evaluated in a mouse sudden death model. In vivo antithrombotic potencies of these compounds were consistent with the in vitro potencies as TXA2 receptor antagonists in platelet systems. Administration of rac-iodoTMQ, (+)-(R)-TMQ and rac-TMQ 15 min before the injection of U46619 (800 micrograms/kg, iv) protected mice against U46619-induced sudden death. On the other hand, (-)-(S)-TMQ did not protect animals against death. Protection of U46619-induced cardiopulmonary thrombosis by TMQ analogs was seen at doses of 3-100 mg/kg.« less

  4. Macrophage A2A Adenosinergic Receptor Modulates Oxygen-Induced Augmentation of Murine Lung Injury

    PubMed Central

    D’Alessio, Franco R.; Eto, Yoshiki; Chau, Eric; Avalos, Claudia; Waickman, Adam T.; Garibaldi, Brian T.; Mock, Jason R.; Files, Daniel C.; Sidhaye, Venkataramana; Polotsky, Vsevolod Y.; Powell, Jonathan; Horton, Maureen; King, Landon S.

    2013-01-01

    Acute respiratory distress syndrome (ARDS) causes significant morbidity and mortality. Exacerbating factors increasing the risk of ARDS remain unknown. Supplemental oxygen is often necessary in both mild and severe lung disease. The potential effects of supplemental oxygen may include augmentation of lung inflammation by inhibiting anti-inflammatory pathways in alveolar macrophages. We sought to determine oxygen-derived effects on the anti-inflammatory A2A adenosinergic (ADORA2A) receptor in macrophages, and the role of the ADORA2A receptor in lung injury. Wild-type (WT) and ADORA2A−/− mice received intratracheal lipopolysaccharide (IT LPS), followed 12 hours later by continuous exposure to 21% oxygen (control mice) or 60% oxygen for 1 to 3 days. We measured the phenotypic endpoints of lung injury and the alveolar macrophage inflammatory state. We tested an ADORA2A-specific agonist, CGS-21680 hydrochloride, in LPS plus oxygen-exposed WT and ADORA2A−/− mice. We determined the specific effects of myeloid ADORA2A, using chimera experiments. Compared with WT mice, ADORA2A−/− mice exposed to IT LPS and 60% oxygen demonstrated significantly more histologic lung injury, alveolar neutrophils, and protein. Macrophages from ADORA2A−/− mice exposed to LPS plus oxygen expressed higher concentrations of proinflammatory cytokines and cosignaling molecules. CGS-21680 prevented the oxygen-induced augmentation of lung injury after LPS only in WT mice. Chimera experiments demonstrated that the transfer of WT but not ADORA2A−/− bone marrow cells into irradiated ADORA2A−/− mice reduced lung injury after LPS plus oxygen, demonstrating myeloid ADORA2A protection. ADORA2A is protective against lung injury after LPS and oxygen. Oxygen after LPS increases macrophage activation to augment lung injury by inhibiting the ADORA2A pathway. PMID:23349051

  5. Membrane omega-3 fatty acids modulate the oligomerisation kinetics of adenosine A2A and dopamine D2 receptors

    NASA Astrophysics Data System (ADS)

    Guixà-González, Ramon; Javanainen, Matti; Gómez-Soler, Maricel; Cordobilla, Begoña; Domingo, Joan Carles; Sanz, Ferran; Pastor, Manuel; Ciruela, Francisco; Martinez-Seara, Hector; Selent, Jana

    2016-01-01

    Membrane levels of docosahexaenoic acid (DHA), an essential omega-3 polyunsaturated fatty acid (ω-3 PUFA), are decreased in common neuropsychiatric disorders. DHA modulates key cell membrane properties like fluidity, thereby affecting the behaviour of transmembrane proteins like G protein-coupled receptors (GPCRs). These receptors, which have special relevance for major neuropsychiatric disorders have recently been shown to form dimers or higher order oligomers, and evidence suggests that DHA levels affect GPCR function by modulating oligomerisation. In this study, we assessed the effect of membrane DHA content on the formation of a class of protein complexes with particular relevance for brain disease: adenosine A2A and dopamine D2 receptor oligomers. Using extensive multiscale computer modelling, we find a marked propensity of DHA for interaction with both A2A and D2 receptors, which leads to an increased rate of receptor oligomerisation. Bioluminescence resonance energy transfer (BRET) experiments performed on living cells suggest that this DHA effect on the oligomerisation of A2A and D2 receptors is purely kinetic. This work reveals for the first time that membrane ω-3 PUFAs play a key role in GPCR oligomerisation kinetics, which may have important implications for neuropsychiatric conditions like schizophrenia or Parkinson’s disease.

  6. A2B Adenosine Receptor Agonist Improves Erectile Function in Diabetic Rats.

    PubMed

    Wen, Jiaming; Wang, Bohan; Du, Chuanjun; Xu, Gang; Zhang, Zhewei; Li, Yi; Zhang, Nan

    2015-10-01

    Diabetes is an important risk factor for erectile dysfunction (ED). Recent studies have indicated that A2B adenosine receptor (ADORA2B) signaling is essential for penile erection. Thus, we hypothesize that diabetic ED may be attributed to impaired A2B adenosine signaling. To test this hypothesis, we generated diabetic rats by injecting streptozocin as animal model. After 12 weeks, immunohistochemistry staining was used to localize the expression of ADORA2B. Western Blot and quantitative PCR were employed to determine ADORA2B expression level. Intracavernosal pressure (ICP) measurement was used to evaluate erectile function. Diabetic rats received a single intravenous injection of BAY 60-6583, an ADORA2B agonist, or vehicle solution, at 60 min before the ICP measurement. The results showed that ADORA2B expressed in the nerve bundle, smooth muscle, and endothelium in penile tissue of control mice. Western Blot and quantitative PCR results indicated that the expression levels of ADORA2B protein and mRNA were significantly reduced in penile tissues of diabetic rats. Functional studies showed that the erectile response induced by electrical stimulation was remarkably decreased in diabetic rats, compared with age-matched control rats. However, at 60 min after BAY 60-6583 treatment, the erectile function was improved in diabetic rats, suggesting that enhancement of ADORA2B signaling may improve erectile function in diabetic ED. This preclinical study has revealed a previously unrecognized therapeutic possibility of BAY 60-6583 as an effective and mechanism-based drug to treat diabetic ED. In conclusion, we propose that impaired A2B adenosine signaling is one of the pathological mechanisms of diabetic ED.

  7. The Sam Domain of EphA2 Receptor and its Relevance to Cancer: A Novel Challenge for Drug Discovery?

    PubMed

    Mercurio, Flavia A; Leone, Marilisa

    2016-01-01

    Eph receptors play important functions in developmental processes and diseases and among them EphA2 is well known for its controversial role in cancer. Drug discovery strategies are mainly centered on EphA2 extracellular ligand-binding domain however, the receptor also contains a largely unexplored cytosolic Sam (Sterile alpha motif) domain at the C-terminus. EphA2-Sam binds the Sam domain from the lipid phosphatase Ship2 and the first Sam domain of Odin. Sam-Sam interactions may be important to regulate ligand-induced receptor endocytosis and degradation i.e., processes that could be engaged against tumor malignancy. We critically analyzed literature related to a) Eph receptors with particular emphasis on EphA2 and its role in cancer, b) Sam domains, c) heterotypic Sam-Sam interactions involving EphA2-Sam. While literature data indicate that binding of EphA2-Sam to Ship2-Sam should largely generate pro-oncogenic effects in cancer cells, the correlation between EphA2- Sam/Odin-Sam1 complex and the disease is unclear. Recently a few linear peptides encompassing binding interfaces from either Ship2-Sam and Odin-Sam1 have been characterized but failed to efficiently block heterotypic Sam-Sam interactions involving EphA2-Sam due to the lack of a native like fold. Molecule antagonists of heterotypic EphA2-Sam associations could work as potential anticancer agents or be implemented as tools to further clarify receptor functions and eventually validate its role as a novel target in the field of anti-cancer drug discovery. Due to the failure of linear peptides there is a crucial need for novel approaches, based on cyclic or helical molecules, to target Sam-Sam interfaces.

  8. Equol is more active than soy isoflavone itself to compete for binding to thromboxane A(2) receptor in human platelets.

    PubMed

    Muñoz, Yenny; Garrido, Argelia; Valladares, Luis

    2009-03-01

    Several dietary intervention studies examining the health effect of soy isoflavones allude to the importance of equol in establishing the cardiovascular response to soy protein. Although, the specific mechanism by which this action occurs has not been established. The aim of this study was to investigate the inhibitory effect of soy-isoflavones and the metabolite of daidzein, equol, on agonist-induced platelet responses dependent on thromboxane A(2) (TxA(2)) receptor. Competitive radioligand binding assay was used to screen for affinity of these compounds to the TxA(2) receptor. The effect of equol on platelet activation, evaluate through of release of the ATP, by analogs of TxA(2) was analyzed. The effect of equol on platelet aggregation was investigated with ADP, U46619 (a TxA(2) mimic) and the calcium ionophore A23187. The data showed that aglycone isoflavones and equol bind to TxA(2) receptor in the micromol/L range, whereas their glucoside derivates had very low binding activity for this receptor. Under equilibrium conditions, the following order of the relative affinity in inhibiting [(3)H]-SQ29585 binding was: equol>genistein>daidzein>glycitein>genistin, daidzin, glycitin. Equol interaction was reversible and competitive for labeled-SQ29548 with not apparent decrease in the number of TxA(2) binding sites. In addition, from platelet activation studies, equol effectively inhibited ATP secretion elicited by the TxA(2) analog U46619. On the other hand, equol inhibited the platelet aggregation induced by U46619 and A23187, while it failed to inhibit that induced by ADP. The aglycone isoflavones from soy, and particularly equol, have been found to have biological effects attributable to thromboxane A(2) receptor antagonism. These findings may help elucidate how dietary isoflavone modulate platelet function and explain why soy-rich foods are claimed to have beneficial effects in the prevention of thrombotic events.

  9. Neuroprotection by caffeine in the MPTP model of parkinson's disease and its dependence on adenosine A2A receptors.

    PubMed

    Xu, K; Di Luca, D G; Orrú, M; Xu, Y; Chen, J-F; Schwarzschild, M A

    2016-05-13

    Considerable epidemiological and laboratory data have suggested that caffeine, a nonselective adenosine receptor antagonist, may protect against the underlying neurodegeneration of parkinson's disease (PD). Although both caffeine and more specific antagonists of the A2A subtype of adenosine receptor (A2AR) have been found to confer protection in animal models of PD, the dependence of caffeine's neuroprotective effects on the A2AR is not known. To definitively determine its A2AR dependence, the effect of caffeine on 1-methyl-4-phenyl-1,2,3,6 tetra-hydropyridine (MPTP) neurotoxicity was compared in wild-type (WT) and A2AR gene global knockout (A2A KO) mice, as well as in central nervous system (CNS) cell type-specific (conditional) A2AR knockout (cKO) mice that lack the receptor either in postnatal forebrain neurons or in astrocytes. In WT and in heterozygous A2AR KO mice caffeine pretreatment (25mg/kgip) significantly attenuated MPTP-induced depletion of striatal dopamine. By contrast in homozygous A2AR global KO mice caffeine had no effect on MPTP toxicity. In forebrain neuron A2AR cKO mice, caffeine lost its locomotor stimulant effect, whereas its neuroprotective effect was mostly preserved. In astrocytic A2AR cKO mice, both caffeine's locomotor stimulant and protective properties were undiminished. Taken together, these results indicate that neuroprotection by caffeine in the MPTP model of PD relies on the A2AR, although the specific cellular localization of these receptors remains to be determined. Copyright © 2016 IBRO. All rights reserved.

  10. Nucleus Accumbens Adenosine A2A Receptors Regulate Exertion of Effort by Acting on the Ventral Striatopallidal Pathway

    PubMed Central

    Mingote, Susana; Font, Laura; Farrar, Andrew M.; Vontell, Regina; Worden, Lila T.; Stopper, Colin M.; Port, Russell G.; Sink, Kelly S.; Bunce, Jamie G.; Chrobak, James J.; Salamone, John D.

    2009-01-01

    Goal-directed actions are sensitive to work-related response costs, and dopamine in nucleus accumbens is thought to modulate the exertion of effort in motivated behavior. Dopamine-rich striatal areas such as nucleus accumbens also contain high numbers of adenosine A2A receptors, and, for that reason, the behavioral and neurochemical effects of the adenosine A2A receptor agonist CGS 21680 [2-p-(2-carboxyethyl) phenethylamino-5′-N-ethylcarboxamidoadenosine] were investigated. Stimulation of accumbens adenosine A2A receptors disrupted performance of an instrumental task with high work demands (i.e., an interval lever-pressing schedule with a ratio requirement attached) but had little effect on a task with a lower work requirement. Immunohistochemical studies revealed that accumbens neurons that project to the ventral pallidum showed adenosine A2A receptors immunoreactivity. Moreover, activation of accumbens A2A receptors by local injections of CGS 21680 increased extracellular GABA levels in the ventral pallidum. Combined contralateral injections of CGS 21680 into the accumbens and the GABAA agonist muscimol into ventral pallidum (i.e., “disconnection” methods) also impaired response output, indicating that these structures are part of a common neural circuitry regulating the exertion of effort. Thus, accumbens adenosine A2A receptors appear to regulate behavioral activation and effort-related processes by modulating the activity of the ventral striatopallidal pathway. Research on the effort-related functions of these forebrain systems may lead to a greater understanding of pathological features of motivation, such as psychomotor slowing, anergia, and fatigue in depression. PMID:18768698

  11. TP53 mutations, expression and interaction networks in human cancers

    PubMed Central

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-01

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers. PMID:27880943

  12. TP53 mutations, expression and interaction networks in human cancers.

    PubMed

    Wang, Xiaosheng; Sun, Qingrong

    2017-01-03

    Although the associations of p53 dysfunction, p53 interaction networks and oncogenesis have been widely explored, a systematic analysis of TP53 mutations and its related interaction networks in various types of human cancers is lacking. Our study explored the associations of TP53 mutations, gene expression, clinical outcomes, and TP53 interaction networks across 33 cancer types using data from The Cancer Genome Atlas (TCGA). We show that TP53 is the most frequently mutated gene in a number of cancers, and its mutations appear to be early events in cancer initiation. We identified genes potentially repressed by p53, and genes whose expression correlates significantly with TP53 expression. These gene products may be especially important nodes in p53 interaction networks in human cancers. This study shows that while TP53-truncating mutations often result in decreased TP53 expression, other non-truncating TP53 mutations result in increased TP53 expression in some cancers. Survival analyses in a number of cancers show that patients with TP53 mutations are more likely to have worse prognoses than TP53-wildtype patients, and that elevated TP53 expression often leads to poor clinical outcomes. We identified a set of candidate synthetic lethal (SL) genes for TP53, and validated some of these SL interactions using data from the Cancer Cell Line Project. These predicted SL genes are promising candidates for experimental validation and the development of personalized therapeutics for patients with TP53-mutated cancers.

  13. The Role of Adenosine A2A Receptor, CYP450s, and PPARs in the Regulation of Vascular Tone

    PubMed Central

    Khayat, Maan T.

    2017-01-01

    Adenosine is an endogenous mediator involved in a myriad of physiologic functions, including vascular tone regulation. It is also implicated in some pathologic conditions. Four distinct receptor subtypes mediate the effects of adenosine, such as its role in the regulation of the vascular tone. Vascular tone regulation is a complex and continuous process which involves many mechanisms and mediators that are not fully disclosed. The vascular endothelium plays a pivotal role in regulating blood flow to and from all body organs. Also, the vascular endothelium is not merely a physical barrier; it is a complex tissue with numerous functions. Among adenosine receptors, A2A receptor subtype (A2AAR) stands out as the primary receptor responsible for the vasodilatory effects of adenosine. This review focuses on important effectors of the vascular endothelium, including adenosine, adenosine receptors, EETs (epoxyeicosatrienoic acids), HETEs (hydroxyeicosatetraenoic acids), PPARs (peroxisome proliferator-activated receptors), and KATP channels. Given the impact of vascular tone regulation in cardiovascular physiology and pathophysiology, better understanding of the mechanisms affecting it could have a significant potential for developing therapeutic agents for cardiovascular diseases. PMID:28884118

  14. Protein kinase A can block EphA2 receptor-mediated cell repulsion by increasing EphA2 S897 phosphorylation.

    PubMed

    Barquilla, Antonio; Lamberto, Ilaria; Noberini, Roberta; Heynen-Genel, Susanne; Brill, Laurence M; Pasquale, Elena B

    2016-09-01

    The EphA2 receptor tyrosine kinase plays key roles in tissue homeostasis and disease processes such as cancer, pathological angiogenesis, and inflammation through two distinct signaling mechanisms. EphA2 "canonical" signaling involves ephrin-A ligand binding, tyrosine autophosphorylation, and kinase activity; EphA2 "noncanonical" signaling involves phosphorylation of serine 897 (S897) by AKT and RSK kinases. To identify small molecules counteracting EphA2 canonical signaling, we developed a high-content screening platform measuring inhibition of ephrin-A1-induced PC3 prostate cancer cell retraction. Surprisingly, most hits from a screened collection of pharmacologically active compounds are agents that elevate intracellular cAMP by activating G protein-coupled receptors such as the β2-adrenoceptor. We found that cAMP promotes phosphorylation of S897 by protein kinase A (PKA) as well as increases the phosphorylation of several nearby serine/threonine residues, which constitute a phosphorylation hotspot. Whereas EphA2 canonical and noncanonical signaling have been viewed as mutually exclusive, we show that S897 phosphorylation by PKA can coexist with EphA2 tyrosine phosphorylation and block cell retraction induced by EphA2 kinase activity. Our findings reveal a novel paradigm in EphA2 function involving the interplay of canonical and noncanonical signaling and highlight the ability of the β2-adrenoceptor/cAMP/PKA axis to rewire EphA2 signaling in a subset of cancer cells. © 2016 Barquilla et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  15. Exploring an interaction of adenosine A2A receptor variability with coffee and tea intake in Parkinson's disease.

    PubMed

    Tan, E K; Lu, Z Y; Fook-Chong, S M C; Tan, E; Shen, H; Chua, E; Yih, Y; Teo, Y Y; Zhao, Y

    2006-09-05

    Caffeine is an adenosine receptor A1 and A2A receptor antagonist and a putative functional genetic variant of the A2A receptor (2592C > Tins) mediates caffeine-induced anxiety. Here we investigated the potential interaction of this A2A genetic variant with the quantity of coffee and tea intake and their relationship with the risk of PD. A total of 441 subjects consisting of 222 PD and 219 race, gender and age matched controls were included. A multivariate analysis of the variables including the 2592C > Tins A2A genotypes, age of onset, gender, and the quantity of tea and coffee intake, interaction of the A2A genotypes with coffee intake, interaction of A2A genotypes with tea intake demonstrated the quantity of coffee intake to be significantly associated with PD (P < 0.0005, OR = 0.922, 95% CI: 0.881, 0.964). However, there was no significant interaction of the A2A genotypes with the quantity of coffee and tea intake in modulating the risk of PD. The dose dependent protective effect of coffee intake in PD was independent of the 2592C > Tins A2A genotype suggesting that the pharmacogenetic action of caffeine in PD may be mediated differently from other caffeine-induced neurologic syndromes.

  16. SKCa Channels Blockage Increases the Expression of Adenosine A2A Receptor in Jurkat Human T Cells

    PubMed Central

    Regaya, Imed; Aidi-Knani, Sabrine; By, Youlet; Condo, Jocelyne; Gerolami, Victoria; Berge-Lefranc, Jean-Louis; Ben Hamida, Jeannette; Sabatier, Jean-Marc; Fenouillet, Emmanuel; Guieu, Régis

    2013-01-01

    Abstract Adenosine is a nucleoside displaying various biological effects via stimulation of four G-protein–coupled receptors, A1, A2A, A2B, and A3. Adenosine also modulates voltage-gated (Kv) and small conductance calcium-activated (SKCa) potassium channels. The effect of these potassium channels on the expression of adenosine receptors is poorly understood. We evaluated the action of BgK (a natural Kv channel blocker) and Lei-Dab7 (a synthetic SKCa channel blocker) on the expression of adenosine A2A receptors (A2AR) in Jurkat human T cells. We found that Lei-Dab7, but not BgK, increased the maximal binding value of the tritiated ligand ZM241385 to A2AR in a dose-dependent manner (+45% at 5 nM; +70% at 50 nM as compared to control). These results were further confirmed by Western blotting using a specific monoclonal antibody to human A2AR. The ligand affinity-related dissociation constant and A2AR mRNA amount were not significantly modified by either drug. We suggest that modulation of SKCa channels can influence membrane expression of A2AR and thus has a therapeutic potential. PMID:23593569

  17. Release inhibitory receptors activation favours the A2A-adenosine receptor-mediated facilitation of noradrenaline release in isolated rat tail artery

    PubMed Central

    Fresco, Paula; Diniz, Carmen; Queiroz, Glória; Gonçalves, Jorge

    2002-01-01

    Interactions between A2A-adenosine receptors and α2-, A1- and P2- release-inhibitory receptors, on the modulation of noradrenaline release were studied in isolated rat tail artery. Preparations were labelled with [3H]-noradrenaline, superfused with desipramine-containing medium, and stimulated electrically (100 pulses at 5 Hz or 20 pulses at 50 Hz).Blockade of α2-autoreceptors with yohimbine (1 μM) increased tritium overflow elicited by 100 pulses at 5 Hz but not by 20 pulses at 50 Hz.The selective A2A-receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680; 1 – 100 nM) enhanced tritium overflow elicited by 100 pulses at 5 Hz. Yohimbine prevented the effect of CGS 21680, which was restored by the A1-receptor agonist N6-cyclopentyladenosine (CPA; 100 nM) or by the P2-receptor agonist 2-methylthioadenosine triphosphate (2-MeSATP; 80 μM).CGS 21680 (100 nM) failed to increase tritium overflow elicited by 20 pulses at 50 Hz. The α2-adrenoceptor agonist 5-bromo-6-(2-imidazolin-2-ylamino)-quinoxaline (UK 14304; 30 nM), the A1-receptor agonist CPA (100 nM) or the P2-receptor agonist 2-MeSATP (80 μM) reduced tritium overflow. In the presence of these agonists CGS 21680 elicited a facilitation of tritium overflow.Blockade of potassium channels with tetraethylammonium (TEA; 5 mM) increased tritium overflow elicited by 100 pulses at 5 Hz to values similar to those obtained in the presence of yohimbine but did not prevent the effect of CGS 21680 (100 nM) on tritium overflow.It is concluded that, in isolated rat tail artery, the facilitation of noradrenaline release mediated by A2A-adenosine receptors is favoured by activation of release inhibitory receptors. PMID:12010771

  18. Genome-wide characterization reveals complex interplay between TP53 and TP63 in response to genotoxic stress

    PubMed Central

    McDade, Simon S.; Patel, Daksha; Moran, Michael; Campbell, James; Fenwick, Kerry; Kozarewa, Iwanka; Orr, Nicholas J.; Lord, Christopher J.; Ashworth, Alan A.; McCance, Dennis J.

    2014-01-01

    In response to genotoxic stress the TP53 tumour suppressor activates target gene expression to induce cell cycle arrest or apoptosis depending on the extent of DNA damage. These canonical activities can be repressed by TP63 in normal stratifying epithelia to maintain proliferative capacity or drive proliferation of squamous cell carcinomas, where TP63 is frequently overexpressed/amplified. Here we use ChIP-sequencing, integrated with microarray analysis, to define the genome-wide interplay between TP53 and TP63 in response to genotoxic stress in normal cells. We reveal that TP53 and TP63 bind to overlapping, but distinct cistromes of sites through utilization of distinctive consensus motifs and that TP53 is constitutively bound to a number of sites. We demonstrate that cisplatin and adriamycin elicit distinct effects on TP53 and TP63 binding events, through which TP53 can induce or repress transcription of an extensive network of genes by direct binding and/or modulation of TP63 activity. Collectively, this results in a global TP53-dependent repression of cell cycle progression, mitosis and DNA damage repair concomitant with activation of anti-proliferative and pro-apoptotic canonical target genes. Further analyses reveal that in the absence of genotoxic stress TP63 plays an important role in maintaining expression of DNA repair genes, loss of which results in defective repair. PMID:24823795

  19. Adenosine A2A Receptor in the Monkey Basal Ganglia: Ultrastructural Localization and Colocalization With the Metabotropic Glutamate Receptor 5 in the Striatum

    PubMed Central

    Bogenpohl, James W.; Ritter, Stefanie L.; Hall, Randy A.; Smith, Yoland

    2012-01-01

    The adenosine A2A receptor (A2AR) is a potential drug target for the treatment of Parkinson’s disease and other neurological disorders. In rodents, the therapeutic efficacy of A2AR modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A2AR localization in the primate basal ganglia and to assess the degree of A2AR/mGluR5 colocalization in the striatum. A2AR immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A2AR immunoreactivity. At the electron microscopic level, striatal A2AR labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A2AR immunoreactivity. Striatal A2AR/mGluR5 colocalization was commonly found; 60–70% of A2AR-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A2AR in the primate basal ganglia and demonstrate that A2AR and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A2AR could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism. PMID:21858817

  20. Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.

    PubMed

    Piirainen, Henni; Hellman, Maarit; Tossavainen, Helena; Permi, Perttu; Kursula, Petri; Jaakola, Veli-Pekka

    2015-02-17

    Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  1. Comparative Analysis of Virtual Screening Approaches in the Search for Novel EphA2 Receptor Antagonists.

    PubMed

    Callegari, Donatella; Pala, Daniele; Scalvini, Laura; Tognolini, Massimiliano; Incerti, Matteo; Rivara, Silvia; Mor, Marco; Lodola, Alessio

    2015-09-17

    The EphA2 receptor and its ephrin-A1 ligand form a key cell communication system, which has been found overexpressed in many cancer types and involved in tumor growth. Recent medicinal chemistry efforts have identified bile acid derivatives as low micromolar binders of the EphA2 receptor. However, these compounds suffer from poor physicochemical properties, hampering their use in vivo. The identification of compounds able to disrupt the EphA2-ephrin-A1 complex lacking the bile acid scaffold may lead to new pharmacological tools suitable for in vivo studies. To identify the most promising virtual screening (VS) protocol aimed at finding novel EphA2 antagonists, we investigated the ability of both ligand-based and structure-based approaches to retrieve known EphA2 antagonists from libraries of decoys with similar molecular properties. While ligand-based VSs were conducted using UniPR129 and ephrin-A1 ligand as reference structures, structure-based VSs were performed with Glide, using the X-ray structure of the EphA2 receptor/ephrin-A1 complex. A comparison of enrichment factors showed that ligand-based approaches outperformed the structure-based ones, suggesting ligand-based methods using the G-H loop of ephrin-A1 ligand as template as the most promising protocols to search for novel EphA2 antagonists.

  2. Hypoxia increases pulmonary arterial thromboxane receptor internalization independent of receptor sensitization.

    PubMed

    Fediuk, J; Sikarwar, A S; Lizotte, P P; Hinton, M; Nolette, N; Dakshinamurti, S

    2015-02-01

    Persistent Pulmonary Hypertension of the Newborn (PPHN) is characterized by sustained vasospasm and an increased thromboxane:prostacyclin ratio. Thromboxane (TP) receptors signal via Gαq to mobilize IP3 and Ca(2+), causing pulmonary arterial constriction. We have previously reported increased TP internalization in hypoxic pulmonary arterial (PA) myocytes. Serum-deprived PA myocytes were grown in normoxia (NM) or hypoxia (HM) for 72 h. TP localization was visualized in agonist-naïve and -challenged NM and HM by immunocytochemistry. Pathways for agonist-induced TP receptor internalization were determined by inhibiting caveolin- or clathrin-mediated endocytosis, and caveolar fractionation. Roles of actin and tubulin in TP receptor internalization were assessed using inhibitors of tubulin, actin-stabilizing or -destabilizing agents. PKA, PKC or GRK activation and inhibition were used to determine the kinase responsible for post-agonist receptor internalization. Agonist-naïve HM had decreased cell surface TP, and greater TP internalization after agonist challenge. TP protein did not sort with caveolin-rich fractions. Inhibition of clathrin prevented TP internalization. Both actin-stabilizing and -destabilizing agents prevented TP endocytosis in NM, while normalizing TP internalization in HM. Velocity of TP internalization was unaffected by PKA activity, but PKC activation normalized TP receptor internalization in HM. GRK inhibition had no effect. We conclude that in hypoxic myocytes, TP is internalized faster and to a greater extent than in normoxic controls. Internalization of the agonist-challenged TP requires clathrin, dynamic actin and is sensitive to PKC activity. TP receptor trafficking and signaling in hypoxia are pivotal to understanding increased vasoconstrictor sensitivity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Impact of purification conditions and history on A 2A adenosine receptor activity: The role of CHAPS and lipids

    SciTech Connect

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John

    The adenosine A 2A receptor (A 2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A 2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A 2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilizationmore » in DDM, DDM/CHAPS, or DHPC micelles, although A 2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A 2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  4. Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum.

    PubMed

    Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

    2018-04-01

    The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A 2A receptor (A 2A R) and cannabinoid CB 1 receptor (CB 1 R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A 2A R and CB 1 R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A 2A R-CB 1 R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A 2A R-CB 1 R heteromers in the dorsal striatum. Specifically, our data unveil that the A 2A R-CB 1 R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

  5. Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

    PubMed Central

    Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

    2018-01-01

    The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases. PMID:28102227

  6. Impact of purification conditions and history on A 2A adenosine receptor activity: The role of CHAPS and lipids

    DOE PAGES

    Naranjo, Andrea N.; McNeely, Patrick M.; Katsaras, John; ...

    2016-05-27

    The adenosine A 2A receptor (A 2AR) is a much-studied class A G protein-coupled receptor (GPCR). For biophysical studies, A 2AR is commonly purified in a detergent mixture of dodecylmaltoside (DDM), 3-(3-cholamidopropyl) dimethylammoniopropane sulfonate (CHAPS), and cholesteryl hemisuccinate (CHS). Here we studied the effects of CHAPS on the ligand binding activity and stability of wild type, full-length human A 2AR. We also tested the cholesterol requirement for maintaining the active conformation of the receptor when solubilized in detergent micelles. To this end, the receptor was purified using DDM, DDM/CHAPS, or the short hydrocarbon chain lipid 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC, di-6:0PC). After solubilizationmore » in DDM, DDM/CHAPS, or DHPC micelles, although A 2AR was found to retain its native-like fold, its binding ability was significantly compromised compared to DDM or DDM/CHAPS with CHS. It therefore appears that although cholesterol is not needed for A 2AR to retain a native-like, α-helical conformation, it may be a critical component for high affinity ligand binding. Further, this result suggests that the conformational differences between the active and inactive protein may be so subtle that commonly used spectroscopic methods are unable to differentiate between the two forms, highlighting the need for activity measurements. Furthermore, the studies presented in this paper also underline the importance of the protein’s purification history; i.e., detergents that interact with the protein during purification affect the ligand binding properties of the receptor in an irreversible manner.« less

  7. Binding and Function of Phosphotyrosines of the Ephrin A2 (EphA2) Receptor Using Synthetic Sterile α Motif (SAM) Domains*

    PubMed Central

    Borthakur, Susmita; Lee, HyeongJu; Kim, SoonJeung; Wang, Bing-Cheng; Buck, Matthias

    2014-01-01

    The sterile α motif (SAM) domain of the ephrin receptor tyrosine kinase, EphA2, undergoes tyrosine phosphorylation, but the effect of phosphorylation on the structure and interactions of the receptor is unknown. Studies to address these questions have been hindered by the difficulty of obtaining site-specifically phosphorylated proteins in adequate amounts. Here, we describe the use of chemically synthesized and specifically modified domain-length peptides to study the behavior of phosphorylated EphA2 SAM domains. We show that tyrosine phosphorylation of any of the three tyrosines, Tyr921, Tyr930, and Tyr960, has a surprisingly small effect on the EphA2 SAM structure and stability. However, phosphorylation at Tyr921 and Tyr930 enables differential binding to the Src homology 2 domain of the adaptor protein Grb7, which we propose will lead to distinct functional outcomes. Setting up different signaling platforms defined by selective interactions with adaptor proteins thus adds another level of regulation to EphA2 signaling. PMID:24825902

  8. Aberrant methylation of the M-type phospholipase A2 receptor gene in leukemic cells

    PubMed Central

    2012-01-01

    Background The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. Methods Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5`-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. Results Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2´-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. Conclusions The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis. PMID:23217014

  9. Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma

    PubMed Central

    Nolin, James D.; Ogden, H. Luke; Lai, Ying; Altemeier, William A.; Frevert, Charles W.; Bollinger, James G.; Naika, Gajendra S.; Kicic, Anthony; Stick, Stephen M.; Lambeau, Gerard; Henderson, William R.; Gelb, Michael H.

    2016-01-01

    Secreted phospholipase A2s (sPLA2s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA2s function as enzymes, some of the sPLA2s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA2s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1−/−) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1−/− mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1−/− mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation. PMID:27448109

  10. Identification of Epithelial Phospholipase A2 Receptor 1 as a Potential Target in Asthma.

    PubMed

    Nolin, James D; Ogden, H Luke; Lai, Ying; Altemeier, William A; Frevert, Charles W; Bollinger, James G; Naika, Gajendra S; Kicic, Anthony; Stick, Stephen M; Lambeau, Gerard; Henderson, William R; Gelb, Michael H; Hallstrand, Teal S

    2016-12-01

    Secreted phospholipase A 2 s (sPLA 2 s) regulate eicosanoid formation and have been implicated in asthma. Although sPLA 2 s function as enzymes, some of the sPLA 2 s bind with high affinity to a C-type lectin receptor, called PLA2R1, which has functions in both cellular signaling and clearance of sPLA 2 s. We sought to examine the expression of PLA2R1 in the airway epithelium of human subjects with asthma and the function of the murine Pla2r1 gene in a model of asthma. Expression of PLA2R1 in epithelial brushings was assessed in two distinct cohorts of children with asthma by microarray and quantitative PCR, and immunostaining for PLA2R1 was conducted on endobronchial tissue and epithelial brushings from adults with asthma. C57BL/129 mice deficient in Pla2r1 (Pla2r1 -/- ) were characterized in an ovalbumin (OVA) model of allergic asthma. PLA2R1 was differentially overexpressed in epithelial brushings of children with atopic asthma in both cohorts. Immunostaining for PLA2R1 in endobronchial tissue localized to submucosal glandular epithelium and columnar epithelial cells. After OVA sensitization and challenge, Pla2r1 -/- mice had increased airway hyperresponsiveness, as well as an increase in cellular trafficking of eosinophils to the peribronchial space and bronchoalveolar lavage fluid, and an increase in airway permeability. In addition, Pla2r1 -/- mice had more dendritic cells in the lung, higher levels of OVA-specific IgG, and increased production of both type-1 and type-2 cytokines by lung leukocytes. PLA2R1 is increased in the airway epithelium in asthma, and serves as a regulator of airway hyperresponsiveness, airway permeability, antigen sensitization, and airway inflammation.

  11. Synthesis and properties of a new water-soluble prodrug of the adenosine A 2A receptor antagonist MSX-2.

    PubMed

    Vollmann, Karl; Qurishi, Ramatullah; Hockemeyer, Jörg; Müller, Christa E

    2008-02-12

    The compound L-valine-3-{8-[(E)-2-[3-methoxyphenyl)ethenyl]-7-methyl-1-propargylxanthine-3-yl}propyl ester hydrochloride (MSX-4) was synthesized as an amino acid ester prodrug of the adenosine A2A receptor antagonist MSX-2. It was found to be stable in artificial gastric acid, but readily cleaved by pig liver esterase.

  12. Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

    PubMed

    Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

    2016-04-01

    The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Extrinsic Tryptophans as NMR Probes of Allosteric Coupling in Membrane Proteins: Application to the A2A Adenosine Receptor.

    PubMed

    Eddy, Matthew T; Gao, Zhan-Guo; Mannes, Philip; Patel, Nilkanth; Jacobson, Kenneth A; Katritch, Vsevolod; Stevens, Raymond C; Wüthrich, Kurt

    2018-06-20

    Tryptophan indole 15 N- 1 H signals are well separated in nuclear magnetic resonance (NMR) spectra of proteins. Assignment of the indole 15 N- 1 H signals therefore enables one to obtain site-specific information on complex proteins in supramacromolecular systems, even when extensive assignment of backbone 15 N- 1 H resonances is challenging. Here we exploit the unique indole 15 N- 1 H chemical shift by introducing extrinsic tryptophan reporter residues at judiciously chosen locations in a membrane protein for increased coverage of structure and function by NMR. We demonstrate this approach with three variants of the human A 2A adenosine receptor (A 2A AR), a class A G protein-coupled receptor, each containing a single extrinsic tryptophan near the receptor intracellular surface, in helix V, VI, or VII, respectively. We show that the native A 2A AR global protein fold and ligand binding activity are preserved in these A 2A AR variants. The indole 15 N- 1 H signals from the extrinsic tryptophan reporter residues show different responses to variable efficacy of drugs bound to the receptor orthosteric cavity, and the indole 15 N- 1 H chemical shift of the tryptophan introduced at the intracellular end of helix VI is sensitive to conformational changes resulting from interactions with a polypeptide from the carboxy terminus of the Gα S intracellular partner protein. Introducing extrinsic tryptophans into proteins in complex supramolecular systems thus opens new avenues for NMR investigations in solution.

  14. Evaluation of Tp-E Interval and Tp-E/QT Ratio in Patients with Aortic Stenosis.

    PubMed

    Yayla, Çağrı; Bilgin, Murat; Akboğa, Mehmet Kadri; Gayretli Yayla, Kadriye; Canpolat, Uğur; Dinç Asarcikli, Lale; Doğan, Mehmet; Turak, Osman; Çay, Serkan; Özeke, Özcan; Akyel, Ahmet; Yeter, Ekrem; Aydoğdu, Sinan

    2016-05-01

    The risk of syncope and sudden cardiac death due to ventricular arrhythmias increased in patients with aortic stenosis (AS). Recently, it was shown that Tp-e interval, Tp-e/QT, and Tp-e/QTc ratio can be novel indicators for prediction of ventricular arrhythmias and mortality. We aimed to investigate the association between AS and ventricular repolarization using Tp-e interval and Tp-e/QT ratio. Totally, 105 patients with AS and 60 control subjects were enrolled to this study. The severity of AS was defined by transthoracic echocardiographic examination. Tp-e interval, Tp-e/QT, and Tp-e/QTc ratios were measured from the 12-lead electrocardiogram. Tp-e interval, Tp-e/QT, and Tp-e/QTc ratios were significantly increased in parallel to the severity of AS (P < 0.001, P = 0.001, and P = 0.001, respectively). Also, it was shown that Tp-e/QTc ratio had significant positive correlation with mean aortic gradient (r = 0.192, P = 0.049). In multivariate logistic regression analysis, Tp-e/QTc ratio and left ventricular mass were found to be independent predictors of severe AS (P = 0.03 and P = 0.04, respectively). Our study showed that Tp-e interval, Tp-e/QT, and Tp-e/QTc ratios were increased in patients with severe AS. Tp-e/QTc ratio and left ventricular mass were found as independent predictors of severe AS. © 2015 Wiley Periodicals, Inc.

  15. Presynaptic facilitatory adenosine A2A receptors mediate fade induced by neuromuscular relaxants that exhibit anticholinesterase activity.

    PubMed

    Bornia, Elaine Cs; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson

    2011-03-01

    1. Pancuronium, cisatracurium and vecuronium are antinicotinic agents that, in contrast with d-tubocurarine and hexamethonium, exhibit anticholinesterase activity. Pancuronium-, cisatracurium- and vecuronium-induced fade results from blockade of facilitatory nicotinic receptors on motor nerves, but fade produced by such agents also depends on the presynaptic activation of inhibitory muscarinic M2 receptors by acetylcholine released from motor nerve terminals and activation of inhibitory adenosine A1 receptors by adenosine released from motor nerves and muscles. The participation of presynaptic facilitatory A2A receptors in fade caused by pancuronium, cisatracurium and vecuronium has not yet been investigated. In the present study, we determined the effects of ZM241385, an antagonist of presynaptic facilitatory A2A receptors, on fade produced by these neuromuscular relaxants in the rat phrenic nerve-diaphragm (PND) preparation. 2. The muscles were stimulated indirectly at 75±3Hz to induce a sustained tetanizing muscular contraction. The lowest concentration at which each antinicotinic agent produced fade without modifying initial tetanic tension (presynaptic action) was determined. 3. d-Tubocurarine-induced fade occurred only at 55 nmol/L, a concentration that also reduced maximal tetanic tension (post-synaptic action). At 10 nmol/L, ZM 241385 alone did not produce fade, but it did attenuate pancuronium (0.32 μmol/L)-, cisatracurium (0.32 μmol/L)- and vecuronium (0.36 μmol/L)-induced fade. 4. The fade induced by the 'pure' antinicotinic agents d-tubocurarine (55 nmol/L) and hexamethonium (413 μmol/L) was not altered by 10 nmol/L ZM 241385, indicating that presynaptic adenosine A2A receptors play a significant role in the fade produced by antinicotinic agents when such agents have anticholinesterase activity. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

  16. Adenosine A2A receptors modulate the dopamine D2 receptor-mediated inhibition of synaptic transmission in the mouse prefrontal cortex.

    PubMed

    Real, Joana I; Simões, Ana Patrícia; Cunha, Rodrigo A; Ferreira, Samira G; Rial, Daniel

    2018-05-01

    Prefrontal cortex (PFC) circuits are modulated by dopamine acting on D 1 - and D 2 -like receptors, which are pharmacologically exploited to manage neuropsychiatric conditions. Adenosine A 2A receptors (A 2 A R) also control PFC-related responses and A 2 A R antagonists are potential anti-psychotic drugs. As tight antagonistic A 2 A R-D 2 R and synergistic A 2 A R-D 1 R interactions occur in other brain regions, we now investigated the crosstalk between A 2 A R and D 1 /D 2 R controlling synaptic transmission between layers II/III and V in mouse PFC coronal slices. Dopamine decreased synaptic transmission, a presynaptic effect based on the parallel increase in paired-pulse responses. Dopamine inhibition was prevented by the D 2 R-like antagonist sulpiride but not by the D 1 R antagonist SCH23390 and was mimicked by the D 2 R agonist sumanirole, but not by the agonists of either D 4 R (A-412997) or D 3 R (PD128907). Dopamine inhibition was prevented by the A 2 A R antagonist, SCH58261, and attenuated in A 2 A R knockout mice. Accordingly, triple-labelling immunocytochemistry experiments revealed the co-localization of A 2 A R and D 2 R immunoreactivity in glutamatergic (vGluT1-positive) nerve terminals of the PFC. This reported positive A 2 A R-D 2 R interaction controlling PFC synaptic transmission provides a mechanistic justification for the anti-psychotic potential of A 2 A R antagonists. © 2018 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  17. The blood-brain barrier internalises Cryptococcus neoformans via the EphA2-tyrosine kinase receptor.

    PubMed

    Aaron, Phylicia A; Jamklang, Mantana; Uhrig, John P; Gelli, Angie

    2018-03-01

    Cryptococcus neoformans is an opportunistic fungal pathogen that causes life-threatening meningitis most commonly in populations with impaired immunity. Here, we resolved the transcriptome of the human brain endothelium challenged with C. neoformans to establish whether C. neoformans invades the CNS by co-opting particular signalling pathways as a means to promote its own entry. Among the 5 major pathways targeted by C. neoformans, the EPH-EphrinA1 (EphA2) tyrosine kinase receptor-signalling pathway was examined further. Silencing the EphA2 receptor transcript in a human brain endothelial cell line or blocking EphA2 activity with an antibody or chemical inhibitor prevented transmigration of C. neoformans in an in vitro model of the blood-brain barrier (BBB). In contrast, treating brain endothelial cells with an EphA2 chemical agonist or an EphA2 ligand promoted greater migration of fungal cells across the BBB. C. neoformans activated the EPH-tyrosine kinase pathway through a CD44-dependent phosphorylation of EphA2, promoting clustering and internalisation of EphA2 receptors. Moreover, HEK293T cells expressing EphA2 revealed an association between EphA2 and C. neoformans that boosted internalisation of C. neoformans. Collectively, the results suggest that C. neoformans promotes EphA2 activity via CD44, and this in turn creates a permeable barrier that facilitates the migration of C. neoformans across the BBB. © 2017 John Wiley & Sons Ltd.

  18. Deletion of striatal adenosine A2A receptor spares latent inhibition and prepulse inhibition but impairs active avoidance learning

    PubMed Central

    Singer, Philipp; Wei, Catherine J.; Chen, Jiang-Fan; Boison, Detlev; Yee, Benjamin K.

    2013-01-01

    Following early clinical leads, the adenosine A2AR receptor (A2AR) has continued to attract attention as a potential novel target for treating schizophrenia; especially against the negative and cognitive symptoms of the disease because of A2AR’s unique modulatory action over glutamatergic in addition to dopaminergic signaling. Through the antagonistic interaction with the dopamine D2 receptor, and by regulating glutamate release and N-methyl-d-aspartate receptor function, striatal A2AR is ideally positioned to fine-tune the dopamine-glutamate balance whose disturbance is implicated in the pathophysiology of schizophrenia. However, the precise function of striatal A2ARsin the regulation of schizophrenia-relevant behavior is poorly understood. Here, we tested the impact of conditional striatum-specific A2AR knockout (st-A2AR-KO) on latent inhibition (LI) and prepulse inhibition (PPI) – behavior that is tightly regulated by striatal dopamine and glutamate. These are two common cross-species translational tests for the assessment of selective attention and sensorimotor gating deficits reported in schizophrenia patients; and enhanced performance in these tests is associated with antipsychotic drug action. We found that neither LI nor PPI was significantly affected in st-A2AR-KO mice; although a deficit in active avoidance learning was identified in these animals. The latter phenotype, however, was not replicated in another form of aversive conditioning – conditioned taste aversion. Hence, the present study shows that neither learned inattention (as measured by LI) nor sensory gating (as indexed by PPI) requires the integrity of striatal A2ARs– a finding that may undermine the hypothesized importance of A2AR in the genesis and/or treatment of schizophrenia. PMID:23276608

  19. Antidepressant activity of the adenosine A2A receptor antagonist, istradefylline (KW-6002) on learned helplessness in rats.

    PubMed

    Yamada, Koji; Kobayashi, Minoru; Shiozaki, Shizuo; Ohta, Teruko; Mori, Akihisa; Jenner, Peter; Kanda, Tomoyuki

    2014-07-01

    Istradefylline, an adenosine A2A receptor antagonist, improves motor function in animal models of Parkinson's disease (PD) and in patients with PD. In addition, some A2A antagonists exert antidepressant-like activity in rodent models of depression, such as the forced swim and the tail suspension tests. We have investigated the effect of istradefylline on depression-like behaviors using the rat learned helplessness (LH) model. Acute, as well as chronic, oral administration of istradefylline significantly improved the inescapable shock (IES)-induced escape deficit with a degree of efficacy comparable to chronic treatment with the tricyclic antidepressant desipramine and the selective serotonin (5-HT) reuptake inhibitor, fluoxetine. Both the A1/A2A receptor nonspecific antagonist theophylline and the moderately selective antagonist CGS15943, but not the A1 selective antagonist DPCPX, ameliorated the IES-induced escape deficit. The enhancement of escape response by istradefylline was reversed by a local injection of the A2A specific agonist CGS21680 either into the nucleus accumbens, the caudate-putamen, or the paraventricular nucleus of the hypothalamus, but not by the A1 specific agonist R-PIA into the nucleus accumbens. Moreover, neither the 5-HT2A/2C receptor antagonist methysergide or the adrenergic α 2 antagonist yohimbine, nor the β-adrenergic antagonist propranolol, affected the improvement of escape response induced by istradefylline. Istradefylline exerts antidepressant-like effects via modulation of A2A receptor activity which is independent of monoaminergic transmission in the brain. Istradefylline may represent a novel treatment option for depression in PD as well as for the motor symptoms.

  20. Pancreatic and snake venom presynaptically active phospholipases A2 inhibit nicotinic acetylcholine receptors.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Kryukova, Elena V; Spirova, Ekaterina N; Shelukhina, Irina V; Starkov, Vladislav G; Andreeva, Tatyana V; Faure, Grazyna; Zouridakis, Marios; Tsetlin, Victor I; Utkin, Yuri N

    2017-01-01

    Phospholipases A2 (PLA2s) are enzymes found throughout the animal kingdom. They hydrolyze phospholipids in the sn-2 position producing lysophospholipids and unsaturated fatty acids, agents that can damage membranes. PLA2s from snake venoms have numerous toxic effects, not all of which can be explained by phospholipid hydrolysis, and each enzyme has a specific effect. We have earlier demonstrated the capability of several snake venom PLA2s with different enzymatic, cytotoxic, anticoagulant and antiproliferative properties, to decrease acetylcholine-induced currents in Lymnaea stagnalis neurons, and to compete with α-bungarotoxin for binding to nicotinic acetylcholine receptors (nAChRs) and acetylcholine binding protein. Since nAChRs are implicated in postsynaptic and presynaptic activities, in this work we probe those PLA2s known to have strong presynaptic effects, namely β-bungarotoxin from Bungarus multicinctus and crotoxin from Crotalus durissus terrificus. We also wished to explore whether mammalian PLA2s interact with nAChRs, and have examined non-toxic PLA2 from porcine pancreas. It was found that porcine pancreatic PLA2 and presynaptic β-bungarotoxin blocked currents mediated by nAChRs in Lymnaea neurons with IC50s of 2.5 and 4.8 μM, respectively. Crotoxin competed with radioactive α-bungarotoxin for binding to Torpedo and human α7 nAChRs and to the acetylcholine binding protein. Pancreatic PLA2 interacted similarly with these targets; moreover, it inhibited radioactive α-bungarotoxin binding to the water-soluble extracellular domain of human α9 nAChR, and blocked acetylcholine induced currents in human α9α10 nAChRs heterologously expressed in Xenopus oocytes. These and our earlier results show that all snake PLA2s, including presynaptically active crotoxin and β-bungarotoxin, as well as mammalian pancreatic PLA2, interact with nAChRs. The data obtained suggest that this interaction may be a general property of all PLA2s, which should be proved by

  1. Selective inactivation of adenosine A2A receptors in striatal neurons enhances working memory and reversal learning

    PubMed Central

    Wei, Catherine J.; Singer, Philipp; Coelho, Joana; Boison, Detlev; Feldon, Joram; Yee, Benjamin K.; Chen, Jiang-Fan

    2011-01-01

    The adenosine A2A receptor (A2AR) is highly enriched in the striatum where it is uniquely positioned to integrate dopaminergic, glutamatergic, and other signals to modulate cognition. Although previous studies support the hypothesis that A2AR inactivation can be pro-cognitive, analyses of A2AR's effects on cognitive functions have been restricted to a small subset of cognitive domains. Furthermore, the relative contribution of A2ARs in distinct brain regions remains largely unknown. Here, we studied the regulation of multiple memory processes by brain region-specific populations of A2ARs. Specifically, we evaluated the cognitive impacts of conditional A2AR deletion restricted to either the entire forebrain (i.e., cerebral cortex, hippocampus, and striatum, fb-A2AR KO) or to striatum alone (st-A2AR KO) in recognition memory, working memory, reference memory, and reversal learning. This comprehensive, comparative analysis showed for the first time that depletion of A2AR-dependent signaling in either the entire forebrain or striatum alone is associated with two specific phenotypes indicative of cognitive flexibility—enhanced working memory and enhanced reversal learning. These selective pro-cognitive phenotypes seemed largely attributed to inactivation of striatal A2ARs as they were captured by A2AR deletion restricted to striatal neurons. Neither spatial reference memory acquisition nor spatial recognition memory were grossly affected, and no evidence for compensatory changes in striatal or cortical D1, D2, or A1 receptor expression was found. This study provides the first direct demonstration that targeting striatal A2ARs may be an effective, novel strategy to facilitate cognitive flexibility under normal and pathologic conditions. PMID:21693634

  2. Selective inactivation of adenosine A(2A) receptors in striatal neurons enhances working memory and reversal learning.

    PubMed

    Wei, Catherine J; Singer, Philipp; Coelho, Joana; Boison, Detlev; Feldon, Joram; Yee, Benjamin K; Chen, Jiang-Fan

    2011-01-01

    The adenosine A(2A) receptor (A(2A)R) is highly enriched in the striatum where it is uniquely positioned to integrate dopaminergic, glutamatergic, and other signals to modulate cognition. Although previous studies support the hypothesis that A(2A)R inactivation can be pro-cognitive, analyses of A(2A)R's effects on cognitive functions have been restricted to a small subset of cognitive domains. Furthermore, the relative contribution of A(2A)Rs in distinct brain regions remains largely unknown. Here, we studied the regulation of multiple memory processes by brain region-specific populations of A(2A)Rs. Specifically, we evaluated the cognitive impacts of conditional A(2A)R deletion restricted to either the entire forebrain (i.e., cerebral cortex, hippocampus, and striatum, fb-A(2A)R KO) or to striatum alone (st-A(2A)R KO) in recognition memory, working memory, reference memory, and reversal learning. This comprehensive, comparative analysis showed for the first time that depletion of A(2A)R-dependent signaling in either the entire forebrain or striatum alone is associated with two specific phenotypes indicative of cognitive flexibility-enhanced working memory and enhanced reversal learning. These selective pro-cognitive phenotypes seemed largely attributed to inactivation of striatal A(2A)Rs as they were captured by A(2A)R deletion restricted to striatal neurons. Neither spatial reference memory acquisition nor spatial recognition memory were grossly affected, and no evidence for compensatory changes in striatal or cortical D(1), D(2), or A(1) receptor expression was found. This study provides the first direct demonstration that targeting striatal A(2A)Rs may be an effective, novel strategy to facilitate cognitive flexibility under normal and pathologic conditions.

  3. Adenosine enhances sweet taste through A2B receptors in the taste bud

    PubMed Central

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (Type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca2+ mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 µM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (Type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 µM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell RT-PCR on isolated vallate taste cells, we show that many Receptor cells express adenosine receptors, Adora2b, while Presynaptic (Type III) and Glial-like (Type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5′-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase (ACPP). Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste. PMID:22219293

  4. Adenosine enhances sweet taste through A2B receptors in the taste bud.

    PubMed

    Dando, Robin; Dvoryanchikov, Gennady; Pereira, Elizabeth; Chaudhari, Nirupa; Roper, Stephen D

    2012-01-04

    Mammalian taste buds use ATP as a neurotransmitter. Taste Receptor (type II) cells secrete ATP via gap junction hemichannels into the narrow extracellular spaces within a taste bud. This ATP excites primary sensory afferent fibers and also stimulates neighboring taste bud cells. Here we show that extracellular ATP is enzymatically degraded to adenosine within mouse vallate taste buds and that this nucleoside acts as an autocrine neuromodulator to selectively enhance sweet taste. In Receptor cells in a lingual slice preparation, Ca(2+) mobilization evoked by focally applied artificial sweeteners was significantly enhanced by adenosine (50 μM). Adenosine had no effect on bitter or umami taste responses, and the nucleoside did not affect Presynaptic (type III) taste cells. We also used biosensor cells to measure transmitter release from isolated taste buds. Adenosine (5 μM) enhanced ATP release evoked by sweet but not bitter taste stimuli. Using single-cell reverse transcriptase (RT)-PCR on isolated vallate taste cells, we show that many Receptor cells express the adenosine receptor, Adora2b, while Presynaptic (type III) and Glial-like (type I) cells seldom do. Furthermore, Adora2b receptors are significantly associated with expression of the sweet taste receptor subunit, Tas1r2. Adenosine is generated during taste stimulation mainly by the action of the ecto-5'-nucleotidase, NT5E, and to a lesser extent, prostatic acid phosphatase. Both these ecto-nucleotidases are expressed by Presynaptic cells, as shown by single-cell RT-PCR, enzyme histochemistry, and immunofluorescence. Our findings suggest that ATP released during taste reception is degraded to adenosine to exert positive modulation particularly on sweet taste.

  5. Role of A1 and A2A adenosine receptor agonists in adipose tissue inflammation induced by obesity in mice.

    PubMed

    DeOliveira, Caroline Candida; Paiva Caria, Cintia Rabelo E; Ferreira Gotardo, Erica Martins; Ribeiro, Marcelo Lima; Gambero, Alessandra

    2017-03-15

    Adenosine receptors are expressed in adipose tissue and control physiological and pathological events such as lipolysis and inflammation. The aim of this study was to evaluate the activity of N 6 -cyclopentyladenosine (CPA), a potent and selective A 1 adenosine receptor agonist; 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxyamidoadenosine hydrochloride (CGS-21680), an A 2A adenosine receptor agonist; and 5'-N-ethylcarboxamidoadenosine (NECA), a potent non-selective adenosine receptor agonist on adipose tissue inflammatory alterations induced by obesity in mice. Swiss mice were fed with a high-fat diet for 12 weeks and agonists were administered in the last two weeks. Body weight, adiposity and glucose homeostasis were evaluated. Inflammation in adipose tissue was assessed by evaluation of adipokine production and macrophage infiltration. Adenosine receptor signaling in adipose tissue was also evaluated. Mice that received CGS21680 presented an improvement in glucose homeostasis in association with systemically reduced inflammatory markers (TNF-α, PAI-1) and in the visceral adipose tissue (TNF-α, MCP-1, macrophage infiltration). Activation of p38 signaling was found in adipose tissue of this group of mice. NECA-treated mice presented some improvements in glucose homeostasis associated with an observed weight loss. Mice that received CPA presented only a reduction in the ex vivo basal lipolysis rate measured within visceral adipose tissue. In conclusion, administration of the A 2A receptor agonist to obese mice resulted in improvements in glucose homeostasis and adipose tissue inflammation, corroborating the idea that new therapeutics to treat obesity could emerge from these compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Cocaine self-administration differentially affects allosteric A2A-D2 receptor-receptor interactions in the striatum. Relevance for cocaine use disorder.

    PubMed

    Pintsuk, Julia; Borroto-Escuela, Dasiel O; Pomierny, Bartosz; Wydra, Karolina; Zaniewska, Magdalena; Filip, Malgorzata; Fuxe, Kjell

    2016-05-01

    In the current study behavioral and biochemical experiments were performed to study changes in the allosteric A2AR-D2R interactions in the ventral and dorsal striatum after cocaine self-administration versus corresponding yoked saline control. By using ex vivo [(3)H]-raclopride/quinpirole competition experiments, the effects of the A2AR agonist CGS 21680 (100 nM) on the KiH and KiL values of the D2-like receptor (D2-likeR) were determined. One major result was a significant reduction in the D2-likeR agonist high affinity state observed with CGS 21680 after cocaine self-administration in the ventral striatum compared with the yoked saline group. The results therefore support the hypothesis that A2AR agonists can at least in part counteract the motivational actions of cocaine. This action is mediated via the D2-likeR by targeting the A2AR protomer of A2AR-D2-like R heteroreceptor complexes in the ventral striatum, which leads to the reduction of D2-likeR protomer recognition through the allosteric receptor-receptor interaction. In contrast, in the dorsal striatum the CGS 21680-induced antagonistic modulation in the D2-likeR agonist high affinity state was abolished after cocaine self-administration versus the yoked saline group probably due to a local dysfunction/disruption of the A2AR-D2-like R heteroreceptor complexes. Such a change in the dorsal striatum in cocaine self-administration can contribute to the development of either locomotor sensitization, habit-forming learning and/or the compulsive drug seeking by enhanced D2-likeR protomer signaling. Potential differences in the composition and stoichiometry of the A2AR-D2R heteroreceptor complexes, including differential recruitment of sigma 1 receptor, in the ventral and dorsal striatum may explain the differential regional changes observed in the A2A-D2-likeR interactions after cocaine self-administration. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Evaluation of Tp-Te Interval and Tp-Te/QT Ratio in Patients with Coronary Slow Flow Tp-Te/QT Ratio and Coronary Slow Flow.

    PubMed

    Tenekecioglu, Erhan; Karaagac, Kemal; Yontar, Osman Can; Agca, Fahriye Vatansever; Ozluk, Ozlem Arican; Tutuncu, Ahmet; Arslan, Burhan; Yilmaz, Mustafa

    2015-06-01

    Coronary slow flow (CSF) phenomenon is described by angiographically normal coronary arteries with delayed opacification of the distal vasculature. Several studies have suggested that the interval from the peak to the end of the electrocardiographic T wave (Tp-Te) may correspond to the transmural dispersion of the repolarization and that increased Tp-Te interval and Tp-Te/QT ratio are associated with malignant ventricular arrhythmias. The aim of this study was to evaluate the ventricular repolarization by using Tp-Te interval and Tp-Te/QT ratio in patients with CSF. This study included 50 CSF patients (40 male, mean age 48.6±12.5 years) and 40 control individuals (23 male, mean age 47.8±12.5 years). Tp-Te interval and Tp-Te/QT ratio were measured from the 12-lead electrocardiogram. These parameters were compared in groups. Baseline characteristics of the study groups were comparable. In electrocardiographic parameters analysis, QT and corrected QT were similar in CSF patients compared to the controls (357±35.2 vs 362±38.0 milliseconds and 419±25.8 vs 430±44.2 milliseconds, all p value >0.05). Tp-Te interval, Tp-Te/QT and Tp-Te/QTc ratio were significantly higher in CSF patients (85±13.7 vs 74±9.9 milliseconds and 0.24±0.03 vs 0.20±0.02 and 0.20±0.03 vs 0.17±0.02 all p value <0.001). Our study revealed that QTd, Tp-Te interval and Tp-Te/QT ratio are prolonged in patients with CSF.

  8. Structure and dynamics of AMPA receptor GluA2 in resting, pre-open and desensitized states

    PubMed Central

    Dürr, Katharina L.; Chen, Lei; Stein, Richard A.; De Zorzi, Rita; MihaelaFolea, I.; Walz, Thomas; Mchaourab, Hassane S.; Gouaux, Eric

    2014-01-01

    Summary Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory signaling in the nervous system. Despite the profound importance of iGluRs in the nervous system, little is known about the structures and dynamics of intact receptors in distinct functional states. Here we elucidate the structures of the intact GluA2 AMPA receptor in an apo resting/closed state, in an activated/pre-open state bound with the partial agonists and a positive allosteric modulator and in a desensitized/closed state in complex with FW alone. To probe the conformational properties of these states, we carried out double electron-electron resonance experiments on cysteine mutants and cryo-electron microscopy studies. We show how agonist binding modulates the conformation of the ligand binding domain 'layer' of the intact receptors and how, upon desensitization, the receptor undergoes large conformational rearrangements of amino-terminal and ligand-binding domains. We define mechanistic principles by which to understand antagonism, activation and desensitization in AMPA iGluRs. PMID:25109876

  9. Adenosine A2A receptor deletion affects social behaviors and anxiety in mice: Involvement of anterior cingulate cortex and amygdala.

    PubMed

    López-Cruz, Laura; Carbó-Gas, Maria; Pardo, Marta; Bayarri, Pilar; Valverde, Olga; Ledent, Catherine; Salamone, John D; Correa, Mercè

    2017-03-15

    Blockade of adenosine A 2A receptors can potentiate motivation to work for natural reinforcers such as food. Conspecific interaction is a potent natural reinforcer in social animals that can be manifested as preference for social exploration versus other sources of novel stimulation. Deficiencies in this type of motivated behavior (social withdrawal) have been seen in several pathologies such as autism and depression. However, the role of A 2A receptors in motivation for social interaction has not been widely explored. Social interaction paradigms evaluate the natural preference of animals for exploring other conspecifics, and the ability to differentiate between familiar versus novel ones. Anxiety is one of the factors that can induce avoidance of social interaction. In the present study, adenosine A 2A knockout (A 2A KO) and wild-type (WT) mice were assessed for social and anxiety-related behaviors. c-Fos immunoreactivity was evaluated as a measure of neuronal activation in brain areas involved in different aspects of motivation and emotional processes. Although A 2A KO mice showed an anxious profile, they displayed higher levels of sociability and were less sensitive to social novelty. WT mice displayed a typical pattern of social recognition 24h later, but not A 2A KO mice, which explored equally both conspecifics. There were no differences between strains in aggressiveness, perseverance or social odor preferences. c-Fos immunoreactivity in A 2A KO mice was higher in anterior cingulate and amygdala compared to WT mice. Thus, A 2A receptors appear to be potential targets for the improvement of pathologies related to social function. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Receptor Protein Tyrosine Phosphatase-Receptor Tyrosine Kinase Substrate Screen Identifies EphA2 as a Target for LAR in Cell Migration

    PubMed Central

    Lee, Hojin

    2013-01-01

    Receptor tyrosine kinases (RTKs) exist in equilibrium between tyrosyl-phosphorylated and dephosphorylated states. Despite a detailed understanding of how RTKs become tyrosyl phosphorylated, much less is known about RTK tyrosyl dephosphorylation. Receptor protein tyrosine phosphatases (RPTPs) can play essential roles in the dephosphorylation of RTKs. However, a complete understanding of the involvement of the RPTP subfamily in RTK tyrosyl dephosphorylation has not been established. In this study, we have employed a small interfering RNA (siRNA) screen to identify RPTPs in the human genome that serve as RTK phosphatases. We observed that each RPTP induced a unique fingerprint of tyrosyl phosphorylation among 42 RTKs. We identified EphA2 as a novel LAR substrate. LAR dephosphorylated EphA2 at phosphotyrosyl 930, uncoupling Nck1 from EphA2 and thereby attenuating EphA2-mediated cell migration. These results demonstrate that each RPTP exerts a unique regulatory fingerprint of RTK tyrosyl dephosphorylation and suggest a complex signaling interplay between RTKs and RPTPs. Furthermore, we observed that LAR modulates cell migration through EphA2 site-specific dephosphorylation. PMID:23358419

  11. Renal protection from ischemia mediated by A2A adenosine receptors on bone marrow–derived cells

    PubMed Central

    Day, Yuan-Ji; Huang, Liping; McDuffie, Marcia J.; Rosin, Diane L.; Ye, Hong; Chen, Jiang-Fan; Schwarzschild, Michael A.; Fink, J. Stephen; Linden, Joel; Okusa, Mark D.

    2003-01-01

    Activation of A2A adenosine receptors (A2ARs) protects kidneys from ischemia-reperfusion injury (IRI). A2ARs are expressed on bone marrow–derived (BM-derived) cells and renal smooth muscle, epithelial, and endothelial cells. To measure the contribution of A2ARs on BM-derived cells in suppressing renal IRI, we examined the effects of a selective agonist of A2ARs, ATL146e, in chimeric mice in which BM was ablated by lethal radiation and reconstituted with donor BM cells derived from GFP, A2AR-KO, or WT mice to produce GFP→WT, A2A-KO→WT, or WT→WT mouse chimera. We found little or no repopulation of renal vascular endothelial cells by donor BM with or without renal IRI. ATL146e had no effect on IRI in A2A-KO mice or A2A-KO→WT chimera, but reduced the rise in plasma creatinine from IRI by 75% in WT mice and by 60% in WT→WT chimera. ATL146e reduced the induction of IL-6, IL-1β, IL-1ra, and TGF-α mRNA in WT→WT mice but not in A2A-KO→WT mice. Plasma creatinine was significantly greater in A2A-KO than in WT mice after IRI, suggesting some renal protection by endogenous adenosine. We conclude that protection from renal IRI by A2AR agonists or endogenous adenosine requires activation of receptors expressed on BM-derived cells. PMID:12975473

  12. Impact and benefit of A(2B)-adenosine receptor agonists for the respiratory tract: mucociliary clearance, ciliary beat frequency, trachea muscle tonus and cytokine release.

    PubMed

    Walaschewski, Robin; Begrow, Frank; Verspohl, Eugen J

    2013-01-01

    Adenosine is known to induce a bronchospasm in asthma- and COPD patients. The role of A(2B) receptors was investigated with respect to several parameters of the respiratory tract: tonus of smooth muscle, ciliary beat frequency as measured by high-speed video camera connected to a microscope (both in rats) and mucociliary clearance (MCC; transport of a fluorescent dye using a microdialysis procedure) in mice.  NECA (5'-N-ethylcarboxamidoadenosine) (a non-selective adenosine receptor agonist) was able to acutely induce a contraction, which was reversed to a relaxation after repeated dosing. This relaxation was completely abolished by PSB-1115, an A(2B) receptor antagonist. IL-13 (cytokine) was not involved mediating acute contractility effects. MCC was increased by BAY 60-6583 (A(2B) receptor agonist) and NECA (counteracted by the A(2B) receptor antagonist PSB-1115). Activation of A(2B) adenosine receptors by BAY 60-6583 induced an increase of the ciliary beat frequency, which could be reduced by administration of PSB-1115. Several cytokines were increased by NECA although only some are relevant because they are not blocked by A(2B) receptor antagonism. The A(2B) receptors are involved in airway relaxation, MCC improvement and ciliary beat frequency. A(2B) receptor agonists may be of therapeutic value and should be developed. © 2012 The Authors. JPP © 2012 Royal Pharmaceutical Society.

  13. TP53 dysfunction in CLL: Implications for prognosis and treatment.

    PubMed

    Te Raa, Gera D; Kater, Arnon P

    2016-03-01

    Despite the availability of novel targeted agents, TP53 defects remain the most important adverse prognostic factor in chronic lymphocytic leukemia (CLL). Detection of deletion of TP53 locus (17p deletion) by fluorescent in situ hybridization (FISH) has become standard and performed prior to every line of treatment as the incidence dramatically increases as relapses occur. As monoallelic mutations of TP53 equally affect outcome, novel methods are being developed to improve detection of TP53 defects and include next-generation sequencing (NGS) and functional assays. TP53 defects highly affect outcome of immunochemotherapy but also alter response durations of tyrosine kinase inhibitors. Although BCR-targeting agents and Bcl-2-inhibitos have achieved durable responses in some patients with TP53 defects, long-term follow-up is currently lacking. In this review biological and clinical consequences of TP53 dysfunction as well as applicability of currently available methods to detect TP53 defects are described. In addition, proposed novel therapeutic strategies specifically for patients with TP53 dysfunction are discussed. In summary, the only curative treatment option for TP53-defective CLL is still allogeneic hematopoietic stem cell transplantation. Other treatment strategies such as rationale combinations of agents with different (TP53 independent) targets, including kinase inhibitors and inhibitors of anti-apoptotic molecules but also immunomodulatory agents need to be further explored. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress

    PubMed Central

    Kaster, Manuella P.; Machado, Nuno J.; Silva, Henrique B.; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E.; Rodrigues, Ana Lúcia S.; Porciúncula, Lisiane O.; Chen, Jiang Fan; Tomé, Ângelo R.; Agostinho, Paula; Canas, Paula M.; Cunha, Rodrigo A.

    2015-01-01

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function. PMID:26056314

  15. Caffeine acts through neuronal adenosine A2A receptors to prevent mood and memory dysfunction triggered by chronic stress.

    PubMed

    Kaster, Manuella P; Machado, Nuno J; Silva, Henrique B; Nunes, Ana; Ardais, Ana Paula; Santana, Magda; Baqi, Younis; Müller, Christa E; Rodrigues, Ana Lúcia S; Porciúncula, Lisiane O; Chen, Jiang Fan; Tomé, Ângelo R; Agostinho, Paula; Canas, Paula M; Cunha, Rodrigo A

    2015-06-23

    The consumption of caffeine (an adenosine receptor antagonist) correlates inversely with depression and memory deterioration, and adenosine A2A receptor (A2AR) antagonists emerge as candidate therapeutic targets because they control aberrant synaptic plasticity and afford neuroprotection. Therefore we tested the ability of A2AR to control the behavioral, electrophysiological, and neurochemical modifications caused by chronic unpredictable stress (CUS), which alters hippocampal circuits, dampens mood and memory performance, and enhances susceptibility to depression. CUS for 3 wk in adult mice induced anxiogenic and helpless-like behavior and decreased memory performance. These behavioral changes were accompanied by synaptic alterations, typified by a decrease in synaptic plasticity and a reduced density of synaptic proteins (synaptosomal-associated protein 25, syntaxin, and vesicular glutamate transporter type 1), together with an increased density of A2AR in glutamatergic terminals in the hippocampus. Except for anxiety, for which results were mixed, CUS-induced behavioral and synaptic alterations were prevented by (i) caffeine (1 g/L in the drinking water, starting 3 wk before and continued throughout CUS); (ii) the selective A2AR antagonist KW6002 (3 mg/kg, p.o.); (iii) global A2AR deletion; and (iv) selective A2AR deletion in forebrain neurons. Notably, A2AR blockade was not only prophylactic but also therapeutically efficacious, because a 3-wk treatment with the A2AR antagonist SCH58261 (0.1 mg/kg, i.p.) reversed the mood and synaptic dysfunction caused by CUS. These results herald a key role for synaptic A2AR in the control of chronic stress-induced modifications and suggest A2AR as candidate targets to alleviate the consequences of chronic stress on brain function.

  16. Arousal effect of caffeine depends on adenosine A2A receptors in the shell of the nucleus accumbens

    PubMed Central

    Lazarus, Michael; Shen, Hai-Ying; Cherasse, Yoan; Qu, Wei-Min; Huang, Zhi-Li; Bass, Caroline E.; Winsky-Sommerer, Raphaelle; Semba, Kazue; Fredholm, Bertil B.; Boison, Detlev; Hayaishi, Osamu; Urade, Yoshihiro; Chen, Jiang-Fan

    2011-01-01

    Caffeine, the most widely used psychoactive compound, is an adenosine receptor antagonist. It promotes wakefulness by blocking adenosine A2A receptors (A2ARs) in the brain, but the specific neurons on which caffeine acts to produce arousal have not been identified. Using selective gene deletion strategies based on the Cre/loxP technology in mice and focal RNA interference to silence the expression of A2ARs in rats by local infection with adeno-associated virus carrying short-hairpin RNA, we report that the A2ARs in the shell region of the nucleus accumbens (NAc) are responsible for the effect of caffeine on wakefulness. Caffeine-induced arousal was not affected in rats when A2ARs were focally removed from the NAc core or other A2AR-positive areas of the basal ganglia. Our observations suggest that caffeine promotes arousal by activating pathways that traditionally have been associated with motivational and motor responses in the brain. PMID:21734299

  17. Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator.

    PubMed

    Montesinos, M Carmen; Desai-Merchant, Avani; Cronstein, Bruce N

    2015-12-01

    Impaired wound healing, as it occurs in diabetes mellitus or long-term corticoid treatment, is commonly associated with disability, diminished quality of life, and high economic costs. Selective agonists of the A2A receptor subtype of adenosine, an endogenous regulator of inflammation, promote tissue repair in animal models, both healthy and with impaired healing. Plasmin-mediated proteolysis of fibrin and other matrix proteins is essential for cell migration at sites of injury. Since adenosine A2A receptor activation increases plasminogen activator release from macrophages and mast cells, we studied the effect of a selective agonist, CGS-21680, on full-thickness excisional wound closure in wild-type, urokinase plasminogen activator (uPA)-deficient, and tissue plasminogen activator (tPA)-deficient mice. Wound closure was impaired in tPA- and uPA-deficient mice as compared with wild-type mice, and topical application of CGS-21680 significantly increased the rate at which wounds closed in wild-type mice and uPA-deficient mice, but not in tPA-deficient mice. Immunostaining of tissue sections showed that tPA was present in endothelial cells and histiocytes by day 3 post-wound and also by day 6. In contrast, uPA was more prominent in these cell types only by day 6 post-wound. Our results confirm that plasminogen activation contributes to wound repair and are consistent with the hypothesis that adenosine A2A receptor activation promotes wound closure by a mechanism that depends upon tPA, but not uPA. Moreover, our results suggest that topical adenosine A2A receptor agonists may be useful in promotion of wound closure in patients with impaired wound healing.

  18. High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor

    PubMed Central

    O’Malley, Michelle A.; Lazarova, Tzvetana; Britton, Zachary T.; Robinson, Anne S.

    2007-01-01

    The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (~4 mg/L of culture) of functional human adenosine A2a receptor fused to a green fluorescent protein (A2aR-GFP) from Saccharomyces cerevisiae. In this work we re-engineered A2aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A2aR and A2aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A2aR-GFP-His10. One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-β-D-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A2aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A2a-His10 receptor was purified in quantities of 6 +/− 2 mg/L of culture, with ligand-binding yields of 1 mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A2aR yet achieved from any heterologous expression system. PMID:17591446

  19. The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

    PubMed

    Vecchio, Elizabeth A; Chuo, Chung Hui; Baltos, Jo-Anne; Ford, Leigh; Scammells, Peter J; Wang, Bing H; Christopoulos, Arthur; White, Paul J; May, Lauren T

    2016-10-01

    We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Chronic and acute adenosine A2A receptor blockade prevents long-term episodic memory disruption caused by acute cannabinoid CB1 receptor activation.

    PubMed

    Mouro, Francisco M; Batalha, Vânia L; Ferreira, Diana G; Coelho, Joana E; Baqi, Younis; Müller, Christa E; Lopes, Luísa V; Ribeiro, Joaquim A; Sebastião, Ana M

    2017-05-01

    Cannabinoid-mediated memory impairment is a concern in cannabinoid-based therapies. Caffeine exacerbates cannabinoid CB 1 receptor (CB 1 R)-induced memory deficits through an adenosine A 1 receptor-mediated mechanism. We now evaluated how chronic or acute blockade of adenosine A 2A receptors (A 2A Rs) affects long-term episodic memory deficits induced by a single injection of a selective CB 1 R agonist. Long-term episodic memory was assessed by the novel object recognition (NOR) test. Mice received an intraperitoneal (i.p.) injection of the CB 1 /CB 2 receptor agonist WIN 55,212-2 (1 mg/kg) immediately after the NOR training, being tested for novelty recognition 24 h later. Anxiety levels were assessed by the Elevated Plus Maze test, immediately after the NOR. Mice were also tested for exploratory behaviour at the Open Field. For chronic A 2A R blockade, KW-6002 (istradefylline) (3 mg/kg/day) was administered orally for 30 days; acute blockade of A 2A Rs was assessed by i.p. injection of SCH 58261 (1 mg/kg) administered either together with WIN 55,212-2 or only 30 min before the NOR test phase. The involvement of CB 1 Rs was assessed by using the CB 1 R antagonist, AM251 (3 mg/kg, i.p.). WIN 55,212-2 caused a disruption in NOR, an action absent in mice also receiving AM251, KW-6002 or SCH 58261 during the encoding/consolidation phase; SCH 58251 was ineffective if present during retrieval only. No effects were detected in the Elevated Plus maze or Open Field Test. The finding that CB 1 R-mediated memory disruption is prevented by antagonism of adenosine A 2A Rs, highlights a possibility to prevent cognitive side effects when therapeutic application of CB 1 R drugs is desired. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. The Receptor Tyrosine Kinase EphA2 Is a Direct Target Gene of Hypermethylated in Cancer 1 (HIC1)*

    PubMed Central

    Foveau, Bénédicte; Boulay, Gaylor; Pinte, Sébastien; Van Rechem, Capucine; Rood, Brian R.; Leprince, Dominique

    2012-01-01

    The tumor suppressor gene hypermethylated in cancer 1 (HIC1), which encodes a transcriptional repressor, is epigenetically silenced in many human tumors. Here, we show that ectopic expression of HIC1 in the highly malignant MDA-MB-231 breast cancer cell line severely impairs cell proliferation, migration, and invasion in vitro. In parallel, infection of breast cancer cell lines with a retrovirus expressing HIC1 also induces decreased mRNA and protein expression of the tyrosine kinase receptor EphA2. Moreover, chromatin immunoprecipitation (ChIP) and sequential ChIP experiments demonstrate that endogenous HIC1 proteins are bound, together with the MTA1 corepressor, to the EphA2 promoter in WI38 cells. Taken together, our results identify EphA2 as a new direct target gene of HIC1. Finally, we observe that inactivation of endogenous HIC1 through RNA interference in normal breast epithelial cells results in the up-regulation of EphA2 and is correlated with increased cellular migration. To conclude, our results involve the tumor suppressor HIC1 in the transcriptional regulation of the tyrosine kinase receptor EphA2, whose ligand ephrin-A1 is also a HIC1 target gene. Thus, loss of the regulation of this Eph pathway through HIC1 epigenetic silencing could be an important mechanism in the pathogenesis of epithelial cancers. PMID:22184117

  2. Tp-e Interval, Tp-e/QTc Ratio, and Fragmented QRS Are Correlated with the Severity of Liver Cirrhosis.

    PubMed

    Akboga, Mehmet Kadri; Yuksel, Mahmut; Balci, Kevser Gulcihan; Kaplan, Mustafa; Cay, Serkan; Gokbulut, Volkan; Yayla, Cagri; Ertem, Ahmet Goktug; Ayhan, Meral Akdogan; Topaloglu, Serkan; Aras, Dursun

    2017-01-01

    Arrhythmias and electrocardiographic changes are reported in several noncardiac diseases, including liver cirrhosis (LC). We intended to evaluate the interval from the peak to the end of the electrocardiographic T wave (Tp-e), Tp-e/QTc ratio, and fQRS as presumed markers of arrhythmias in LC. In this cross-sectional study, a total of 88 consecutive patients with LC according to clinical, biological, ultrasonographic, or histological criteria and 73 control subjects were enrolled. The severity of cirrhosis was classified according to Pugh-Child's classification and Model for End-Stage Liver Disease (MELD) score. Tp-e interval, Tp-e/QTc ratio, and fQRS rates were measured from the 12-lead electrocardiogram. Tp-e interval, Tp-e/QTc ratio and fQRS rates were significantly increased in parallel to the severity of LC (P < 0.001, P < 0.001, and P = 0.003, respectively). In correlation analysis, Pugh-Child stage showed a significantly positive correlation with Tp-e interval (r = 0.462, P < 0.001), QTc interval (r = 0.373, P < 0.001), Tp-e/QTc ratio (r = 0.352, P < 0.001), and fQRS (r = 0.407, P < 0.001). Furthermore, Tp-e interval (r = 0.414, P < 0.001) and Tp-e/QTc ratio (r = 0.426, P< 0.001) had significant positive correlation with MELD score. Our study demonstrated that Tp-e interval, Tp-e/QTc ratios, and fQRS rates were significantly increased in parallel to the severity of LC. Thus, these findings may implicate that Tp-e interval, Tp-e/QTc ratio, and fQRS may be novel and useful indicators for prediction of arrhythmias in LC. © 2016 Wiley Periodicals, Inc.

  3. Deletion of the EphA2 receptor exacerbates myocardial injury and the progression of ischemic cardiomyopathy.

    PubMed

    O'Neal, Wesley T; Griffin, William F; Kent, Susan D; Faiz, Filza; Hodges, Jonathan; Vuncannon, Jackson; Virag, Jitka A I

    2014-01-01

    EphrinA1-EphA-receptor signaling is protective during myocardial infarction (MI). The EphA2-receptor (EphA2-R) potentially mediates cardiomyocyte survival. To determine the role of the EphA2-R in acute non-reperfused myocardial injury in vivo, infarct size, inflammatory cell density, NF-κB, p-AKT/Akt, and MMP-2 protein levels, and changes in ephrinA1/EphA2-R gene expression profile were assessed 4 days post-MI in B6129 wild-type (WT) and EphA2-R-mutant (EphA2-R-M) mice lacking a functional EphA2-R. Fibrosis, capillary density, morphometry of left ventricular chamber and infarct dimensions, and cardiac function also were measured 4 weeks post-MI to determine the extent of ventricular remodeling. EphA2-R-M infarct size and area of residual necrosis were 31.7% and 113% greater than WT hearts, respectively. Neutrophil and macrophage infiltration were increased by 46% and 84% in EphA2-R-M hearts compared with WT, respectively. NF-κB protein expression was 1.9-fold greater in EphA2-R-M hearts at baseline and 56% less NF-κB after infarction compared with WT. EphA6 gene expression was 2.5-fold higher at baseline and increased 9.8-fold 4 days post-MI in EphA2-R-M hearts compared with WT. EphrinA1 gene expression in EphA2-R-M hearts was unchanged at baseline and decreased by 42% 4 days post-MI compared with WT hearts. EphA2-R-M hearts had 66.7% less expression of total Akt protein and 59% less p-Akt protein than WT hearts post-MI. EphA2-R-M hearts 4 weeks post-MI had increased chamber dilation and interstitial fibrosis and decreased MMP-2 expression and capillary density compared with WT. In conclusion, the EphA2-R is necessary to appropriately modulate the inflammatory response and severity of early injury during acute MI, thereby influencing the progression of ischemic cardiomyopathy.

  4. Deletion of the EphA2 receptor exacerbates myocardial injury and the progression of ischemic cardiomyopathy

    PubMed Central

    O'Neal, Wesley T.; Griffin, William F.; Kent, Susan D.; Faiz, Filza; Hodges, Jonathan; Vuncannon, Jackson; Virag, Jitka A. I.

    2014-01-01

    EphrinA1-EphA-receptor signaling is protective during myocardial infarction (MI). The EphA2-receptor (EphA2-R) potentially mediates cardiomyocyte survival. To determine the role of the EphA2-R in acute non-reperfused myocardial injury in vivo, infarct size, inflammatory cell density, NF-κB, p-AKT/Akt, and MMP-2 protein levels, and changes in ephrinA1/EphA2-R gene expression profile were assessed 4 days post-MI in B6129 wild-type (WT) and EphA2-R-mutant (EphA2-R-M) mice lacking a functional EphA2-R. Fibrosis, capillary density, morphometry of left ventricular chamber and infarct dimensions, and cardiac function also were measured 4 weeks post-MI to determine the extent of ventricular remodeling. EphA2-R-M infarct size and area of residual necrosis were 31.7% and 113% greater than WT hearts, respectively. Neutrophil and macrophage infiltration were increased by 46% and 84% in EphA2-R-M hearts compared with WT, respectively. NF-κB protein expression was 1.9-fold greater in EphA2-R-M hearts at baseline and 56% less NF-κB after infarction compared with WT. EphA6 gene expression was 2.5-fold higher at baseline and increased 9.8-fold 4 days post-MI in EphA2-R-M hearts compared with WT. EphrinA1 gene expression in EphA2-R-M hearts was unchanged at baseline and decreased by 42% 4 days post-MI compared with WT hearts. EphA2-R-M hearts had 66.7% less expression of total Akt protein and 59% less p-Akt protein than WT hearts post-MI. EphA2-R-M hearts 4 weeks post-MI had increased chamber dilation and interstitial fibrosis and decreased MMP-2 expression and capillary density compared with WT. In conclusion, the EphA2-R is necessary to appropriately modulate the inflammatory response and severity of early injury during acute MI, thereby influencing the progression of ischemic cardiomyopathy. PMID:24795639

  5. Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

    PubMed

    Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

    2015-01-01

    Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

  6. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development.

    PubMed

    Nissim, Sahar; Weeks, Olivia; Talbot, Jared C; Hedgepeth, John W; Wucherpfennig, Julia; Schatzman-Bone, Stephanie; Swinburne, Ian; Cortes, Mauricio; Alexa, Kristen; Megason, Sean; North, Trista E; Amacher, Sharon L; Goessling, Wolfram

    2016-10-01

    The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic vs. pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease. Copyright © 2016

  7. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development

    PubMed Central

    Nissim, Sahar; Weeks, Olivia; Talbot, Jared C.; Hedgepeth, John W.; Wucherpfennig, Julia; Schatzman-Bone, Stephanie; Swinburne, Ian; Cortes, Mauricio; Alexa, Kristen; Megason, Sean; North, Trista E.; Amacher, Sharon L.; Goessling, Wolfram

    2016-01-01

    The stepwise progression of common endoderm progenitors into differentiated liver and pancreas organs is regulated by a dynamic array of signals that are not well understood. The nuclear receptor subfamily 5, group A, member 2 gene nr5a2, also known as Liver receptor homolog-1 (Lrh-1) is expressed in several tissues including the developing liver and pancreas. Here, we interrogate the role of Nr5a2 at multiple developmental stages using genetic and chemical approaches and uncover novel pleiotropic requirements during zebrafish liver and pancreas development. Zygotic loss of nr5a2 in a targeted genetic null mutant disrupted the development of the exocrine pancreas and liver, while leaving the endocrine pancreas intact. Loss of nr5a2 abrogated exocrine pancreas markers such as trypsin, while pancreas progenitors marked by ptf1a or pdx1 remained unaffected, suggesting a role for Nr5a2 in regulating pancreatic acinar cell differentiation. In the developing liver, Nr5a2 regulates hepatic progenitor outgrowth and differentiation, as nr5a2 mutants exhibited reduced hepatoblast markers hnf4α and prox1 as well as differentiated hepatocyte marker fabp10a. Through the first in vivo use of Nr5a2 chemical antagonist Cpd3, the iterative requirement for Nr5a2 for exocrine pancreas and liver differentiation was temporally elucidated: chemical inhibition of Nr5a2 function during hepatopancreas progenitor specification was sufficient to disrupt exocrine pancreas formation and enhance the size of the embryonic liver, suggesting that Nr5a2 regulates hepatic versus pancreatic progenitor fate choice. Chemical inhibition of Nr5a2 at a later time during pancreas and liver differentiation was sufficient to block the formation of mature acinar cells and hepatocytes. These findings define critical iterative and pleiotropic roles for Nr5a2 at distinct stages of pancreas and liver organogenesis, and provide novel perspectives for interpreting the role of Nr5a2 in disease. PMID:27474396

  8. TP53-based interaction analysis identifies cis-eQTL variants for TP53BP2, FBXO28, and FAM53A that associate with survival and treatment outcome in breast cancer

    PubMed Central

    Fagerholm, Rainer; Khan, Sofia; Schmidt, Marjanka K.; GarcClosas, Montserrat; Heikkilä, Päivi; Saarela, Jani; Beesley, Jonathan; Jamshidi, Maral; Aittomäki, Kristiina; Liu, Jianjun; Raza Ali, H.; Andrulis, Irene L.; Beckmann, Matthias W.; Behrens, Sabine; Blows, Fiona M.; Brenner, Hermann; Chang-Claude, Jenny; Couch, Fergus J.; Czene, Kamila; Fasching, Peter A.; Figueroa, Jonine; Floris, Giuseppe; Glendon, Gord; Guo, Qi; Hall, Per; Hallberg, Emily; Hamann, Ute; Holleczek, Bernd; Hooning, Maartje J.; Hopper, John L.; Jager, Agnes; Kabisch, Maria; Investigators, kConFab/AOCS; Keeman, Renske; Kosma, Veli-Matti; Lambrechts, Diether; Lindblom, Annika; Mannermaa, Arto; Margolin, Sara; Provenzano, Elena; Shah, Mitul; Southey, Melissa C.; Dennis, Joe; Lush, Michael; Michailidou, Kyriaki; Wang, Qin; Bolla, Manjeet K.; Dunning, Alison M.; Easton, Douglas F.; Pharoah, Paul D.P .; Chenevix-Trench, Georgia; Blomqvist, Carl; Nevanlinna, Heli

    2017-01-01

    TP53 overexpression is indicative of somatic TP53 mutations and associates with aggressive tumors and poor prognosis in breast cancer. We utilized a two-stage SNP association study to detect variants associated with breast cancer survival in a TP53-dependent manner. Initially, a genome-wide study (n = 575 cases) was conducted to discover candidate SNPs for genotyping and validation in the Breast Cancer Association Consortium (BCAC). The SNPs were then tested for interaction with tumor TP53 status (n = 4,610) and anthracycline treatment (n = 17,828). For SNPs interacting with anthracycline treatment, siRNA knockdown experiments were carried out to validate candidate genes. In the test for interaction between SNP genotype and TP53 status, we identified one locus, represented by rs10916264 (p(interaction) = 3.44 05E010-5; FDR-adjusted p = 0.0011) in estrogen receptor (ER) positive cases. The rs10916264 AA genotype associated with worse survival among cases with ER-positive, TP53-positive tumors (hazard ratio [HR] 2.36, 95% confidence interval [C.I] 1.45 - 3.82). This is a cis-eQTL locus for FBXO28 and TP53BP2; expression levels of these genes were associated with patient survival specifically in ER-positive, TP53-mutated tumors. Additionally, the SNP rs798755 was associated with survival in interaction with anthracycline treatment (p(interaction) = 9.57 05E010-5, FDR-adjusted p = 0.0130). RNAi-based depletion of a predicted regulatory target gene, FAM53A, indicated that this gene can modulate doxorubicin sensitivity in breast cancer cell lines. If confirmed in independent data sets, these results may be of clinical relevance in the development of prognostic and predictive marker panels for breast cancer. PMID:28179588

  9. Deletion of the distal COOH-terminus of the A2B adenosine receptor switches internalization to an arrestin- and clathrin-independent pathway and inhibits recycling.

    PubMed

    Mundell, S J; Matharu, A-L; Nisar, S; Palmer, T M; Benovic, J L; Kelly, E

    2010-02-01

    We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.

  10. Nutlin‐3a selects for cells harbouring TP 53 mutations

    PubMed Central

    Hollstein, Monica; Arlt, Volker M.; Phillips, David H.

    2016-01-01

    TP53 mutations occur in half of all human tumours. Mutagen‐induced or spontaneous TP53 mutagenesis can be studied in vitro using the human TP53 knock‐in (Hupki) mouse embryo fibroblast (HUF) immortalisation assay (HIMA). TP53 mutations arise in up to 30% of mutagen‐treated, immortalised HUFs; however, mutants are not identified until TP53 sequence analysis following immortalisation (2–5 months) and much effort is expended maintaining TP53‐WT cultures. In order to improve the selectivity of the HIMA for HUFs harbouring TP53 mutations, we explored the use of Nutlin‐3a, an MDM2 inhibitor that leads to stabilisation and activation of wild‐type (WT) p53. First, we treated previously established immortal HUF lines carrying WT or mutated TP53 with Nutlin‐3a to examine the effect on cell growth and p53 activation. Nutlin‐3a induced the p53 pathway in TP53‐WT HUFs and inhibited cell growth, whereas most TP53‐mutated HUFs were resistant to Nutlin‐3a. We then assessed whether Nutlin‐3a treatment could discriminate between TP53‐WT and TP53‐mutated cells during the HIMA (n = 72 cultures). As immortal clones emerged from senescent cultures, each was treated with 10 µM Nutlin‐3a for 5 days and observed for sensitivity or resistance. TP53 was subsequently sequenced from all immortalised clones. We found that all Nutlin‐3a‐resistant clones harboured TP53 mutations, which were diverse in position and functional impact, while all but one of the Nutlin‐3a‐sensitive clones were TP53‐WT. These data suggest that including a Nutlin‐3a counter‐screen significantly improves the specificity and efficiency of the HIMA, whereby TP53‐mutated clones are selected prior to sequencing and TP53‐WT clones can be discarded. PMID:27813088

  11. Adenosine through the A2A adenosine receptor increases IL-1β in the brain contributing to anxiety

    PubMed Central

    Chiu, Gabriel S.; Darmody, Patrick T.; Walsh, John P.; Moon, Morgan L.; Kwakwa, Kristin A.; Bray, Julie K.; McCusker, Robert H.; Freund, Gregory G.

    2014-01-01

    Anxiety is one of the most commonly reported psychiatric conditions, but its pathogenesis is poorly understood. Ailments associated with activation of the innate immune system, however, are increasingly linked to anxiety disorders. In adult male mice, we found that adenosine doubled caspase-1 activity in brain by a pathway reliant on ATP-sensitive potassium (KATP) channels, protein kinase A (PKA) and the A2A adenosine receptor (AR). In addition, adenosine-dependent activation of caspase-1 increased interleukin (IL)-1β in the brain by two-fold. Peripheral administration of adenosine in wild-type (WT) mice led to a 2.3-fold increase in caspase-1 activity in the amygdala and to a 33% and 42% reduction in spontaneous locomotor activity and food intake, respectively, that were not observed in caspase-1 knockout (KO), IL-1 receptor type 1 (IL-1R1) KO and A2A AR KO mice or in mice administered a caspase-1 inhibitor centrally. Finally, adenosine administration increased anxiety-like behaviors in WT mice by 28% in the open field test and by 55% in the elevated zero-maze. Caspase-1 KO mice, IL-1R1 KO mice, A2A AR KO mice and WT mice treated with the KATP channel blocker, glyburide, were resistant to adenosine-induced anxiety-like behaviors. Thus, our results indicate that adenosine can act as an anxiogenic by activating caspase-1 and increasing IL-1β in the brain. PMID:24907587

  12. The EphA2 Receptor and EphrinA1 Ligand in Solid Tumors: Function and Therapeutic Targeting

    PubMed Central

    Wykosky, Jill; Debinski, Waldemar

    2013-01-01

    The Eph receptor tyrosine kinases and ephrin ligands have been studied extensively for their roles in developmental processes. In recent years, Eph receptors and ephrins have been found to be integral players in cancer formation and progression. Among these are EphA2 and ephrinA1, which are involved in the development and maintenance of many different types of solid tumors. The function of EphA2 and ephrinA1 in tumorigenesis and tumor progression is complex and seems to be dependent on cell type and microenvironment. These variables affect the expression of the EphA2 and ephrinA1 proteins, the pathways through which they induce signaling, and the functional consequences of that signaling on the behavior of tumor cells and tumor-associated cells. This review will specifically focus on the roles that EphA2 and ephrinA1 play in the different cell types that contribute to the malignancy of solid tumors, with emphasis on the opportunities for therapeutic targeting. PMID:19074825

  13. Activation of microglial cells triggers a release of brain-derived neurotrophic factor (BDNF) inducing their proliferation in an adenosine A2A receptor-dependent manner: A2A receptor blockade prevents BDNF release and proliferation of microglia

    PubMed Central

    2013-01-01

    Background Brain-derived neurotrophic factor (BDNF) has been shown to control microglial responses in neuropathic pain. Since adenosine A2A receptors (A2ARs) control neuroinflammation, as well as the production and function of BDNF, we tested to see if A2AR controls the microglia-dependent secretion of BDNF and the proliferation of microglial cells, a crucial event in neuroinflammation. Methods Murine N9 microglial cells were challenged with lipopolysaccharide (LPS, 100 ng/mL) in the absence or in the presence of the A2AR antagonist, SCH58261 (50 nM), as well as other modulators of A2AR signaling. The BDNF cellular content and secretion were quantified by Western blotting and ELISA, A2AR density was probed by Western blotting and immunocytochemistry and cell proliferation was assessed by BrdU incorporation. Additionally, the A2AR modulation of LPS-driven cell proliferation was also tested in primary cultures of mouse microglia. Results LPS induced time-dependent changes of the intra- and extracellular levels of BDNF and increased microglial proliferation. The maximal LPS-induced BDNF release was time-coincident with an LPS-induced increase of the A2AR density. Notably, removing endogenous extracellular adenosine or blocking A2AR prevented the LPS-mediated increase of both BDNF secretion and proliferation, as well as exogenous BDNF-induced proliferation. Conclusions We conclude that A2AR activation plays a mandatory role controlling the release of BDNF from activated microglia, as well as the autocrine/paracrine proliferative role of BDNF. PMID:23363775

  14. The A2b adenosine receptor antagonist PSB-603 promotes oxidative phosphorylation and ROS production in colorectal cancer cells via adenosine receptor-independent mechanism.

    PubMed

    Mølck, Christina; Ryall, James; Failla, Laura M; Coates, Janine L; Pascussi, Jean-Marc; Heath, Joan K; Stewart, Gregory; Hollande, Frédéric

    2016-12-01

    Adenosine is a multifaceted regulator of tumor progression. It modulates immune cell activity as well as acting directly on tumor cells. The A 2b adenosine receptor (A 2b -AR) is thought to be an important mediator of these effects. In this study we sought to analyze the contribution of the A 2b -AR to the behavior of colorectal cancer cells. The A 2b -AR antagonist PSB-603 changed cellular redox state without affecting cellular viability. Quantification of cellular bioenergetics demonstrated that PSB-603 increased basal oxygen consumption rates, indicative of enhanced mitochondrial oxidative phosphorylation. Unexpectedly, pharmacological and genetic approaches to antagonize AR-related signalling of PSB-603 did not abolish the response, suggesting that it was AR-independent. PSB-603 also induced acute increases in reactive oxygen species, and PSB-603 synergized with chemotherapy treatment to increase colorectal cancer cell death, consistent with the known link between cellular metabolism and chemotherapy response. PSB-603 alters cellular metabolism in colorectal cancer cells and increases their sensitivity to chemotherapy. Although requiring more mechanistic insight into its A 2b -AR-independent activity, our results show that PSB-603 may have clinical value as an anti-colorectal cancer therapeutic. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  15. Adenosine A2A receptor inhibition restores the normal transport of endothelial glutamate transporters in the brain.

    PubMed

    Bai, Wei; Li, Ping; Ning, Ya-Lei; Peng, Yan; Xiong, Ren-Ping; Yang, Nan; Chen, Xing; Zhou, Yuan-Guo

    2018-04-15

    Excitatory amino acid transporters (EAATs) on cerebral vascular endothelial cells play an important role in maintaining glutamate homeostasis in the brain. The dysfunction of endothelial EAATs is an important reason for the dramatically elevated brain glutamate levels after brain injury, such as traumatic brain injury (TBI). The adenosine A 2A receptor (A 2A R) plays an important role in regulating the brain glutamate level after brain injury; however, researchers have not clearly determined whether this role was related to its ability to regulate endothelial EAATs. Activation of A 2A R in vitro not only decreased the PKA- and glutamate level-dependent strengthening of the interaction between NKA-α1 and the FXYD1 subunit and the subsequent decrease in the activity of Na + /K + -ATPases (NKAs) but also enhanced its interaction with EAATs and ultimately aggravated the reverse transport function of endothelial EAATs under oxygen-glucose deprivation (OGD) conditions. Conversely, inhibition of A 2A R restored the normal transport of EAAT. Moreover, A 2A R inhibition increased NKA activity and decreased its interaction with EAATs in isolated brain capillaries after TBI, further confirming its role in endothelial EAATs in vivo. Based on our results, A 2A R played an important role in regulating endothelial EAAT function, and strategies that restore the normal transport of endothelial EAATs through the inhibition of A 2A R might serve as an effective treatment for brain injury. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Evaluation of distribution of adenosine A2A receptors in normal human brain measured with [11C]TMSX PET.

    PubMed

    Mishina, Masahiro; Ishiwata, Kiichi; Kimura, Yuichi; Naganawa, Mika; Oda, Keiichi; Kobayashi, Shiro; Katayama, Yasuo; Ishii, Kenji

    2007-09-01

    Adenosine A(2A) receptor (A2AR) is thought to interact with dopamine D(2) receptor. Selective A2AR antagonists have attracted attention as the treatment of Parkinson's disease. In this study, we investigated the distribution of the A2ARs in the living human brain using positron emission tomography (PET) and [7-methyl-(11)C]-(E)-8-(3,4,5-trimethoxystyryl)-1,3,7-trimethylxanthine ([(11)C]TMSX). We recruited five normal male subjects. A dynamic series of PET scans was performed for 60 min, and the arterial blood was sampled during the scan to measure radioactivity of the parent compound and labeled metabolites. Circular regions of interest of 10-mm diameter were placed in the PET images over the cerebellum, brainstem, thalamus, head of caudate nucleus, anterior and posterior putamen, frontal lobe, temporal lobe, parietal lobe, occipital lobe, and posterior cingulate gyrus for each subject. A two-tissue, three-compartment model was used to estimate K(1), k(2), k(3), and k(4) between metabolite-corrected plasma and tissue time activity of [(11)C]TMSX. The binding potential (BP) was the largest in the anterior (1.25) and posterior putamen (1.20), was next largest in the head of caudate nucleus (1.05) and thalamus (1.03), and was small in the cerebral cortex, especially frontal lobe (0.46). [(11)C]TMSX PET showed the largest BP in the striatum in which A2ARs were enriched as in postmortem and nonhuman studies reported, but that the binding of [(11)C]TMSX was relatively larger in the thalamus to compare with other mammals. To date, [(11)C]TMSX is the only promising PET ligand, which is available to clinical use for mapping the A2ARs in the living human brain.

  17. Thermodynamics and structural analysis of positive allosteric modulation of the ionotropic glutamate receptor GluA2.

    PubMed

    Krintel, Christian; Frydenvang, Karla; Olsen, Lars; Kristensen, Maria T; de Barrios, Oriol; Naur, Peter; Francotte, Pierre; Pirotte, Bernard; Gajhede, Michael; Kastrup, Jette S

    2012-01-01

    Positive allosteric modulators of the ionotropic glutamate receptor-2 (GluA2) are promising compounds for the treatment of cognitive disorders, e.g. Alzheimer's disease. These modulators bind within the dimer interface of the LBD (ligand-binding domain) and stabilize the agonist-bound conformation slowing receptor desensitization and/or deactivation. In the present study, we employ isothermal titration calorimetry to determine binding affinities and thermodynamic details of binding of modulators of GluA2. A mutant of the LBD of GluA2 (LBD-L483Y-N754S) that forms a stable dimer in solution was used. The potent GluA2 modulator BPAM-97 was used as a reference compound. Evidence that BPAM-97 binds in the same pocket as the well-known GluA2 modulator cyclothiazide was obtained from X-ray structures. The LBD-L483Y-N754S:BPAM-97 complex has a Kd of 5.6 μM (ΔH=-4.9 kcal/mol, -TΔS=-2.3 kcal/mol; where 1 kcal≈4.187 kJ). BPAM-97 was used in a displacement assay to determine a Kd of 0.46 mM (ΔH=-1.2 kcal/mol, -TΔS=-3.3 kcal/mol) for the LBD-L483Y-N754S:IDRA-21 complex. The major structural factors increasing the potency of BPAM-97 over IDRA-21 are the increased van der Waals contacts to, primarily, Met496 in GluA2 imposed by the ethyl substituent of BPAM-97. These results add important information on binding affinities and thermodynamic details, and provide a new tool in the development of drugs against cognitive disorders.

  18. EphA2 and ephrin-A5 are not a receptor-ligand pair in the ocular lens.

    PubMed

    Cheng, Catherine; Fowler, Velia M; Gong, Xiaohua

    2017-09-01

    Eph-ephrin bidirectional signaling is essential for eye lens transparency in humans and mice. Our previous studies in mouse lenses demonstrate that ephrin-A5 is mainly expressed in the anterior epithelium, where it is required for maintaining the anterior epithelial monolayer. In contrast, EphA2 is localized in equatorial epithelial and fiber cells where it is essential for equatorial epithelial and fiber cell organization and hexagonal cell shape. Immunostaining of lens epithelial and fiber cells reveals that EphA2 and ephrin-A5 are also co-expressed in anterior fiber cell tips, equatorial epithelial cells and newly formed lens fibers, although they are not precisely colocalized. Due to this complex expression pattern and the promiscuous interactions between Eph receptors and ephrin ligands, as well as their complex bidirectional signaling pathways, cataracts in ephrin-A5(-/-) or EphA2(-/-) lenses may arise from loss of function or abnormal signaling mechanisms. To test whether abnormal signaling mechanisms may play a role in cataractogenesis in ephrin-A5(-/-) or EphA2(-/-) lenses, we generated EphA2 and ephrin-A5 double knockout (DKO) mice. We compared the phenotypes of EphA2(-/-) and ephrin-A5(-/-) lenses to that of DKO lenses. DKO lenses displayed an additive lens phenotype that was not significantly different from the two single KO lens phenotypes. Similar to ephrin-A5(-/-) lenses, DKO lenses had abnormal anterior epithelial cells leading to a large mass of epithelial cells that invade into the underlying fiber cell layer, directly resulting in anterior cataracts in ephrin-A5(-/-) and DKO lenses. Yet, similar to EphA2(-/-) lenses, DKO lenses also had abnormal packing of equatorial epithelial cells with disorganized meridional rows, lack of a lens fulcrum and disrupted fiber cells. The DKO lens phenotype rules out abnormal signaling by EphA2 in ephrin-A5(-/-) lenses or by ephrin-A5 in EphA2(-/-) lenses as possible cataract mechanisms. Thus, these results

  19. The Corticostriatal Adenosine A2A Receptor Controls Maintenance and Retrieval of Spatial Working Memory.

    PubMed

    Li, Zhihui; Chen, Xingjun; Wang, Tao; Gao, Ying; Li, Fei; Chen, Long; Xue, Jin; He, Yan; Li, Yan; Guo, Wei; Zheng, Wu; Zhang, Liping; Ye, Fenfen; Ren, Xiangpeng; Feng, Yue; Chan, Piu; Chen, Jiang-Fan

    2018-03-15

    Working memory (WM) taps into multiple executive processes including encoding, maintenance, and retrieval of information, but the molecular and circuit modulation of these WM processes remains undefined due to the lack of methods to control G protein-coupled receptor signaling with temporal resolution of seconds. By coupling optogenetic control of the adenosine A 2A receptor (A 2A R) signaling, the Cre-loxP-mediated focal A 2A R knockdown with a delayed non-match-to-place (DNMTP) task, we investigated the effect of optogenetic activation and focal knockdown of A 2A Rs in the dorsomedial striatum (n = 8 to 14 per group) and medial prefrontal cortex (n = 16 to 22 per group) on distinct executive processes of spatial WM. We also evaluated the therapeutic effect of the A 2A R antagonist KW6002 on delayed match-to-sample/place tasks in 6 normal and 6 MPTP-treated cynomolgus monkeys. Optogenetic activation of striatopallidal A 2A Rs in the dorsomedial striatum selectively at the delay and choice (not sample) phases impaired DNMTP performance. Optogenetic activation of A 2A Rs in the medial prefrontal cortex selectively at the delay (not sample or choice) phase improved DNMTP performance. The corticostriatal A 2A R control of spatial WM was specific for a novel but not well-trained DNMTP task. Focal dorsomedial striatum A 2A R knockdown or KW6002 improved DNMTP performance in mice. Last, KW6002 improved spatial WM in delayed match-to-sample and delayed match-to-place tasks of normal and dopamine-depleted cynomolgus monkeys. The A 2A Rs in striatopallidal and medial prefrontal cortex neurons exert distinctive control of WM maintenance and retrieval to achieve cognitive stability and flexibility. The procognitive effect of KW6002 in nonhuman primates provides the preclinical data to translate A 2A R antagonists for improving cognitive impairments in Parkinson's disease. Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  20. Prevention of adenosine A2A receptor activation diminishes beat-to-beat alternation in human atrial myocytes.

    PubMed

    Molina, Cristina E; Llach, Anna; Herraiz-Martínez, Adela; Tarifa, Carmen; Barriga, Montserrat; Wiegerinck, Rob F; Fernandes, Jacqueline; Cabello, Nuria; Vallmitjana, Alex; Benitéz, Raúl; Montiel, José; Cinca, Juan; Hove-Madsen, Leif

    2016-01-01

    Atrial fibrillation (AF) has been associated with increased spontaneous calcium release from the sarcoplasmic reticulum and linked to increased adenosine A2A receptor (A2AR) expression and activation. Here we tested whether this may favor atrial arrhythmogenesis by promoting beat-to-beat alternation and irregularity. Patch-clamp and confocal calcium imaging was used to measure the beat-to-beat response of the calcium current and transient in human atrial myocytes. Responses were classified as uniform, alternating or irregular and stimulation of Gs-protein coupled receptors decreased the frequency where a uniform response could be maintained from 1.0 ± 0.1 to 0.6 ± 0.1 Hz; p < 0.01 for beta-adrenergic receptors and from 1.4 ± 0.1 to 0.5 ± 0.1 Hz; p < 0.05 for A2ARs. The latter was linked to increased spontaneous calcium release and after-depolarizations. Moreover, A2AR activation increased the fraction of non-uniformly responding cells in HL-1 myocyte cultures from 19 ± 3 to 51 ± 9 %; p < 0.02, and electrical mapping in perfused porcine atria revealed that adenosine induced electrical alternans at longer cycle lengths, doubled the fraction of electrodes showing alternation, and increased the amplitude of alternations. Importantly, protein kinase A inhibition increased the highest frequency where uniform responses could be maintained from 0.84 ± 0.12 to 1.86 ± 0.11 Hz; p < 0.001 and prevention of A2AR-activation with exogenous adenosine deaminase selectively increased the threshold from 0.8 ± 0.1 to 1.2 ± 0.1 Hz; p = 0.001 in myocytes from patients with AF. In conclusion, A2AR-activation promotes beat-to-beat irregularities in the calcium transient in human atrial myocytes, and prevention of A2AR activation may be a novel means to maintain uniform beat-to-beat responses at higher beating frequencies in patients with atrial fibrillation.

  1. Selecting an optimal number of binding site waters to improve virtual screening enrichments against the adenosine A2A receptor.

    PubMed

    Lenselink, Eelke B; Beuming, Thijs; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

    2014-06-23

    A major challenge in structure-based virtual screening (VS) involves the treatment of explicit water molecules during docking in order to improve the enrichment of active compounds over decoys. Here we have investigated this in the context of the adenosine A2A receptor, where water molecules have previously been shown to be important for achieving high enrichment rates with docking, and where the positions of some binding site waters are known from a high-resolution crystal structure. The effect of these waters (both their presence and orientations) on VS enrichment was assessed using a carefully curated set of 299 high affinity A2A antagonists and 17,337 decoys. We show that including certain crystal waters greatly improves VS enrichment and that optimization of water hydrogen positions is needed in order to achieve the best results. We also show that waters derived from a molecular dynamics simulation - without any knowledge of crystallographic waters - can improve enrichments to a similar degree as the crystallographic waters, which makes this strategy applicable to structures without experimental knowledge of water positions. Finally, we used decision trees to select an ensemble of structures with different water molecule positions and orientations that outperforms any single structure with water molecules. The approach presented here is validated against independent test sets of A2A receptor antagonists and decoys from the literature. In general, this water optimization strategy could be applied to any target with waters-mediated protein-ligand interactions.

  2. Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

    PubMed

    Bonet, Isis; Franco-Montero, Pedro; Rivero, Virginia; Teijeira, Marta; Borges, Fernanda; Uriarte, Eugenio; Morales Helguera, Aliuska

    2013-12-23

    A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

  3. Regulation of lung endothelial permeability and inflammatory responses by prostaglandin A2: role of EP4 receptor

    PubMed Central

    Ohmura, Tomomi; Tian, Yufeng; Sarich, Nicolene; Ke, Yunbo; Meliton, Angelo; Shah, Alok S.; Andreasson, Katrin; Birukov, Konstantin G.; Birukova, Anna A.

    2017-01-01

    The role of prostaglandin A2 (PGA2) in modulation of vascular endothelial function is unknown. We investigated effects of PGA2 on pulmonary endothelial cell (EC) permeability and inflammatory activation and identified a receptor mediating these effects. PGA2 enhanced the EC barrier and protected against barrier dysfunction caused by vasoactive peptide thrombin and proinflammatory bacterial wall lipopolysaccharide (LPS). Receptor screening using pharmacological and molecular inhibitory approaches identified EP4 as a novel PGA2 receptor. EP4 mediated barrier-protective effects of PGA2 by activating Rap1/Rac1 GTPase and protein kinase A targets at cell adhesions and cytoskeleton: VE-cadherin, p120-catenin, ZO-1, cortactin, and VASP. PGA2 also suppressed LPS-induced inflammatory signaling by inhibiting the NFκB pathway and expression of EC adhesion molecules ICAM1 and VCAM1. These effects were abolished by pharmacological or molecular inhibition of EP4. In vivo, PGA2 was protective in two distinct models of acute lung injury (ALI): LPS-induced inflammatory injury and two-hit ALI caused by suboptimal mechanical ventilation and injection of thrombin receptor–activating peptide. These protective effects were abolished in mice with endothelial-specific EP4 knockout. The results suggest a novel role for the PGA2–EP4 axis in vascular EC protection that is critical for improvement of pathological states associated with increased vascular leakage and inflammation. PMID:28428256

  4. Genetic Deletion of the Adenosine A2A Receptor Confers Postnatal Development of Relative Myopia in Mice

    PubMed Central

    Zhou, Xiangtian; Huang, Qinzhu; An, Jianhong; Lu, Runxia; Qin, Xiaoyi; Jiang, Liqin; Li, Yuan; Wang, Jianhua; Chen, Jiangfan; Qu, Jia

    2010-01-01

    Purpose. To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. Methods. Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. Results. Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. Conclusions. Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development. PMID:20484596

  5. Group IIA secretory phospholipase A2 (GIIA) mediates apoptotic death during NMDA receptor activation in rat primary cortical neurons.

    PubMed

    Chiricozzi, Elena; Fernandez-Fernandez, Seila; Nardicchi, Vincenza; Almeida, Angeles; Bolaños, Juan Pedro; Goracci, Gianfrancesco

    2010-03-01

    Phospholipases A(2) (PLA(2)) participate in neuronal death signalling pathways because of their ability to release lipid mediators, although the contribution of each isoform and mechanism of neurotoxicity are still elusive. Using a novel fluorogenic method to assess changes in a PLA(2) activity by flow cytometry, here we show that the group IIA secretory phospholipase A(2) isoform (GIIA) was specifically activated in cortical neurons following stimulation of N-methyl-d-aspartate glutamate receptor subtype (NMDAR). For activation, GIIA required Ca(2+) and reactive oxygen/nitrogen species, and inhibition of its activity fully prevented NMDAR-mediated neuronal apoptotic death. Superoxide, nitric oxide or peroxynitrite donors stimulated GIIA activity, which mediated neuronal death. Intriguingly, we also found that GIIA activity induced mitochondrial superoxide production after NMDAR stimulation. These results reveal a novel role for GIIA in excitotoxicity both as target and producer of superoxide in a positive-loop of activation that may contribute to the propagation of neurodegeneration.

  6. Resetting microbiota by Lactobacillus reuteri inhibits T reg deficiency–induced autoimmunity via adenosine A2A receptors

    PubMed Central

    Hoang, Thomas K.; Tian, Xiangjun; Luo, Meng; Zhou, Jain; Tatevian, Nina; Molina, Jose G.; Blackburn, Michael R.; Gomez, Thomas H.

    2017-01-01

    Regulatory T (T reg) cell deficiency causes lethal, CD4+ T cell–driven autoimmune diseases. Stem cell transplantation is used to treat these diseases, but this procedure is limited by the availability of a suitable donor. The intestinal microbiota drives host immune homeostasis by regulating the differentiation and expansion of T reg, Th1, and Th2 cells. It is currently unclear if T reg cell deficiency–mediated autoimmune disorders can be treated by targeting the enteric microbiota. Here, we demonstrate that Foxp3+ T reg cell deficiency results in gut microbial dysbiosis and autoimmunity over the lifespan of scurfy (SF) mouse. Remodeling microbiota with Lactobacillus reuteri prolonged survival and reduced multiorgan inflammation in SF mice. L. reuteri changed the metabolomic profile disrupted by T reg cell deficiency, and a major effect was to restore levels of the purine metabolite inosine. Feeding inosine itself prolonged life and inhibited multiorgan inflammation by reducing Th1/Th2 cells and their associated cytokines. Mechanistically, the inhibition of inosine on the differentiation of Th1 and Th2 cells in vitro depended on adenosine A2A receptors, which were also required for the efficacy of inosine and of L. reuteri in vivo. These results reveal that the microbiota–inosine–A2A receptor axis might represent a potential avenue for combatting autoimmune diseases mediated by T reg cell dysfunction. PMID:27994068

  7. Adenosine A(2A) receptor gene (ADORA2A) variants may increase autistic symptoms and anxiety in autism spectrum disorder.

    PubMed

    Freitag, Christine M; Agelopoulos, Konstantin; Huy, Ellen; Rothermundt, Matthias; Krakowitzky, Petra; Meyer, Jobst; Deckert, Jürgen; von Gontard, Alexander; Hohoff, Christa

    2010-01-01

    Autism spectrum disorders (ASDs) are heterogeneous disorders presenting with increased rates of anxiety. The adenosine A(2A) receptor gene (ADORA2A) is associated with panic disorder and is located on chromosome 22q11.23. Its gene product, the adenosine A(2A) receptor, is strongly expressed in the caudate nucleus, which also is involved in ASD. As autistic symptoms are increased in individuals with 22q11.2 deletion syndrome, and large 22q11.2 deletions and duplications have been observed in ASD individuals, in this study, 98 individuals with ASD and 234 control individuals were genotyped for eight single-nucleotide polymorphisms in ADORA2A. Nominal association with the disorder was observed for rs2236624-CC, and phenotypic variability in ASD symptoms was influenced by rs3761422, rs5751876 and rs35320474. In addition, association of ADORA2A variants with anxiety was replicated for individuals with ASD. Findings point toward a possible mediating role of ADORA2A variants on phenotypic expression in ASD that need to be replicated in a larger sample.

  8. 5′-AMP impacts lymphocyte recirculation through activation of A2B receptors

    PubMed Central

    Bouma, Hjalmar R.; Mandl, Judith N.; Strijkstra, Arjen M.; Boerema, Ate S.; Kok, Jan-Willem; van Dam, Annie; IJzerman, Ad; Kroese, Frans G. M.; Henning, Robert H.

    2013-01-01

    Natural hibernation consists of torpid phases with metabolic suppression alternating with euthermic periods. Induction of torpor holds substantial promise in various medical conditions, including trauma, major surgery, and transplantation. Torpor in mice can be induced pharmacologically by 5′-AMP. Previously, we showed that during natural torpor, the reduction in body temperature results in lymphopenia via a reduction in plasma S1P. Here, we show that during torpor induced by 5′-AMP, there is a similar reduction in the number of circulating lymphocytes that is a result of their retention in secondary lymphoid organs. This lymphopenia could be mimicked by engagement of A2BRs by a selective A2BR agonist (LUF6210) in the absence of changes in temperature and prevented by A2BR antagonists during 5′-AMP-induced torpor. In addition, forced cooling of mice led to peripheral blood lymphopenia, independent of A2BR signaling. The induction of torpor using 5′-AMP impacted the migration of lymphocytes within and between secondary lymphoid organs. During torpor, the homing into LNs was impaired, and two-photon intravital microscopy revealed that cell motility was decreased significantly and rapidly upon 5′-AMP administration. Furthermore, the S1P plasma concentration was reduced by 5′-AMP but not by LUF6210. S1P plasma levels restored upon arousal. Likely, the reduced migration in LNs combined with the reduced S1P plasma level substantially reduces lymphocyte egress after injection of 5′-AMP. In conclusion, 5′-AMP induces a state of pharmacological torpor in mice, during which, lymphopenia is governed primarily by body temperature-independent suppression of lymphocyte egress from LNs. PMID:23682128

  9. Identification of novel modulators for ionotropic glutamate receptor, iGluA2 by in-silico screening

    PubMed Central

    2013-01-01

    Background Ionotropic glutamate receptors (iGluAs, IUPHAR nomenclature) are the major excitatory amino acid neurotransmitter receptors in the mammalian central nervous system (CNS). iGluAs are potential therapeutic drug targets for various neurological disorders including ischemia, epilepsy, Parkinson’s and Alzheimer’s diseases. The known iGluA modulators, cyclothiazide (CTZ), IDRA-21, and other benzothiadiazide derivatives (ALTZ, HCTZ, and CLTZ) bind to the ligand-binding domain of flip-form of iGluA2 at the dimer interface, thereby increasing steady-state activation by reducing desensitization. Methods To discover new modulator compounds, we performed virtual screening for the ligand binding domain (LBD) of iGluA2 against NCI Diversity Set III library containing 1597 compounds, and subsequently performed binding-energy analysis for selected compounds. The crystal structure of rat iGluA2 S1S2J (PDB ID: 3IJO) was used for docking studies. Results and conclusion From this study, we obtained four compounds: (1) 10-2(methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, (2) 2-benzo[e]benzotriazol-2-yl-aniline, (3) 9-nitro-6H-indolo-(2,3,-b)quinoxaline, and (4) 1-hydroxy-n-(3-nitrophenyl)-2-napthamide. The binding mode of these four compounds is very similar to that of abovementioned established modulators: two molecules of each compound independently bind to the protein symmetrically at the dimer interface; occupy the subsites B, C, B’ and C’; potentially interact with Ser518 and Ser775. Binding energy analysis shows that all the four hits are comparable to the drug molecule, CTZ, and hence, we propose that the discovered hits may be potential molecules to develop new chemical libraries for modulating the flip form of iGluA2 function. PMID:23855825

  10. Identification of novel modulators for ionotropic glutamate receptor, iGluA2 by in-silico screening.

    PubMed

    Padmanabhan, Balasundaram

    2013-07-15

    Ionotropic glutamate receptors (iGluAs, IUPHAR nomenclature) are the major excitatory amino acid neurotransmitter receptors in the mammalian central nervous system (CNS). iGluAs are potential therapeutic drug targets for various neurological disorders including ischemia, epilepsy, Parkinson's and Alzheimer's diseases. The known iGluA modulators, cyclothiazide (CTZ), IDRA-21, and other benzothiadiazide derivatives (ALTZ, HCTZ, and CLTZ) bind to the ligand-binding domain of flip-form of iGluA2 at the dimer interface, thereby increasing steady-state activation by reducing desensitization. To discover new modulator compounds, we performed virtual screening for the ligand binding domain (LBD) of iGluA2 against NCI Diversity Set III library containing 1597 compounds, and subsequently performed binding-energy analysis for selected compounds. The crystal structure of rat iGluA2 S1S2J (PDB ID: 3IJO) was used for docking studies. From this study, we obtained four compounds: (1) 10-2(methoxyethyl)-3-phenylbenzo[g]pteridine-2,4-dione, (2) 2-benzo[e]benzotriazol-2-yl-aniline, (3) 9-nitro-6H-indolo-(2,3,-b)quinoxaline, and (4) 1-hydroxy-n-(3-nitrophenyl)-2-napthamide. The binding mode of these four compounds is very similar to that of abovementioned established modulators: two molecules of each compound independently bind to the protein symmetrically at the dimer interface; occupy the subsites B, C, B' and C'; potentially interact with Ser518 and Ser775. Binding energy analysis shows that all the four hits are comparable to the drug molecule, CTZ, and hence, we propose that the discovered hits may be potential molecules to develop new chemical libraries for modulating the flip form of iGluA2 function.

  11. Activation of nuclear receptor NR5A2 increases Glut4 expression and glucose metabolism in muscle cells

    SciTech Connect

    Bolado-Carrancio, A.; Riancho, J.A.; Sainz, J.

    Highlights: • NR5A2 expression in C2C12 is associated with myotube differentiation. • DLPC induces an increase in GLUT4 levels and glucose uptake in C2C12 myotubes. • In high glucose conditions the activation of NR5A2 inhibits fatty acids oxidation. - Abstract: NR5A2 is a nuclear receptor which regulates the expression of genes involved in cholesterol metabolism, pluripotency maintenance and cell differentiation. It has been recently shown that DLPC, a NR5A2 ligand, prevents liver steatosis and improves insulin sensitivity in mouse models of insulin resistance, an effect that has been associated with changes in glucose and fatty acids metabolism in liver. Becausemore » skeletal muscle is a major tissue in clearing glucose from blood, we studied the effect of the activation of NR5A2 on muscle metabolism by using cultures of C2C12, a mouse-derived cell line widely used as a model of skeletal muscle. Treatment of C2C12 with DLPC resulted in increased levels of expression of GLUT4 and also of several genes related to glycolysis and glycogen metabolism. These changes were accompanied by an increased glucose uptake. In addition, the activation of NR5A2 produced a reduction in the oxidation of fatty acids, an effect which disappeared in low-glucose conditions. Our results suggest that NR5A2, mostly by enhancing glucose uptake, switches muscle cells into a state of glucose preference. The increased use of glucose by muscle might constitute another mechanism by which NR5A2 improves blood glucose levels and restores insulin sensitivity.« less

  12. Genetic Inactivation of the Adenosine A2A Receptor Attenuates Pathologic but Not Developmental Angiogenesis in the Mouse Retina

    PubMed Central

    Liu, Xiao-Ling; Zhou, Rong; Pan, Qi-Qi; Jia, Xiao-Lin; Gao, Wei-Na; Wu, Jun; Lin, Jing; Chen, Jiang-Fan

    2010-01-01

    Purpose. The adenosine A2A receptor (A2AR) modulates normal vascularization and pathologic angiogenesis in many tissues and may contribute to the pathogenesis of retinopathy of prematurity (ROP) characterized by abnormal retinal vascularization in surviving premature infants. Here, the authors studied the effects of the genetic inactivation of A2AR on normal retinal vascularization and the development of pathologic angiogenesis in oxygen-induced retinopathy (OIR), an animal model of ROP. Methods. After exposure to 75% oxygen for 5 days (postnatal day [P] 7–P12) and subsequently to room air for the next 9 days (P13–P21), we evaluated retinal vascular morphology by ADPase staining in retinal whole mounts, retinal neovascularization response by histochemistry in serial retinal sections, and retinal VEGF gene expression by real-time PCR analysis in A2AR knockout (KO) mice and their wild-type (WT) littermates. Results. At P17, A2AR KO mice displayed attenuated OIR compared with WT littermates, as evidenced by reduced vaso-obliteration and areas of nonperfusion in the center of the retina, reduced pathologic angiogenesis as evident by decreased non-ganglion cells and neovascular nuclei, and inhibited hypoxia-induced retinal VEGF gene expression. Notably, the attenuation of pathologic angiogenesis by A2AR inactivation was selective for OIR because it did not affect normal retinal vascularization during postnatal development. Conclusions. These findings provide the first evidence that A2AR is critical for the development of OIR and suggest a novel therapeutic approach of A2AR inactivation for ROP by selectively targeting pathologic but not developmental angiogenesis in the retina. PMID:20610844

  13. Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor.

    PubMed

    Piirainen, Henni; Taura, Jaume; Kursula, Petri; Ciruela, Francisco; Jaakola, Veli-Pekka

    2017-04-01

    Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A 2A receptor (A 2A R), has an exceptionally long intracellular C terminus (A 2A R-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A 2A R and the role of Ca 2+ in this process. First, we studied the A 2A R-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A 2A R-ct through its distal calmodulin-like domain in a Ca 2+ -independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the A 2A R-calmodulin/Ca 2+ complex. Importantly, calmodulin displaced α-actinin 1 from the A 2A R-ct in a Ca 2+ -dependent fashion, disrupting the A 2A R-α-actinin 1 complex. Finally, we assessed the impact of Ca 2+ on A 2A R internalization in living cells, a function operated by the A 2A R-α-actinin 1 complex. Interestingly, while Ca 2+ influx did not affect constitutive A 2A R endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A 2A R/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A 2A R with calmodulin and α-actinin 1 is fine-tuned by Ca 2+ , a fact that might power agonist-mediated receptor internalization and function. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. TP53 mutation and survival in aggressive B cell lymphoma.

    PubMed

    Zenz, Thorsten; Kreuz, Markus; Fuge, Maxi; Klapper, Wolfram; Horn, Heike; Staiger, Annette M; Winter, Doris; Helfrich, Hanne; Huellein, Jennifer; Hansmann, Martin-Leo; Stein, Harald; Feller, Alfred; Möller, Peter; Schmitz, Norbert; Trümper, Lorenz; Loeffler, Markus; Siebert, Reiner; Rosenwald, Andreas; Ott, German; Pfreundschuh, Michael; Stilgenbauer, Stephan

    2017-10-01

    TP53 is mutated in 20-25% of aggressive B-cell lymphoma (B-NHL). To date, no studies have addressed the impact of TP53 mutations in prospective clinical trial cohorts. To evaluate the impact of TP53 mutation to current risk models in aggressive B-NHL, we investigated TP53 gene mutations within the RICOVER-60 trial. Of 1,222 elderly patients (aged 61-80 years) enrolled in the study and randomized to six or eight cycles of CHOP-14 with or without Rituximab (NCT00052936), 265 patients were analyzed for TP53 mutations. TP53 mutations were demonstrated in 63 of 265 patients (23.8%). TP53 mutation was associated with higher LDH (65% vs. 37%; p < 0.001), higher international prognostic index-Scores (IPI 4/5 27% vs. 12%; p = 0.025) and B-symptoms (41% vs. 24%; p = 0.011). Patients with TP53 mutation were less likely to obtain a complete remission CR/CRu (CR unconfirmed) 61.9% (mut) vs. 79.7% (wt) (p = 0.007). TP53 mutations were associated with decreased event-free (EFS), progression-free (PFS) and overall survival (OS) (median observation time of 40.2 months): the 3 year EFS, PFS and OS were 42% (vs. 60%; p = 0.012), 42% (vs. 67.5%; p < 0.001) and 50% (vs. 76%; p < 0.001) for the TP53 mutation group. In a Cox proportional hazard analysis adjusting for IPI-factors and treatment arms, TP53 mutation was shown to be an independent predictor of EFS (HR 1.5), PFS (HR 2.0) and OS (HR 2.3; p < 0.001). TP53 mutations are independent predictors of survival in untreated patients with aggressive CD20+ lymphoma. TP53 mutations should be considered for risk models in DLBCL and strategies to improve outcome for patients with mutant TP53 must be developed. © 2017 UICC.

  15. Role of Adenosine Receptor A2A in Traumatic Optic Neuropathies (Addendum)

    DTIC Science & Technology

    2016-03-01

    inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT...Neuroimmunology 277 (2014) 96–104were evaluated by analysis of variance (one-way ANOVA), and the significance of differences between groups was assessed by the...Ahmad et al. / Journal of Neuroimmunology 277 (2014) 96–104were evaluated by analysis of variance (one-way ANOVA), and the significance of differences

  16. Insulin-Increased L-Arginine Transport Requires A2A Adenosine Receptors Activation in Human Umbilical Vein Endothelium

    PubMed Central

    Guzmán-Gutiérrez, Enrique; Westermeier, Francisco; Salomón, Carlos; González, Marcelo; Pardo, Fabián; Leiva, Andrea; Sobrevia, Luis

    2012-01-01

    Adenosine causes vasodilation of human placenta vasculature by increasing the transport of arginine via cationic amino acid transporters 1 (hCAT-1). This process involves the activation of A2A adenosine receptors (A2AAR) in human umbilical vein endothelial cells (HUVECs). Insulin increases hCAT-1 activity and expression in HUVECs, and A2AAR stimulation increases insulin sensitivity in subjects with insulin resistance. However, whether A2AAR plays a role in insulin-mediated increase in L-arginine transport in HUVECs is unknown. To determine this, we first assayed the kinetics of saturable L-arginine transport (1 minute, 37°C) in the absence or presence of nitrobenzylthioinosine (NBTI, 10 µmol/L, adenosine transport inhibitor) and/or adenosine receptors agonist/antagonists. We also determined hCAT-1 protein and mRNA expression levels (Western blots and quantitative PCR), and SLC7A1 (for hCAT-1) reporter promoter activity. Insulin and NBTI increased the extracellular adenosine concentration, the maximal velocity for L-arginine transport without altering the apparent K m for L-arginine transport, hCAT-1 protein and mRNA expression levels, and SLC7A1 transcriptional activity. An A2AAR antagonist ZM-241385 blocked these effects. ZM241385 inhibited SLC7A1 reporter transcriptional activity to the same extent in cells transfected with pGL3-hCAT-1−1606 or pGL3-hCAT-1−650 constructs in the presence of NBTI + insulin. However, SLC7A1 reporter activity was increased by NBTI only in cells transfected with pGL3-hCAT-1−1606, and the ZM-241385 sensitive fraction of the NBTI response was similar in the absence or in the presence of insulin. Thus, insulin modulation of hCAT-1 expression and activity requires functional A2AAR in HUVECs, a mechanism that may be applicable to diseases associated with fetal insulin resistance, such as gestational diabetes. PMID:22844517

  17. Coupling to protein kinases A and C of adenosine A2B receptors involved in the facilitation of noradrenaline release in the prostatic portion of rat vas deferens.

    PubMed

    Queiroz, Glória; Quintas, Clara; Talaia, Carlos; Gonçalves, Jorge

    2004-08-01

    In the prostatic portion of rat vas deferens, the non-selective adenosine receptor agonist NECA (0.1-30 microM), but not the A(2A) agonist CGS 21680 (0.001-10 microM), caused a facilitation of electrically evoked noradrenaline release (up to 43 +/- 4%), when inhibitory adenosine A(1) receptors were blocked. NECA-elicited facilitation of noradrenaline release was prevented by the A(2B) receptor-antagonist MRS 1754, enhanced by preventing cyclic-AMP degradation with rolipram, abolished by the protein kinase A inhibitors H-89, KT 5720 and cyclic-AMPS-Rp and attenuated by the protein kinase C inhibitors Ro 32-0432 and calphostin C. The adenosine uptake inhibitor NBTI also elicited a facilitation of noradrenaline release; an effect that was abolished by adenosine deaminase and attenuated by MRS 1754, by inhibitors of the extracellular nucleotide metabolism and by blockade of alpha(1)-adrenoceptors and P2X receptors with prazosin and NF023, respectively. It was concluded that adenosine A(2B) receptors are involved in a facilitation of noradrenaline release in the prostatic portion of rat vas deferens that can be activated by adenosine formed by extracellular catabolism of nucleotides. The receptors seem to be coupled to the adenylyl cyclase-protein kinase A pathway but activation of the protein kinase C by protein kinase A, may also contribute to the adenosine A(2B) receptor-mediated facilitation of noradrenaline release.

  18. Evaluation of Tp-e interval and Tp-e/QT ratio in patients with ankylosing spondylitis.

    PubMed

    Acar, Gurkan; Yorgun, Hikmet; Inci, Mehmet Fatih; Akkoyun, Murat; Bakan, Betul; Nacar, Alper Bugra; Dirnak, Imran; Cetin, Gozde Yildirim; Bozoglan, Orhan

    2014-03-01

    Ankylosing spondylitis (AS) is a chronic multi-systemic inflammatory rheumatic disorder. Several studies have suggested that the interval from the peak to the end of the electrocardiographic T wave (Tp-e) may correspond to the transmural dispersion of repolarization and that increased Tp-e interval and Tp-e/QT ratio are associated with malignant ventricular arrhythmias. The aim of this study was to evaluate ventricular repolarization by using Tp-e interval and Tp-e/QT ratio in patients with AS, and to assess the relation with inflammation. Sixty-two patients with AS and 50 controls were included. Tp-e interval and Tp-e/QT ratio were measured from a 12-lead electrocardiogram, and the Tp-e interval corrected for heart rate. The plasma level of high sensitive C-reactive protein (hsCRP) was measured. These parameters were compared between groups. In electrocardiographic parameters analysis, QT dispersion (QTd) and corrected QTd were significantly increased in AS patients compared to the controls (31.7 ± 9.6 vs 28.2 ± 7.4 and 35.8 ± 11.5 vs 30.6 ± 7.9 ms, P = 0.03 and P = 0.007, respectively). cTp-e interval and Tp-e/QT ratio were also significantly higher in AS patients (92.1 ± 10.2 vs 75.8 ± 8.4 and 0.22 ± 0.02 vs 0.19 ± 0.02 ms, all P values <0.001). cTp-e interval and Tp-e/QT ratio were significantly correlated with hsCRP (r = 0.63, P < 0.001 and r = 0.49, P < 0.001, respectively). Our study revealed that Tp-e interval and Tp-e/QT ratio were increased in AS patients. These electrocardiographic ventricular repolarization indexes were significantly correlated with the plasma level of hsCRP.

  19. Evaluation of Tp-e interval and Tp-e/QT ratio in patients with ankylosing spondylitis.

    PubMed

    Acar, Gurkan; Yorgun, Hikmet; Inci, Mehmet Fatih; Akkoyun, Murat; Bakan, Betul; Nacar, Alper Bugra; Dirnak, Imran; Cetin, Gozde Yildirim; Bozoglan, Orhan

    2013-04-12

    OBJECTIVES: Ankylosing spondylitis (AS) is a chronic multi-systemic inflammatory rheumatic disorder. Several studies have suggested that the interval from the peak to the end of the electrocardiographic T wave (Tp-e) may correspond to the transmural dispersion of repolarization and that increased Tp-e interval and Tp-e/QT ratio are associated with malignant ventricular arrhythmias. The aim of this study was to evaluate ventricular repolarization by using Tp-e interval and Tp-e/QT ratio in patients with AS, and to assess the relation with inflammation. METHODS: Sixty-two patients with AS and 50 controls were included. Tp-e interval and Tp-e/QT ratio were measured from a 12-lead electrocardiogram, and the Tp-e interval corrected for heart rate. The plasma level of high sensitive C-reactive protein (hsCRP) was measured. These parameters were compared between groups. RESULTS: In electrocardiographic parameters analysis, QT dispersion (QTd) and corrected QTd were significantly increased in AS patients compared to the controls (31.7 ± 9.6 vs 28.2 ± 7.4 and 35.8 ± 11.5 vs 30.6 ± 7.9 ms, P = 0.03 and P = 0.007, respectively). cTp-e interval and Tp-e/QT ratio were also significantly higher in AS patients (92.1 ± 10.2 vs 75.8 ± 8.4 and 0.22 ± 0.02 vs 0.19 ± 0.02 ms, all P values <0.001). cTp-e interval and Tp-e/QT ratio were significantly correlated with hsCRP (r = 0.63, P < 0.001 and r = 0.49, P < 0.001, respectively). CONCLUSIONS: Our study revealed that Tp-e interval and Tp-e/QT ratio were increased in AS patients. These electrocardiographic ventricular repolarization indexes were significantly correlated with the plasma level of hsCRP.

  20. Adenosine A2A Receptor Blockade or Deletion Diminishes Fibrocyte Accumulation in the Skin in a Murine Model of Scleroderma, Bleomycin-induced Fibrosis

    PubMed Central

    Katebi, Majid; Fernandez, Patricia; Chan, Edwin S. L.; Cronstein, Bruce N.

    2015-01-01

    Peripheral blood fibrocytes are a newly identified circulating leukocyte subpopulation that migrates into injured tissue where it may display fibroblast-like properties and participate in wound healing and fibrosis of skin and other organs. Previous studies in our lab demonstrated that A2A receptor-deficient and A2A antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis, thus the aim of this study was to determine whether the adenosine A2A receptor regulates recruitment of fibrocytes to the dermis in this bleomycin-induced model of dermal fibrosis. Sections of skin from normal mice and bleomycin-treated wild type, A2A knockout and A2A antagonist-treated mice were stained for Procollagen α2 Type I and CD34 and the double stained cells, fibrocytes, were counted in the tissue sections. There were more fibrocytes in the dermis of bleomycin-treated mice than normal mice and the increase was abrogated by deletion or blockade of adenosine A2A receptors. Because fibrocytes play a central role in tissue fibrosis these results suggest that diminished adenosine A2A receptor-mediated recruitment of fibrocytes into tissue may play a role in the pathogenesis of fibrosing diseases of the skin. Moreover, these results provide further evidence that adenosine A2A receptors may represent a new target for the treatment of such fibrosing diseases as scleroderma or nephrogenic fibrosing dermopathy. PMID:18709547

  1. Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

    PubMed

    Seigfried, Franziska A; Dietmann, Petra; Kühl, Michael; Kühl, Susanne J

    2018-06-01

    The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Epithelial-specific A2B adenosine receptor signaling protects the colonic epithelial barrier during acute colitis

    PubMed Central

    Aherne, CM; Saeedi, B; Collins, CB; Masterson, JC; McNamee, EN; Perrenoud, L; Rapp, CR; Curtis, VF; Bayless, A; Fletcher, A; Glover, LE; Evans, CM; Jedlicka, P; Furuta, GT; de Zoeten, EF; Colgan, SP; Eltzschig, HK

    2015-01-01

    Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b−/− mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2bfl/flVeCadCre+) or intestinal epithelia (Adora2bfl/flVillinCre+) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses. PMID:25850656

  3. Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous‐cell carcinoma

    PubMed Central

    Syed, Nazia; Barbhuiya, Mustafa A.; Pinto, Sneha M.; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K.; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T. S. Keshava; Kumar, M. Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh

    2015-01-01

    Esophageal squamous‐cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early‐stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non‐neoplastic Het‐1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry‐based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA‐based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation. PMID:25366905

  4. Adenosine A(2B) receptor antagonist PSB603 suppresses tumor growth and metastasis by inhibiting induction of regulatory T cells.

    PubMed

    Kaji, Wakako; Tanaka, Satomi; Tsukimoto, Mitsutoshi; Kojima, Shuji

    2014-04-01

    Regulatory T cells (Treg) play a role in suppression of immune response, including anti-tumor immunity. We have recently reported that treatment of naïve CD4 T cells with adenosine A(2B) receptor antagonist PSB603 under Treg-skewing conditions inhibits expression of Foxp3, a marker of differentiation to Treg, without blocking IL-2 production or CD25 expression, which are activation markers, in CD4 T cells. We hypothesized that PSB603 suppresses cancer growth and metastasis by inhibiting induction of Treg, thereby facilitating anti-tumor immunity. In this study, we first examined the effect of PSB603 on tumor growth in B16 melanoma-bearing C57BL/6 mice. Administration of PSB603 significantly suppressed the increase of tumor volume as well as the increase of Treg population in these mice. The populations of CD4 and CD8 T cells were higher and splenic lymphocyte-mediated cytotoxicity towards B16 melanoma was significantly increased in PSB603-treated mice. We confirmed that PSB603 did not reduce the viability of B16 melanoma cells in vitro. Moreover, we also examined the effect of PSB603 on tumor metastasis in pulmonary metastasis model mice intravenously injected with B16 melanoma cells. The metastasis was also suppressed in PSB603-treated mice, in which the population of Treg was significantly lower. Overall, our results suggest that A(2B) receptor antagonist PSB603 enhances anti-tumor immunity by inhibiting differentiation to Treg, resulting in a delay of tumor growth and a suppression of metastasis.

  5. Ligand Recognition by A-Class Eph Receptors: Crystal Structures of the EphA2 Ligand-Binding Domain and the EphA2/ephrin-A1 Complex

    SciTech Connect

    Himanen, J.; Goldgur, Y; Miao, H

    2009-01-01

    Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B-class receptors. Here, we present the crystal structures of an A-class complex between EphA2 and ephrin-A1 and of unbound EphA2. Although these structures are similar overall to their B-class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A-class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a 'lock-and-key'-type binding mechanism, in contrast to the 'induced fit' mechanism defining the B-class molecules.more » This model is supported by structure-based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell-based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2-specific homotypic cell adhesion interactions.« less

  6. Guanosine may increase absence epileptic activity by means of A2A adenosine receptors in Wistar Albino Glaxo Rijswijk rats.

    PubMed

    Lakatos, Renáta Krisztina; Dobolyi, Árpád; Todorov, Mihail Ivilinov; Kékesi, Katalin A; Juhász, Gábor; Aleksza, Magdolna; Kovács, Zsolt

    2016-06-01

    The non-adenosine nucleoside guanosine (Guo) was demonstrated to decrease quinolinic acid(QA)-induced seizures, spontaneously emerged absence epileptic seizures and lipopolysaccharide(LPS)-evoked induction of absence epileptic seizures suggesting its antiepileptic potential. It was also described previously that intraperitoneal (i.p.) injection of 20 and 50mg/kg Guo decreased the number of spike-wave discharges (SWDs) in a well investigated model of human absence epilepsy, the Wistar Albino Glaxo Rijswijk (WAG/Rij) rats during 4th (20mg/kg Guo) and 3rd as well as 4th (50mg/kg Guo) measuring hours. Guanosine can potentially decrease SWD number by means of its putative receptors but absence epileptic activity changing effects of Guo by means of increased extracellular adenosine (Ado) cannot be excluded. An increase in the dose of i.p. injected Guo is limited by its low solubility in saline, therefore, we addressed in the present study whether higher doses of Guo, diluted in sodium hydroxide (NaOH) solution, have more potent antiepileptic effect in WAG/Rij rats. We confirmed that i.p. 50mg/kg Guo decreased but, surprisingly, i.p. 100mg/kg Guo enhanced the number of SWDs in WAG/Rij rats. Combined i.p. injection of a non-selective Ado receptor antagonist theophylline (5mg/kg) or a selective Ado A2A receptor (A2AR) antagonist SCH 58261 (7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine) (1mg/kg) and a cyclooxygenase 1 and 2/COX-1 and COX-2 inhibitor indomethacin (10mg/kg) with 100mg/kg Guo decreased the SWD number compared to i.p. 100mg/kg Guo alone. The results suggest that i.p. 100mg/kg Guo can increase SWD number by means of the adenosinergic system. Copyright © 2016 Elsevier Inc. All rights reserved.

  7. Adenosine A2A receptor blockade attenuates spatial memory deficit and extent of demyelination areas in lyolecithin-induced demyelination model.

    PubMed

    Akbari, Atefeh; Khalili-Fomeshi, Mohsen; Ashrafpour, Manouchehr; Moghadamnia, Ali Akbar; Ghasemi-Kasman, Maryam

    2018-05-03

    In recent years, inactivation of A 2A adenosine receptors has been emerged as a novel strategy for treatment of several neurodegenerative diseases. Although numerous studies have shown the beneficial effects of A 2A receptors blockade on spatial memory, the impacts of selective adenosine A 2A receptors on memory performance has not yet been examined in the context of demyelination. In the present study, we evaluated the effect of A 2A receptor antagonist SCH58261 on spatial memory and myelination in an experimental model of focal demyelination in rat fimbria. Demyelination was induced by local injection of lysolecithin (LPC) 1% (2 μl) into the hippocampus fimbria. SCH58261 (20 μg/0.5 μl or 40 μg/0.5 μl) was daily injected intracerebroventricularly (i.c.v.) for 10 days post LPC injection. The Morris water maze test was used to assess the spatial learning and memory on day 6 post lesion. Myelin staining and immunostaining against astrocytes/microglia were carried out 10 days post LPC injection. The administration of adenosine A 2A receptor antagonist prevented the spatial memory impairment in LPC receiving animals. Myelin staining revealed that application of SCH58261 reduces the extent of demyelination areas in the fimbria. Furthermore, the level of astrocytes and microglia activation was attenuated following administration of A 2A receptor antagonist. Collectively, the results of this study suggest that A 2A receptor blockade can improve the spatial memory and protect myelin sheath, which might be considered as a novel therapeutic approach for multiple sclerosis disease. Copyright © 2017. Published by Elsevier Inc.

  8. Effect of long term caffeine treatment on A1 and A2 adenosine receptor binding and on mRNA levels in rat brain.

    PubMed

    Johansson, B; Ahlberg, S; van der Ploeg, I; Brené, S; Lindefors, N; Persson, H; Fredholm, B B

    1993-04-01

    The effect of long-term oral treatment with caffeine on A1 and A2 receptors in the rat brain was studied. Caffeine was added to the drinking water and the animals were sacrificed after a 12 day treatment period. The plasma caffeine concentration was close to 100 microM. A1 receptors were studied using quantitative autoradiography with [3H]cyclohexyladenosine (CHA). Caffeine treatment increased the number of A1 receptors in the CA3 subfield of the hippocampus from 337 to 393 fmol/mg with no change in KD (0.692 vs. 0.675 nM). A1 mRNA was measured using Northern blots and quantitative in situ hybridization. There was no increase in A1 mRNA. A2a receptors, located in dopamine rich regions of the rat brain, were studied with quantitative autoradiography using [3H]CGS 21680 as the ligand, and the A2a mRNA was determined using quantitative in situ hybridization. Caffeine treatment produced no significant change in either receptor number or mRNA, even though the apparent Bmax tended to increase from 322 +/- 8 to 352 +/- 8 fmol/mg. The results show that treatment with caffeine in a dose that causes tolerance to several effects of caffeine and increases some effects of adenosine analogues increases the number of A1 receptors without any change in A1 mRNA, suggesting that the adaptive changes are at a post-translational level. There were no significant changes in A2 receptors indicating that the two types are regulated differently and/or that the amount of endogenous agonist is sufficient to regulate A1, but not A2 receptors.

  9. Wound healing effects of noni (Morinda citrifolia L.) leaves: a mechanism involving its PDGF/A2A receptor ligand binding and promotion of wound closure.

    PubMed

    Palu, Afa; Su, Chen; Zhou, Bing-Nan; West, Brett; Jensen, Jarakae

    2010-10-01

    Morinda citrifolia L. (Rubiaceae) commonly known as noni, has been used in Polynesia by traditional healers for the treatment of cuts, bruises and wounds. Our objective was to investigate the wound-healing mechanisms of the noni leaf. The investigations of its wound-healing mechanisms were carried out using fresh noni leaf juice (NLJ), noni leaf ethanol extract (NLEE) and its methanol (MFEE) and hexane (HFEE) fractions on the PDGF and A(2A) receptors in vitro and topically in mice. Fresh noni leaf juice showed significant affinity to PDGF receptors, and displayed 166% binding inhibition of the ligand binding to its receptors, while at the same concentration, it only had 7% inhibition of the ligand binding to the A(2A) receptors. NLEE, HFEE and MFEE showed significant affinity to A(2A) receptors, concentration dependently, with IC(50) values of 34.1, 42.9 and 86.7 μg/mL, respectively. However, MFEE significantly increased wound closure and reduced the half closure time in mice with a CT(50) of 5.4 ± 0.2 days compared with control (p < 0.05). These results suggest that noni leaf significantly accelerated wound healing in mice via its ligand binding to the PDGF and A(2A) receptors as its probable mechanisms of wound-healing and also support its traditional usage for wound-healing in Polynesia. Copyright © 2010 John Wiley & Sons, Ltd.

  10. Hsp90 is an essential regulator of EphA2 receptor stability and signaling: Implications for cancer cell migration and metastasis

    PubMed Central

    Annamalai, Balasubramaniam; Liu, Xueguang; Gopal, Udhayakumar; Isaacs, Jennifer

    2011-01-01

    A subset of Eph receptors and their corresponding ligands are commonly expressed in tumor cells, where they mediate biological processes such as cell migration and adhesion, while their expression in endothelial cells promotes angiogenesis. In particular, the tumor-specific upregulation of EphA2 confers properties of increased cellular motility, invasiveness, tumor angiogenesis, and tumor progression, and its overexpression correlates with poor prognosis in several cancer types. The cellular chaperone Hsp90 also plays a significant role in regulating cell migration and angiogenesis, although the full repertoire of motility driving proteins dependent upon Hsp90 function remain poorly defined. We explored the hypothesis that Hsp90 may regulate the activity of EphA2 and examined the potential relationship between EphA2 receptor signaling and chaperone function. We demonstrate that geldanamycin (GA), an Hsp90 antagonist, dramatically destabilizes newly synthesized EphA2 protein and diminishes receptor levels in a proteasome-dependent pathway. In addition, GA treatment impairs EphA2 signaling, as evidenced by a decrease in ligand-dependent receptor phosphorylation and subsequent cell rounding. Therefore, Hsp90 exerts a dual role in regulating the stability of nascent EphA2 protein, and maintaining the signaling capacity of the mature receptor. Our findings also suggest that the GA-dependent mitigation of EphA2 signaling in receptor-overexpressing cancer cells may be sufficient to recapitulate the anti-motility effects of this drug. Finally, the identification of a pharmacologic approach to suppress EphA2 expression and signaling highlights the attractive possibility that Hsp90 inhibitors may have clinical utility in antagonizing EphA2-dependent tumorigenic progression. PMID:19567782

  11. MicroRNA‑663b mediates TAM resistance in breast cancer by modulating TP73 expression.

    PubMed

    Jiang, Hua; Cheng, Lin; Hu, Pan; Liu, Renbin

    2018-05-23

    Breast cancer is the second leading cause of cancer‑associated mortalities in women. Tamoxifen (TAM) is an endocrine therapy commonly used in the treatment of patients with breast cancer expressing estrogen receptor α. However, treatment often ends in failure due to the emergence of drug resistance. MicroRNAs (miRNAs), a family of small non‑coding RNAs, serve critical roles in the regulation of gene expression and cell events. To date, whether miRNA‑663b could mediate TAM resistance in breast cancer remains unknown. Therefore, the aim of the present study was to investigate the role of miRNA‑663b in TAM resistance in breast cancer. The results demonstrated that miRNA‑663b was upregulated in breast cancer with TAM resistance. Tumor protein 73 (TP73) was a direct target of miRNA‑663b, and was negatively regulated by miRNA‑663b in MCF‑7 cells. Furthermore, it was identified that downregulation of miRNA‑663b inhibited cell proliferation ability and promoted cell apoptosis, resulting in enhanced TAM sensitivity. In addition, these findings suggested that TP73 silencing may have eliminated the effects of miRNA‑663b inhibitor on breast cancer cells. In conclusion, the present study verified a novel molecular link between miRNA‑663b and TP73, and indicated that miRNA‑663b may be a critical therapeutic target in breast cancer.

  12. NR4A2: Effects of an “Orphan” Receptor on Sustained Attention in a Schizophrenic Population

    PubMed Central

    Ancín, Inés; Cabranes, José A.; Vázquez-Álvarez, Blanca; Santos, José Luis; Sánchez-Morla, Eva; Alaerts, Maaike; Del-Favero, Jurgen; Barabash, Ana

    2013-01-01

    NR4A2 (nuclear receptor subfamily 4 group A member 2) or Nurr1 is a transcription factor implied in the differentiation, maturation, and survival of dopaminergic neurons. It also has a role in the expression of several proteins that are necessary for the synthesis and regulation of dopamine (DA), such as tyrosine hidroxilase, dopamine transporter, vesicular monoamine transporter 2, and cRET. DA is an important neurotransmitter in attentional pathways. Our aim was to evaluate the influence of NR4A2 gene in the performance of schizophrenia (SZ) patients and healthy subjects on a sustained attention task. For this study, we collected 188 SZ subjects (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) and 100 control individuals. We genotyped 5 tag SNPs in NR4A2 gene: rs1150143 (C/G), rs1150144 (A/G), rs834830 (A/G), rs1466408 (T/A), and rs707132 (A/G). We also analyzed the influence of its haplotypes (frequency >5%). To examine sustained attention, all the individuals completed the Degraded Stimulus Continuous Performance Test. We evaluated “hits,” “reaction time,” “sensibility a,” and “false alarms.” In the schizophrenic group, recessive genotypes of rs1150143, rs1150144, rs834830, and rs707132 were associated with a worse performance. SZ subjects who carried GGGTG haplotype showed less hits (P < .004), lower sensibility a scores (P < .009), and a higher reaction time (P = .013). We observed a sex effect of the gene: genotype and haplotype associations were only present in the male group. We conclude that NR4A2 gene is involved in attentional deficits of SZ patients, modifying hits, sensibility a, and reaction time. PMID:22294735

  13. Distinct domains of the AVRPM3A2/F2 avirulence protein from wheat powdery mildew are involved in immune receptor recognition and putative effector function

    USDA-ARS?s Scientific Manuscript database

    Recognition of the AVRPM3A2/F2 avirulence protein from powdery mildew by the wheat PM3A/F immune receptor induces a hypersensitive response after coexpression in Nicotiana benthamiana. The molecular determinants of this interaction and how they shape natural AvrPm3a2/f2 allelic diversity is unknown....

  14. Adenosine A2A receptors are required for glutamate mGluR5- and dopamine D1 receptor-evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor.

    PubMed

    Krania, Paraskevi; Dimou, Eleni; Bantouna, Maria; Kouvaros, Stylianos; Tsiamaki, Eirini; Papatheodoropoulos, Costas; Sarantis, Konstantinos; Angelatou, Fevronia

    2018-05-01

    Interaction between mGluR5 and NMDA receptors (NMDAR) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A 2A receptors (A 2A R) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDARs co-stimulation synergistically activate ERK1/2 signaling leading to c-Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A 2A Rs. Moreover, mGluR5-mediated ERK1/2 phosphorylation depends on NMDAR, which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR-evoked ERK1/2 activation correlates well with the mGluR5/NMDAR-evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A 2A Rs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA1 region of hippocampus, the theta burst stimulation (TBS)-induced long-term potentiation coincides temporally with an increase in ERK1/2 activation and both phenomena are dependent on the tripartite A 2A , mGlu5, and NMDARs. Furthermore, we show that the dopamine D1 receptors evoked ERK1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A 2A Rs. In conclusion, our results highlight the A 2A Rs as a crucial regulator not only for NMDAR responses, but also for regulating ERK1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation. © 2017 International

  15. Enthalpy-Entropy Compensation in the Binding of Modulators at Ionotropic Glutamate Receptor GluA2.

    PubMed

    Krintel, Christian; Francotte, Pierre; Pickering, Darryl S; Juknaitė, Lina; Pøhlsgaard, Jacob; Olsen, Lars; Frydenvang, Karla; Goffin, Eric; Pirotte, Bernard; Kastrup, Jette S

    2016-06-07

    The 1,2,4-benzothiadiazine 1,1-dioxide type of positive allosteric modulators of the ionotropic glutamate receptor A2 (GluA2) are promising lead compounds for the treatment of cognitive disorders, e.g., Alzheimer's disease. The modulators bind in a cleft formed by the interface of two neighboring ligand binding domains and act by stabilizing the agonist-bound open-channel conformation. The driving forces behind the binding of these modulators can be significantly altered with only minor substitutions to the parent molecules. In this study, we show that changing the 7-fluorine substituent of modulators BPAM97 (2) and BPAM344 (3) into a hydroxyl group (BPAM557 (4) and BPAM521 (5), respectively), leads to a more favorable binding enthalpy (ΔH, kcal/mol) from -4.9 (2) and -7.5 (3) to -6.2 (4) and -14.5 (5), but also a less favorable binding entropy (-TΔS, kcal/mol) from -2.3 (2) and -1.3 (3) to -0.5 (4) and 4.8 (5). Thus, the dissociation constants (Kd, μM) of 4 (11.2) and 5 (0.16) are similar to those of 2 (5.6) and 3 (0.35). Functionally, 4 and 5 potentiated responses of 10 μM L-glutamate at homomeric rat GluA2(Q)i receptors with EC50 values of 67.3 and 2.45 μM, respectively. The binding mode of 5 was examined with x-ray crystallography, showing that the only change compared to that of earlier compounds was the orientation of Ser-497 pointing toward the hydroxyl group of 5. The favorable enthalpy can be explained by the formation of a hydrogen bond from the side-chain hydroxyl group of Ser-497 to the hydroxyl group of 5, whereas the unfavorable entropy might be due to desolvation effects combined with a conformational restriction of Ser-497 and 5. In summary, this study shows a remarkable example of enthalpy-entropy compensation in drug development accompanied with a likely explanation of the underlying structural mechanism. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Use of molecular modeling aided design to dial out hERG liability in adenosine A(2A) receptor antagonists.

    PubMed

    Deng, Qiaolin; Lim, Yeon-Hee; Anand, Rajan; Yu, Younong; Kim, Jae-hun; Zhou, Wei; Zheng, Junying; Tempest, Paul; Levorse, Dorothy; Zhang, Xiaoping; Greene, Scott; Mullins, Deborra; Culberson, Chris; Sherborne, Brad; Parker, Eric M; Stamford, Andrew; Ali, Amjad

    2015-08-01

    Molecular modeling was performed on a triazolo quinazoline lead compound to help develop a series of adenosine A2A receptor antagonists with improved hERG profile. Superposition of the lead compound onto MK-499, a benchmark hERG inhibitor, combined with pKa calculations and measurement, identified terminal fluorobenzene to be responsible for hERG activity. Docking of the lead compound into an A2A crystal structure suggested that this group is located at a flexible, spacious, and solvent-exposed opening of the binding pocket, making it possible to tolerate various functional groups. Transformation analysis (MMP, matched molecular pair) of in-house available experimental data on hERG provided suggestions for modifications in order to mitigate this liability. This led to the synthesis of a series of compounds with significantly reduced hERG activity. The strategy used in the modeling work can be applied to other medicinal chemistry programs to help improve hERG profile. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Beneficial effects of a novel agonist of the adenosine A2A receptor on monocrotaline-induced pulmonary hypertension in rats

    PubMed Central

    Alencar, Allan K N; Pereira, Sharlene L; Montagnoli, Tadeu L; Maia, Rodolfo C; Kümmerle, Arthur E; Landgraf, Sharon S; Caruso-Neves, Celso; Ferraz, Emanuelle B; Tesch, Roberta; Nascimento, José H M; de Sant'Anna, Carlos M R; Fraga, Carlos A M; Barreiro, Eliezer J; Sudo, Roberto T; Zapata-Sudo, Gisele

    2013-01-01

    Background and Purpose Pulmonary arterial hypertension (PAH) is characterized by enhanced pulmonary vascular resistance, right ventricular hypertrophy and increased right ventricular systolic pressure. Here, we investigated the effects of a N-acylhydrazone derivative, 3,4-dimethoxyphenyl-N-methyl-benzoylhydrazide (LASSBio-1359), on monocrotaline (MCT)-induced pulmonary hypertension in rats. Experimental Approach PAH was induced in male Wistar rats by a single i.p. injection of MCT (60 mg·kg−1) and 2 weeks later, oral LASSBio-1359 (50 mg·kg−1) or vehicle was given once daily for 14 days. Echocardiography was used to measure cardiac function and pulmonary artery dimensions, with histological assay of vascular collagen. Studies of binding to human recombinant adenosine receptors (A1, A2A, A3) and of docking with A2A receptors were also performed. Key Results MCT administration induced changes in vascular and ventricular structure and function, characteristic of PAH. These changes were reversed by treatment with LASSBio-1359. MCT also induced endothelial dysfunction in pulmonary artery, as measured by diminished relaxation of pre-contracted arterial rings, and this dysfunction was reversed by LASSBio-1359. In pulmonary artery rings from normal Wistar rats, LASSBio-1359 induced relaxation, which was decreased by the adenosine A2A receptor antagonist, ZM 241385. In adenosine receptor binding studies, LASSBio-1359 showed most affinity for the A2A receptor and in the docking analyses, binding modes of LASSBio-1359 and the A2A receptor agonist, CGS21680, were very similar. Conclusion and Implications In rats with MCT-induced PAH, structural and functional changes in heart and pulmonary artery were reversed by treatment with oral LASSBio-1359, most probably through the activation of adenosine A2A receptors. PMID:23530610

  18. Adenosine A2a blockade prevents synergy between mu-opiate and cannabinoid CB1 receptors and eliminates heroin-seeking behavior in addicted rats.

    PubMed

    Yao, Lina; McFarland, Krista; Fan, Peidong; Jiang, Zhan; Ueda, Takashi; Diamond, Ivan

    2006-05-16

    Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of mu-opiate receptors (MORs) and CB1 receptors in primary NAc/striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP/PKA signaling in NAc/striatal neurons. In addition, we find that synergy requires adenosine and A2a. Importantly, an A2a antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts.

  19. Adenosine A2a blockade prevents synergy between μ-opiate and cannabinoid CB1 receptors and eliminates heroin-seeking behavior in addicted rats

    PubMed Central

    Yao, Lina; McFarland, Krista; Fan, Peidong; Jiang, Zhan; Ueda, Takashi; Diamond, Ivan

    2006-01-01

    Relapse is the most serious limitation of effective medical treatment of opiate addiction. Opiate-related behaviors appear to be modulated by cannabinoid CB1 receptors (CB1) through poorly understood cross-talk mechanisms. Opiate and CB1 receptors are coexpressed in the nucleus accumbens (NAc) and dorsal striatum. These regions also have the highest density of adenosine A2a receptors (A2a) in the brain. We have been investigating the postsynaptic signaling mechanisms of μ-opiate receptors (MORs) and CB1 receptors in primary NAc/striatal neurons. In this article, we present evidence that MOR and CB1 act synergistically on cAMP/PKA signaling in NAc/striatal neurons. In addition, we find that synergy requires adenosine and A2a. Importantly, an A2a antagonist administered either directly into the NAc or indirectly by i.p. injection eliminates heroin-induced reinstatement in rats trained to self-administer heroin, a model of human craving and relapse. These findings suggest that A2a antagonists might be effective therapeutic agents in the management of abstinent heroin addicts. PMID:16684876

  20. Structure-activity relationships and mechanism of action of Eph-ephrin antagonists: interaction of cholanic acid with the EphA2 receptor

    PubMed Central

    Tognolini, Massimiliano; Incerti, Matteo; Mohamed, Iftiin Hassan; Giorgio, Carmine; Russo, Simonetta; Bruni, Renato; Lelli, Barbara; Bracci, Luisa; Noberini, Roberta; Pasquale, Elena B.; Barocelli, Elisabetta; Vicini, Paola; Mor, Marco

    2012-01-01

    The Eph–ephrin system, including the EphA2 receptor and the ephrin-A1 ligand, plays a critical role in tumor and vascular functions during carcinogenesis. We previously identified (3α,5β)-3-hydroxycholan-24-oic acid (lithocholic acid) as an Eph-ephrin antagonist able to inhibit EphA2 receptor activation and therefore potentially useful as a novel EphA2 receptor targeting agent. Here, we explore the structure-activity relationships of a focused set of lithocholic acid derivatives, based on molecular modelling investigation and displacement binding assays. Our exploration shows that while the 3-α-hydroxyl group of lithocholic acid has a negligible role in the recognition of the EphA2 receptor, its carboxylate group is critical for disrupting the binding of ephrin-A1 to the EphA2. As a result of our investigation, we identified (5β)-cholan-24-oic acid (cholanic acid) as a novel compound that competitively inhibits EphA2-ephrin-A1 interaction with higher potency than lithocholic acid. Surface plasmon resonance analysis indicates that cholanic acid binds specifically and reversibly to the ligand-binding domain of EphA2, with a steady-state dissociation constant (KD) in the low micromolar range. Furthermore, cholanic acid blocks the phosphorylation of EphA2 and cell retraction and rounding in PC3 prostate cancer cells, two effects that depend on EphA2 activation by the ephrin-A1 ligand. These findings suggest that cholanic acid can be used as a template structure to design effective EphA2 antagonists, with potential impact in the elucidation of the role played by this receptor in pathological conditions. PMID:22529030

  1. Evaluation of Tp-e interval and Tp-e/QT ratio in patients with non-dipper hypertension.

    PubMed

    Demir, Mehmet; Uyan, Umut

    2014-01-01

    Non-dipper hypertension is associated with increased cardiovascular morbidity and mortality. Several studies have suggested that the interval from the peak to the end of the electrocardiographic T wave (Tp-e) may correspond to the transmural dispersion of repolarization and that increased Tp-e interval and Tp-e/QT ratio are associated with malignant ventricular arrhythmias. The aim of this study was to evaluate ventricular repolarization by using Tp-e interval and Tp-e/QT ratio in patients with non-dipper hypertension. This study included 80 hypertensive patients. Hypertensive patients were divided into two groups: 50 dipper patients (29 male, mean age 51.5 ± 8 years) and 30 non-dipper patients (17 male, mean age 50.6 ± 5.4 years). Tp-e interval and Tp-e/QT ratio were measured from the 12-lead electrocardiogram. These parameters were compared between groups. No statistically significant difference was found between two groups in terms of basic characteristics. In electrocardiographic parameters analysis, QT dispersion (QTd) and corrected QTd were significantly increased in non-dipper patients compared to the dippers (39.4 ± 11.5 versus 27.3 ± 7.5 ms and 37.5 ± 9.5 versus 29.2 ± 6.5 ms, p = 0.001 and p = 0.01, respectively). Tp-e interval and Tp-e/QT ratio were also significantly higher in non-dipper patients (97.5 ± 11.2 versus 84.2 ± 8.3 ms and 0.23 ± 0.02 versus 0.17 ± 0.02, all p value <0.001). Our study revealed that QTd, Tp-e interval and Tp-e/QT ratio are prolonged in patients with non-dipper hypertension.

  2. Evaluation of Tp-e interval and Tp-e/QT ratio in patients with rheumatoid arthritis.

    PubMed

    Acar, Gu Rkan; Akkoyun, Murat; Nacar, Alper Bugra; Dirnak, Imran; Yıldırım Çetin, Gözde; Nur Yıldırım, Makbule; Zencir, Cemil; Karaman, Kayıhan; Cetin, Mustafa; Sayarlıoğlu, Mehmet

    2014-01-01

    Several studies have suggested that the interval from the peak to the end of the electrocardiographic T wave (Tp-e) may correspond to the transmural dispersion of repolarization and that increased Tp-e interval and Tp-e/QT ratio are associated with malignant ventricular arrhythmias. The aim of this study was to evaluate ventricular repolarization by using the Tp-e interval and Tp-e/QT ratio in patients with rheumatoid arthritis (RA), and to assess the relation with inflammation. Ninety-six patients (72 females, 24 males; mean age 43.8±11.8 years) with RA and 50 controls (35 females, 15 males; mean age 44.2±11.1 years) were included. From the 12-lead electrocardiogram, Tp-e interval and Tp-e/QT ratio were measured. Blood samples were taken for erythrocyte sedimentation rate (ESR) and plasma levels of C-reactive protein (CRP). These parameters were compared between groups. The relationship between ventricular repolarization and inflammation was assessed by Pearson correlation coefficients. Tp-e interval and Tp-e/QT ratio were increased in RA patients compared to the controls (72.6±8.2 vs 66.4±8.5 ms, 0.20±0.02 vs 0.18±0.02; p<0.001 and p<0.001, respectively). The Tp-e interval was significantly correlated with CRP, ESR, and disease activity score (DAS-28) (r=0.56, p<0.001, r=0.57, p<0.001, and r=0.29, p=0.02, respectively). The Tp-e/QT ratio was also correlated with CRP, ESR, and DAS-28 score (r=0.43, p<0.001, r=0.53, p<0.001, and r=0.25, p=0.03, respectively). In RA patients, the increased frequency of ventricular arrhythmias may be explained by increased indexes of ventricular repolarization and their relationship with inflammation.

  3. Neuroprotection by caffeine in the MPTP model of Parkinson’s disease and its dependence on adenosine A2A receptors

    PubMed Central

    Xu, Kui; Di Luca, Daniel Garbin; Orrú, Marco; Xu, Yuehang; Chen, Jiang-Fan; Schwarzschild, Michael A.

    2016-01-01

    Considerable epidemiological and laboratory data have suggested that caffeine, a nonselective adenosine receptor antagonist, may protect against the underlying neurodegeneration of Parkinson’s disease (PD). Although both caffeine and more specific antagonists of the A2A subtype of adenosine receptor (A2AR) have been found to confer protection in animal models of PD, the dependence of caffeine’s neuroprotective effects on the A2AR is not known. To definitively determine its A2AR dependence, the effect of caffeine on MPTP neurotoxicity was compared in wild-type (WT) and A2AR gene global knockout (A2A KO) mice, as well as in CNS cell type-specific (conditional) A2AR knockout (cKO) mice that lack the receptor either in postnatal forebrain neurons or in astrocytes. In WT and in heterozygous A2AR KO mice caffeine pretreatment (25 mg/kg ip) significantly attenuated MPTP-induced depletion of striatal dopamine. By contrast in homozygous A2AR global KO mice caffeine had no effect on MPTP toxicity. In forebrain neuron A2AR cKO mice, caffeine lost its locomotor stimulant effect, whereas its neuroprotective effect was mostly preserved. In astrocytic A2AR cKO mice, both caffeine’s locomotor stimulant and protective properties were undiminished. Taken together, these results indicate that neuroprotection by caffeine in the MPTP model of PD relies on the A2AR, although the specific cellular localization of these receptors remains to be determined. PMID:26905951

  4. The A2A adenosine receptor rescues the urea cycle deficiency of Huntington's disease by enhancing the activity of the ubiquitin-proteasome system.

    PubMed

    Chiang, Ming-Chang; Chen, Hui-Mei; Lai, Hsing-Lin; Chen, Hsiao-Wen; Chou, Szu-Yi; Chen, Chiung-Mei; Tsai, Fuu-Jen; Chern, Yijuang

    2009-08-15

    Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g. the liver) and causes devastating neuronal degeneration. Metabolic defects resulting from Htt aggregates in peripheral tissues also contribute to HD pathogenesis. Simultaneous improvement of defects in both the CNS and peripheral tissues is thus the most effective therapeutic strategy and is highly desirable. We earlier showed that an agonist of the A(2A) adenosine receptor (A(2A) receptor), CGS21680 (CGS), attenuates neuronal symptoms of HD. We found herein that the A(2A) receptor also exists in the liver, and that CGS ameliorated the urea cycle deficiency by reducing mHtt aggregates in the liver. By suppressing aggregate formation, CGS slowed the hijacking of a crucial transcription factor (HSF1) and two protein chaperons (Hsp27 and Hsp70) into hepatic Htt aggregates. Moreover, the abnormally high levels of high-molecular-mass ubiquitin conjugates in the liver of an HD mouse model (R6/2) were also ameliorated by CGS. The protective effect of CGS against mHtt-induced aggregate formation was reproduced in two cells lines and was prevented by an antagonist of the A(2A) receptor and a protein kinase A (PKA) inhibitor. Most importantly, the mHtt-induced suppression of proteasome activity was also normalized by CGS through PKA. Our findings reveal a novel therapeutic pathway of A(2A) receptors in HD and further strengthen the concept that the A(2A) receptor can be a drug target in treating HD.

  5. Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

    DOE PAGES

    Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan; ...

    2018-04-10

    Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

  6. Mechanistic insights into allosteric regulation of the A 2A adenosine G protein-coupled receptor by physiological cations

    SciTech Connect

    Ye, Libin; Neale, Chris Andrew; Sljoka, Adnan

    Cations play key roles in regulating G-protein-coupled receptors (GPCRs), although their mechanisms are poorly understood. Here, 19F NMR is used to delineate the effects of cations on functional states of the adenosine A 2A GPCR. While Na + reinforces an inactive ensemble and a partial-agonist stabilized state, Ca 2+ and Mg 2+ shift the equilibrium toward active states. Positive allosteric effects of divalent cations are more pronounced with agonist and a G-protein-derived peptide. In cell membranes, divalent cations enhance both the affinity and fraction of the high affinity agonist-bound state. Molecular dynamics simulations suggest high concentrations of divalent cations bridgemore » specific extracellular acidic residues, bringing TM5 and TM6 together at the extracellular surface and allosterically driving open the G-protein-binding cleft as shown by rigidity-transmission allostery theory. Lastly, an understanding of cation allostery should enable the design of allosteric agents and enhance our understanding of GPCR regulation in the cellular milieu.« less

  7. Activation of farnesoid X receptor promotes triglycerides lowering by suppressing phospholipase A2 G12B expression.

    PubMed

    Liu, Qingli; Yang, Meng; Fu, Xuekun; Liu, Renzhong; Sun, Caijun; Pan, Haobo; Wong, Chi-Wai; Guan, Min

    2016-11-15

    As a novel mediator of hepatic very low-density lipoproteins (VLDL) secretion, phospholipase A2 G12B (PLA2G12B) is transcriptionally regulated by hepatocyte nuclear factor-4 alpha (HNF-4α). Farnesoid X receptor (FXR) plays a critical role in maintaining bile acids and triglycerides (TG) homeostasis. Here we report that FXR regulates serum TG level in part through PLA2G12B. Activation of FXR by chenodeoxycholic acid (CDCA) or GW4064 significantly decreased PLA2G12B expression in HepG2 cells. PLA2G12B expression was transcriptionally repressed due to an FXR-mediated up-regulation of small heterodimer partner (SHP) which functionally suppresses HNF-4α activity. We found that hepatic PLA2G12B expression was suppressed and serum TG level reduced in high fat diet mice treated with CDCA. Concurrently, CDCA treatment lowered hepatic VLDL-TG secretion. Our data demonstrate that activation of FXR promotes TG lowering, not only by decreasing de novo lipogenesis but also reducing hepatic secretion of TG-rich VLDL particles in part through suppressing PLA2G12B expression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. sPLA2 IB induces human podocyte apoptosis via the M-type phospholipase A2 receptor

    PubMed Central

    Pan, Yangbin; Wan, Jianxin; Liu, Yipeng; Yang, Qian; Liang, Wei; Singhal, Pravin C.; Saleem, Moin A.; Ding, Guohua

    2014-01-01

    The M-type phospholipase A2 receptor (PLA2R) is expressed in podocytes in human glomeruli. Group IB secretory phospholipase A2 (sPLA2 IB), which is one of the ligands of the PLA2R, is more highly expressed in chronic renal failure patients than in controls. However, the roles of the PLA2R and sPLA2 IB in the pathogenesis of glomerular diseases are unknown. In the present study, we found that more podocyte apoptosis occurs in the kidneys of patients with higher PLA2R and serum sPLA2 IB levels. In vitro, we demonstrated that human podocyte cells expressed the PLA2R in the cell membrane. After binding with the PLA2R, sPLA2 IB induced podocyte apoptosis in a time- and concentration-dependent manner. sPLA2 IB-induced podocyte PLA2R upregulation was not only associated with increased ERK1/2 and cPLA2α phosphorylation but also displayed enhanced apoptosis. In contrast, PLA2R-silenced human podocytes displayed attenuated apoptosis. sPLA2 IB enhanced podocyte arachidonic acid (AA) content in a dose-dependent manner. These data indicate that sPLA2 IB has the potential to induce human podocyte apoptosis via binding to the PLA2R. The sPLA2 IB-PLA2R interaction stimulated podocyte apoptosis through activating ERK1/2 and cPLA2α and through increasing the podocyte AA content. PMID:25335547

  9. Involvement of adenosine A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of cocaine and methamphetamine in rats.

    PubMed

    Justinova, Zuzana; Ferre, Sergi; Segal, Pavan N; Antoniou, Katerina; Solinas, Marcello; Pappas, Lara A; Highkin, Jena L; Hockemeyer, Jorg; Munzar, Patrik; Goldberg, Steven R

    2003-12-01

    Adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission. We have recently reported that nonselective adenosine receptor antagonists (caffeine and 3,7-dimethyl-1-propargylxanthine) can partially substitute for the discriminative-stimulus effects of methamphetamine. In the present study, by using more selective compounds, we investigated the involvement of A1 and A2A receptors in the adenosinergic modulation of the discriminative-stimulus effects of both cocaine and methamphetamine. The effects of the A1 receptor agonist N6-cyclopentyladenosine (CPA; 0.01-0.1 mg/kg) and antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.3-23.7 mg/kg) and the A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS 21680; 0.03-0.18 mg/kg) and antagonist 3-(3-hydroxypropyl)-8-(3-methoxystyryl)-7-methyl-1-propargylxanthin phosphate disodium salt (MSX-3; 1-56 mg/kg) were evaluated in rats trained to discriminate either 1 mg/kg methamphetamine or 10 mg/kg cocaine from saline under a fixed-ratio 10 schedule of food presentation. The A1 and A2A receptor antagonists (CPT and MSX-3) both produced high levels of drug-lever selection when substituted for either methamphetamine or cocaine and significantly shifted dose-response curves of both psychostimulants to the left. Unexpectedly, the A2A receptor agonist CGS 21680 also produced drug-appropriate responding (although at lower levels) when substituted for the cocaine-training stimulus, and both CGS 21680 and the A1 receptor agonist CPA significantly shifted the cocaine dose-response curve to the left. In contrast, both agonists did not produce significant levels of drug-lever selection when substituted for the methamphetamine-training stimulus and failed to shift the methamphetamine dose-response curve. Therefore, adenosine A1 and A2A receptors appear to play important but differential roles in the modulation of the

  10. Electron heating and Tp/Te variations during magnetic dipolarizations

    NASA Astrophysics Data System (ADS)

    Grigorenko, Elena; Kronberg, Elena; Daly, Patrick; Ganushkina, Natalia; Lavraud, Benoit; Sauvaud, Jean-Andre; Zelenyi, Lev

    2017-04-01

    The proton-to-electron temperature ratio (Tp/Te) in the plasma sheet (PS) of the Earth's magnetotail is studied by using 5 years of Cluster observations (2001-2005). The PS intervals are searched within a region defined with -19Tp/Te varies over a wide range from a few units to several tens of units. In 86 PS intervals the Tp/Te decreases below 3.5. In the majority of these intervals the Tp/Te drops are observed during magnetotail dipolarizations. A superposed epoch analysis applied to these events shows that the minimum value of Tp/Te is observed after the dipolarization onset during the "turbulent phase" of dipolarization, when a number of transient Bz pulses are reduced, but the value of Bz field is still large and an intensification of wave activity is observed. The Tp/Te drops and associated increases of Te often coincide either with bursts of broadband electrostatic emissions, which may include electron cyclotron harmonics, or with broadband electromagnetic emission in a frequency range from proton plasma frequency (fpp) up to the electron gyrofrequency (fce). These findings show that the wave activity developing in the current sheet after dipolarization onset may play a role in the additional electron heating and the associated Tp/Te decrease. This work was supported by the Volskwagen Foundation (grant Az 90 312).

  11. VPAC1 Targeted 64Cu-TP3805 kit preparation and its evaluation.

    PubMed

    Tripathi, Sushil K; Kumar, Pardeep; Trabulsi, Edouard J; Kim, Sung; McCue, Peter A; Intenzo, Charles; Berger, Adam; Gomella, Leonard; Thakur, Mathew L

    2017-08-01

    Previously, our laboratory has shown that 64 Cu-TP3805 can specifically target VPAC1 receptors and be used for positron emission tomography (PET) imaging of breast (BC) and prostate cancer (PC) in humans. Present work is aimed at the formulation of a freeze-dried diaminedithiol-peptide (N 2 S 2 -TP3805) kit and it's evaluation for the preparation of 64 Cu labeled TP3805. Parameters such as pH, temperature and incubation time were examined that influenced the radiolabeling efficiency and stability of the product. Kits were prepared under different conditions and radiolabeling efficiency of TP3805 kit was evaluated for a range of pH3.5-8.5, after addition of 64 Cu in 30μl, 0.1M HCl. Incubation temperature (37-90°C) and time (30-120min.) were also investigated. Kits were stored at -10°C and their long term stability was determined as a function of their radiolabeling efficiency. Further, stability of 64 Cu-TP3805 complex was evaluated in presence of fetal bovine serum and bovine serum albumin by using SDS polyacrylamide gel electrophoresis. Kits were then used for PET imaging of BC and PC following eIND (101550) and institutional approvals. Specificity of 64 Cu-TP3805 for VPAC1 was examined with digital autoradiography (DAR) of prostate tissues obtained after prostatectomy, benign prostatic hyperplasia (BPH) tissue, and benign and malignant lymph nodes. Results were compared with corresponding tissue histology. Radiolabeling efficiency was ≥95% at final pH ~7.2 when incubated at 50°C for 90min. Kits were stable up to 18months when stored at -10°C, and 64 Cu-TP3805 complex exhibited excellent stability for up to 4h at room temperature. 64 Cu-TP3805 complex did not show any transchelation even after 2h incubation at 37°C in 10% FBS as well as in BSA as determined by SDS PAGE analysis. DAR identified ≥95% of malignant lesions 11 new PC lesions, 20 high grade prostatic intraepithelial neoplasia, 2/2 ejaculatory ducts and 5/5 urethra verumontanum not previously

  12. Binding of the Kaposi's Sarcoma-Associated Herpesvirus to the Ephrin Binding Surface of the EphA2 Receptor and Its Inhibition by a Small Molecule

    PubMed Central

    Hahn, Alexander S.

    2014-01-01

    ABSTRACT The ephrin receptor tyrosine kinase A2 (EphA2) is an entry receptor for Kaposi's sarcoma-associated herpesvirus (KSHV) that is engaged by the virus through its gH/gL glycoprotein complex. We describe here that natural ephrin ligands inhibit the gH/gL-EphA2 interaction. The effects of point mutations within EphA2 demonstrated that KSHV gH/gL interacts with EphA2 through a restricted set of the same residues that mediate binding of A-type ephrins. Two previously described inhibitors of the EphA2 interaction with ephrin A5 also inhibited binding of KSHV gH/gL to EphA2. The more potent of the two compounds inhibited KSHV infection of blood vessel and lymphatic endothelial cells in the micromolar concentration range. Our results demonstrate that interaction of KSHV with EphA2 occurs in a fashion similar to that of the natural ephrin ligands. Our data further indicate a new avenue for drug development against KSHV. IMPORTANCE Our study reports two important findings. First, we show that KSHV engages its receptor, the receptor tyrosine kinase EphA2, at a site that overlaps the binding site of the natural ephrin ligands. Second, we demonstrate that KSHV infection of target cells can be blocked by a small-molecule inhibitor of the viral glycoprotein-EphA2 interaction. These findings represent a novel avenue for the development of strategies to treat KSHV-associated diseases. PMID:24899181

  13. Initial Evaluation of an Adenosine A2A Receptor Ligand, 11C-Preladenant, in Healthy Human Subjects.

    PubMed

    Sakata, Muneyuki; Ishibashi, Kenji; Imai, Masamichi; Wagatsuma, Kei; Ishii, Kenji; Zhou, Xiaoyun; de Vries, Erik F J; Elsinga, Philip H; Ishiwata, Kiichi; Toyohara, Jun

    2017-09-01

    11 C-preladenant is a selective antagonist for mapping of cerebral adenosine A 2A receptors (A 2A Rs) by PET. This is a first-in-human study to examine the safety, radiation dosimetry, and brain imaging of 11 C-preladenant in healthy human subjects. Methods: Dynamic 11 C-preladenant PET scans (90 min) were obtained in 5 healthy male subjects. During the scan, arterial blood was sampled at various time intervals, and the fraction of the parent compound in plasma was determined. For anatomic coregistration, T1-weighted MRI was performed. The total distribution volume ( V T ) was estimated using 1- and 2-tissue-compartment models (1T and 2T, respectively). The distribution volume ratio (DVR) was calculated from V T of target and reference region and obtained with a noninvasive Logan graphical reference tissue method ( t * = 30 min). The applicability of a shortened protocol as an alternative to the 90-min PET scan was investigated. Tracer biodistribution and dosimetry were determined in 3 healthy male subjects, using serial whole-body PET scans acquired over 2 h after 11 C-preladenant injection. Results: There were no serious adverse events in any of the subjects throughout the study period. 11 C-preladenat readily entered the brain, with a peak uptake in the putamen and head of the caudate nucleus 30-40 min after tracer injection. Other brain regions showed rapid clearance of radioactivity. The regional distribution of 11 C-preladenant was consistent with known A 2A R densities in the brain. At pseudoequilibrium (reached at 40 min after injection), stable target-to-cerebellar cortex ratios of around 3.8-10.0 were obtained. The 2T fit better than the 1T in the low-density A 2A R regions. In contrast, there were no significant differences between 1T and 2T in the high-A 2A R-density regions. DVRs in the putamen and head of the caudate nucleus were around 3.8-10.3 when estimated using a Logan graphical reference tissue method with cerebellum as the reference region. PET

  14. A Small Molecule Agonist of EphA2 Receptor Tyrosine Kinase Inhibits Tumor Cell Migration In Vitro and Prostate Cancer Metastasis In Vivo

    PubMed Central

    Guo, Hong; Miao, Hui; Tochtrop, Gregory P.; Hsieh, Jer-Tsong; Page, Phillip; Liu, Lili; Lindner, Daniel J.; Acharya, Chayan; MacKerell, Alexander D.; Ficker, Eckhard; Song, Jianxing; Wang, Bingcheng

    2012-01-01

    During tumor progression, EphA2 receptor can gain ligand-independent pro-oncogenic functions due to Akt activation and reduced ephrin-A ligand engagement. The effects can be reversed by ligand stimulation, which triggers the intrinsic tumor suppressive signaling pathways of EphA2 including inhibition of PI3/Akt and Ras/ERK pathways. These observations argue for development of small molecule agonists for EphA2 as potential tumor intervention agents. Through virtual screening and cell-based assays, we report here the identification and characterization of doxazosin as a novel small molecule agonist for EphA2 and EphA4, but not for other Eph receptors tested. NMR studies revealed extensive contacts of doxazosin with EphA2/A4, recapitulating both hydrophobic and electrostatic interactions recently found in the EphA2/ephrin-A1 complex. Clinically used as an α1-adrenoreceptor antagonist (Cardura®) for treating hypertension and benign prostate hyperplasia, doxazosin activated EphA2 independent of α1-adrenoreceptor. Similar to ephrin-A1, doxazosin inhibited Akt and ERK kinase activities in an EphA2-dependent manner. Treatment with doxazosin triggered EphA2 receptor internalization, and suppressed haptotactic and chemotactic migration of prostate cancer, breast cancer, and glioma cells. Moreover, in an orthotopic xenograft model, doxazosin reduced distal metastasis of human prostate cancer cells and prolonged survival in recipient mice. To our knowledge, doxazosin is the first small molecule agonist of a receptor tyrosine kinase that is capable of inhibiting malignant behaviors in vitro and in vivo. PMID:22916121

  15. A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

    PubMed

    Mumford, Andrew D; Dawood, Ban B; Daly, Martina E; Murden, Sherina L; Williams, Michael D; Protty, Majd B; Spalton, Jennifer C; Wheatley, Mark; Mundell, Stuart J; Watson, Steve P

    2010-01-14

    We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.

  16. VPAC1 targeted 64Cu-TP3805 PET imaging of prostate cancer: preliminary evaluation in man

    PubMed Central

    Tripathi, Sushil; Trabulsi, Edouard J; Gomella, Leonard; Kim, Sung; McCue, Peter; Intenzo, Charles; Birbe, Ruth; Gandhe, Ashish; Kumar, Pardeep; Thakur, Mathew

    2015-01-01

    Objectives To evaluate 64Cu-TP3805 as a novel biomolecule, to PET image prostate cancer (PC), at the onset of which VPAC1, the superfamily of G-protein coupled receptors, is expressed in high density on PC cells, but not on normal cells. Methods 25 patients undergoing radical prostatectomy were PET/CT imaged preoperatively with 64Cu-TP3805. Standardized uptake values (SUVmax) were determined, malignant lesions (SUV > 1.0) counted, and compared with histologic findings. Whole mount pathology slides from 6 VPAC1 PET imaged patients, 3 BPH patients, one malignant and one benign lymph node underwent digital autoradiography (DAR) after 64Cu-TP3805 incubation and compared to H&E stained slides. Results In 25 patient PET imaging, 212 prostate gland lesions had SUVmax > 1.0 vs.127 lesions identified by histology of biopsy tissues. The status of the additional 85 PET identified prostate lesions remains to be determined. In 68 histological slides from 6 PET imaged patients, DAR identified 105/107 PC foci, 19/19 HGPIN, and ejaculatory ducts and verumontanum involved with cancer. Additionally, DAR found 9 PC lesions not previously identified histologically. The positive and negative lymph nodes were correctly identified and in 3/3 BPH patients and 5/5 cysts, DAR was negative. Conclusion This feasibility study demonstrated that 64Cu-TP3805 delineates PC in vivo and ex vivo, provided normal images for benign masses, and is worthy of further studies. PMID:26519886

  17. Undiscovered role of endogenous thromboxane A2 in activation of cardiac sympathetic afferents during ischaemia

    PubMed Central

    Fu, Liang-Wu; Guo, Zhi-Ling; Longhurst, John C

    2008-01-01

    Myocardial ischaemia activates blood platelets, which in turn stimulate cardiac sympathetic afferents, leading to chest pain and sympathoexcitatory reflex cardiovascular responses. Previous studies have shown that activated platelets stimulate ischaemically sensitive cardiac sympathetic afferents, and that thromboxane A2 (TxA2) is one of the mediators released from activated platelets during myocardial ischaemia. The present study tested the hypothesis that endogenous TxA2 stimulates cardiac afferents during ischaemia through direct activation of TxA2 (TP) receptors coupled with the phospholipase C–protein kinase C (PLC–PKC) cellular pathway. Nerve activity of single unit cardiac sympathetic afferents was recorded from the left sympathetic chain or rami communicantes (T2–T5) in anaesthetized cats. Single fields of 39 afferents (conduction velocity = 0.27–3.65 m s−1) were identified in the left or right ventricle initially with mechanical stimulation and confirmed with a stimulating electrode. Five minutes of myocardial ischaemia stimulated all 39 cardiac afferents (8 Aδ-, 31 C-fibres) and the responses of these 39 afferents to chemical stimuli were further studied in the following four protocols. In the first protocol, 2.5, 5 and 10 μg of the TxA2 mimetic, U46619, injected into the left atrium (LA), stimulated seven ischaemically sensitive cardiac afferents in a dose-dependent manner. Second, BM13,177, a selective TxA2 receptor antagonist, abolished the responses of six afferents to 5 μg of U46619 injected into the left atrium and attenuated the ischaemia-related increase in activity of seven other afferents by 44%. In contrast, cardiac afferents, in the absence of TP receptor blockade responded consistently to repeated administration of U46619 (n = 6) and to recurrent myocardial ischaemia (n = 7). In the fourth protocol, administration of PKC-(19–36), a selective PKC inhibitor, attenuated the responses of six other cardiac afferents to U46619 by 38

  18. M-type phospholipase A2 receptor (PLA2R) glomerular staining in pediatric idiopathic membranous nephropathy.

    PubMed

    Kanda, Shoichiro; Horita, Shigeru; Yanagihara, Takeshi; Shimizu, Akira; Hattori, Motoshi

    2017-04-01

    Identifying M-type phospholipase A 2 receptor (PLA 2 R) is a landmark breakthrough for understanding adult idiopathic membranous nephropathy (iMN). However, potential roles for PLA 2 R in pediatric iMN have not been well investigated. A total of 34 pediatric iMN patients who underwent kidney biopsy between 1972 and 2015 were enrolled in this study. The study cohort consisted of 15 children aged from 3 to 9 years and 19 aged from 10 to 15 years. In all cases, secondary causes of MN, including infections, autoimmune diseases, and others, were ruled out. We examined PLA 2 R glomerular staining in stored, formalin-fixed, paraffin-embedded kidney biopsy samples. Kidney biopsy specimens obtained from an adult patient with iMN and an adult patient with lupus-associated MN were also examined to assess our PLA 2 R staining procedure. Granular staining of PLA 2 R along glomerular capillary loops was present in two patients: an 11-year-old girl and 12-year-old boy identified during a school urine screening test and who presented with mild proteinuria at the time of biopsy. Interestingly, the intensity of PLA 2 R glomerular staining in these patients was weaker than that of a PLA 2 R-positive adult iMN patient. There were no PLA 2 R-positive patients among our cohort of children younger than 10 years. This preliminary study suggests PLA 2 R may play a role in some adolescent and preteen iMN patients but may be less frequently associated with iMN during childhood.

  19. Adenosine A2A receptor agonist prevents cardiac remodeling and dysfunction in spontaneously hypertensive male rats after myocardial infarction

    PubMed Central

    da Silva, Jaqueline S; Gabriel-Costa, Daniele; Sudo, Roberto T; Wang, Hao; Groban, Leanne; Ferraz, Emanuele B; Nascimento, José Hamilton M; Fraga, Carlos Alberto M; Barreiro, Eliezer J; Zapata-Sudo, Gisele

    2017-01-01

    Background This work evaluated the hypothesis that 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), an agonist of adenosine A2A receptor, could be beneficial for preventing cardiac dysfunction due to hypertension associated with myocardial infarction (MI). Methods Male spontaneously hypertensive rats (SHR) were randomly divided into four groups (six animals per group): sham-operation (SHR-Sham), and myocardial infarction rats (SHR-MI) were treated orally either with vehicle or LASSBio-294 (10 and 20 mg.kg−1.d−1) for 4 weeks. Echocardiography and in vivo hemodynamic parameters measured left ventricle (LV) structure and function. Exercise tolerance was evaluated using a treadmill test. Cardiac remodeling was accessed by LV collagen deposition and tumor necrosis factor α expression. Results Early mitral inflow velocity was significantly reduced in the SHR-MI group, and there was significant recovery in a dose-dependent manner after treatment with LASSBio-294. Exercise intolerance observed in the SHR-MI group was prevented by 10 mg.kg−1.d−1 of LASS-Bio-294, and exercise tolerance exceeded that of the SHR-Sham group at 20 mg.kg−1.d−1. LV end-diastolic pressure increased after MI, and this was prevented by 10 and 20 mg.kg−1.d−1 of LASSBio-294. Sarcoplasmic reticulum Ca2+ ATPase levels were restored in a dose-dependent manner after treatment with LASSBio-294. Fibrosis and inflammatory processes were also counteracted by LASSBio-294, with reductions in LV collagen deposition and tumor necrosis factor α expression. Conclusion In summary, oral administration of LASSBio-294 after MI in a dose-dependent manner prevented the development of cardiac dysfunction, demonstrating this compound’s potential as an alternative treatment for heart failure in the setting of ischemic heart disease with superimposed chronic hypertension. PMID:28293100

  20. Clinical implications of genomic profiles in metastatic breast cancer with a focus on TP53 and PIK3CA, the most frequently mutated genes.

    PubMed

    Kim, Ji-Yeon; Lee, Eunjin; Park, Kyunghee; Park, Woong-Yang; Jung, Hae Hyun; Ahn, Jin Seok; Im, Young-Hyuck; Park, Yeon Hee

    2017-04-25

    Breast cancer (BC) has been genetically profiled through large-scale genome analyses. However, the role and clinical implications of genetic alterations in metastatic BC (MBC) have not been evaluated. Therefore, we conducted whole-exome sequencing (WES) and RNA-Seq of 37 MBC samples and targeted deep sequencing of another 29 MBCs. We evaluated somatic mutations from WES and targeted sequencing and assessed gene expression and performed pathway analysis from RNA-Seq. In this analysis, PIK3CA was the most commonly mutated gene in estrogen receptor (ER)-positive BC, while in ER-negative BC, TP53 was the most commonly mutated gene (p = 0.018 and p < 0.001, respectively). TP53 stopgain/loss and frameshift mutation was related to low expression of TP53 in contrast nonsynonymous mutation was related to high expression. The impact of TP53 mutation on clinical outcome varied with regard to ER status. In ER-positive BCs, wild type TP53 had a better prognosis than mutated TP53 (median overall survival (OS) (wild type vs. mutated): 88.5 ± 54.4 vs. 32.6 ± 10.7 (months), p = 0.002). In contrast, mutated TP53 had a protective effect in ER-negative BCs (median OS: 0.10 vs. 32.6 ± 8.2, p = 0.026). However, PIK3CA mutation did not affect patient survival. In gene expression analysis, CALM1, a potential regulator of AKT, was highly expressed in PIK3CA-mutated BCs. In conclusion, mutation of TP53 was associated with expression status and affect clinical outcome according to ER status in MBC. Although mutation of PIK3CA was not related to survival in this study, mutation of PIK3CA altered the expression of other genes and pathways including CALM1 and may be a potential predictive marker of PI3K inhibitor effectiveness.

  1. Crosstalk of the EphA2 Receptor with a Serine/Threonine Phosphatase Suppresses the Akt-mTORC1 Pathway in Cancer Cells

    PubMed Central

    Yang, Nai-Ying; Fernandez, Carlos; Richter, Melanie; Xiao, Zhan; Valencia, Fatima; Tice, David A.; Pasquale, Elena B.

    2010-01-01

    Receptor tyrosine kinases of the Eph family play multiple roles in the physiological regulation of tissue homeostasis and in the pathogenesis of various diseases, including cancer. The EphA2 receptor is highly expressed in most cancer cell types, where it has disparate activities that are not well understood. It has been reported that interplay of EphA2 with oncogenic signaling pathways promotes cancer cell malignancy independently of ephrin ligand binding and receptor kinase activity. In contrast, stimulation of EphA2 signaling with ephrin-A ligands can suppress malignancy by inhibiting the Ras-MAP kinase pathway, integrin-mediated adhesion, and epithelial to mesenchymal transition. Here we show that ephrin-A1 ligand-dependent activation of EphA2 decreases the growth of PC3 prostate cancer cells and profoundly inhibits the Akt-mTORC1 pathway, which is hyperactivated due to loss of the PTEN tumor suppressor. Our results do not implicate changes in the activity of Akt upstream regulators (such as Ras family GTPases, PI3 kinase, integrins, or the Ship2 lipid phosphatase) in the observed loss of Akt T308 and S473 phosphorylation downstream of EphA2. Indeed, EphA2 can inhibit Akt phosphorylation induced by oncogenic mutations of not only PTEN but also PI3 kinase. Furthermore, it can decrease the hyperphosphorylation induced by constitutive membrane-targeting of Akt. Our data suggest a novel signaling mechanism whereby EphA2 inactivates the Akt-mTORC1 oncogenic pathway through Akt dephosphorylation mediated by a serine/threonine phosphatase. Ephrin-A1-induced Akt dephosphorylation was observed not only in PC3 prostate cancer cells but also in other cancer cell types. Thus, activation of EphA2 signaling represents a possible new avenue for anti-cancer therapies that exploit the remarkable ability of this receptor to counteract multiple oncogenic signaling pathways. PMID:20837138

  2. Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration.

    PubMed

    Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B; van der Hoorn, Frans A

    2016-07-15

    The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Purinergic A2b Receptor Activation by Extracellular Cues Affects Positioning of the Centrosome and Nucleus and Causes Reduced Cell Migration*

    PubMed Central

    Ou, Young; Chan, Gordon; Zuo, Jeremy; Rattner, Jerome B.; van der Hoorn, Frans A.

    2016-01-01

    The tight, relative positioning of the nucleus and centrosome in mammalian cells is important for the regulation of cell migration. Under pathophysiological conditions, the purinergic A2b receptor can regulate cell motility, but the underlying mechanism remains unknown. Expression of A2b, normally low, is increased in tissues experiencing adverse physiological conditions, including hypoxia and inflammation. ATP is released from such cells. We investigated whether extracellular cues can regulate centrosome-nucleus positioning and cell migration. We discovered that hypoxia as well as extracellular ATP cause a reversible increase in the distance between the centrosome and nucleus and reduced cell motility. We uncovered the underlying pathway: both treatments act through the A2b receptor and specifically activate the Epac1/RapGef3 pathway. We show that cells lacking A2b do not respond in this manner to hypoxia or ATP but transfection of A2b restores this response, that Epac1 is critically involved, and that Rap1B is important for the relative positioning of the centrosome and nucleus. Our results represent, to our knowledge, the first report demonstrating that pathophysiological conditions can impact the distance between the centrosome and nucleus. Furthermore, we identify the A2b receptor as a central player in this process. PMID:27226580

  4. Differential effects of presynaptic versus postsynaptic adenosine A2A receptor blockade on Δ9-tetrahydrocannabinol (THC) self-administration in squirrel monkeys.

    PubMed

    Justinová, Zuzana; Redhi, Godfrey H; Goldberg, Steven R; Ferré, Sergi

    2014-05-07

    Different doses of an adenosine A2A receptor antagonist MSX-3 [3,7-dihydro-8-[(1E)-2-(3-ethoxyphenyl)ethenyl]-7 methyl-3-[3-(phosphooxy)propyl-1-(2 propynil)-1H-purine-2,6-dione] were found previously to either decrease or increase self-administration of cannabinoids delta-9-tetrahydrocannabinol (THC) or anandamide in squirrel monkeys. It was hypothesized that the decrease observed with a relatively low dose of MSX-3 was related to blockade of striatal presynaptic A2A receptors that modulate glutamatergic neurotransmission, whereas the increase observed with a higher dose was related to blockade of postsynaptic A2A receptors localized in striatopallidal neurons. This hypothesis was confirmed in the present study by testing the effects of the preferential presynaptic and postsynaptic A2A receptor antagonists SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine] and KW-6002 [(E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione], respectively, in squirrel monkeys trained to intravenously self-administer THC. SCH-442416 produced a significant shift to the right of the THC self-administration dose-response curves, consistent with antagonism of the reinforcing effects of THC. Conversely, KW-6002 produced a significant shift to the left, consistent with potentiation of the reinforcing effects of THC. These results show that selectively blocking presynaptic A2A receptors could provide a new pharmacological approach to the treatment of marijuana dependence and underscore corticostriatal glutamatergic neurotransmission as a possible main mechanism involved in the rewarding effects of THC.

  5. Discovery of indolylpiperazinylpyrimidines with dual-target profiles at adenosine A2A and dopamine D2 receptors for Parkinson's disease treatment

    PubMed Central

    Paira, Priyankar; Tan, Aaron; Herr, Deron Raymond; Lim, Kah Leong; Ng, Chee Hoe; Venkatesan, Gopalakrishnan; Klotz, Karl-Norbert; Federico, Stephanie; Spalluto, Giampiero; Cheong, Siew Lee; Chen, Yu Zong

    2018-01-01

    Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive loss of dopaminergic neurons in the substantia nigra of the human brain, leading to depletion of dopamine production. Dopamine replacement therapy remains the mainstay for attenuation of PD symptoms. Nonetheless, the potential benefit of current pharmacotherapies is mostly limited by adverse side effects, such as drug-induced dyskinesia, motor fluctuations and psychosis. Non-dopaminergic receptors, such as human A2A adenosine receptors, have emerged as important therapeutic targets in potentiating therapeutic effects and reducing the unwanted side effects. In this study, new chemical entities targeting both human A2A adenosine receptor and dopamine D2 receptor were designed and evaluated. Two computational methods, namely support vector machine (SVM) models and Tanimoto similarity-based clustering analysis, were integrated for the identification of compounds containing indole-piperazine-pyrimidine (IPP) scaffold. Subsequent synthesis and testing resulted in compounds 5 and 6, which acted as human A2A adenosine receptor binders in the radioligand competition assay (Ki = 8.7–11.2 μM) as well as human dopamine D2 receptor binders in the artificial cell membrane assay (EC50 = 22.5–40.2 μM). Moreover, compound 5 showed improvement in movement and mitigation of the loss of dopaminergic neurons in Drosophila models of PD. Furthermore, in vitro toxicity studies on compounds 5 and 6 did not reveal any mutagenicity (up to 100 μM), hepatotoxicity (up to 30 μM) or cardiotoxicity (up to 30 μM). PMID:29304113

  6. The Adenosine A2A Receptor Agonist, CGS-21680, Blocks Excessive Rearing, Acquisition of Wheel Running, and Increases Nucleus Accumbens CREB Phosphorylation in Chronically Food-Restricted Rats

    PubMed Central

    de Vaca, Soledad Cabeza; Kannan, Pavitra; Pan, Yan; Jiang, Nancy; Sun, Yanjie; Carr, Kenneth D.

    2007-01-01

    Adenosine A2A receptors are preferentially expressed in rat striatum, where they are concentrated in dendritic spines of striatopallidal medium spiny neurons and exist in a heteromeric complex with D2 dopamine (DA) receptors. Behavioral and biochemical studies indicate an antagonistic relationship between A2A and D2 receptors. Previous studies have demonstrated that food-restricted (FR) rats display behavioral and striatal cellular hypersensitivity to D1 and D2 DA receptor stimulation. These alterations may underlie adaptive, as well as maladaptive, behaviors characteristic of the FR rat. The present study examined whether FR rats are hypersensitive to the A2A receptor agonist, CGS-21680. In Experiment 1, spontaneous horizontal motor activity did not differ between FR and ad libitum fed (AL) rats, while vertical activity was greater in the former. Intracerebroventricular (i.c.v.) administration of CGS-21680 (0.25 and 1.0 nmol) decreased both types of motor activity in FR rats, and returned vertical activity levels to those observed in AL rats. In Experiment 2, FR rats given access to a running wheel for a brief period outside of the home cage rapidly acquired wheel running while AL rats did not. Pretreatment with CGS-21680 (1.0 nmol) blocked the acquisition of wheel running. When administered to FR subjects that had previously acquired wheel running, CGS-21680 suppressed the behavior. In Experiment 3, CGS-21680 (1.0 nmol) activated both ERK 1/2 and CREB in caudate-putamen with no difference between feeding groups. However, in nucleus accumbens (NAc), CGS-21680 failed to activate ERK 1/2 and selectively activated CREB in FR rats. These results indicate that FR subjects are hypersensitive to several effects of an adenosine A2A agonist, and suggest the involvement of an upregulated A2A receptor-linked signaling pathway in NAc. Medications targeting the A2A receptor may have utility in the treatment of maladaptive behaviors associated with FR, including substance abuse

  7. In search of novel ligands using a structure-based approach: a case study on the adenosine A2A receptor.

    PubMed

    Lenselink, Eelke B; Beuming, Thijs; van Veen, Corine; Massink, Arnault; Sherman, Woody; van Vlijmen, Herman W T; IJzerman, Adriaan P

    2016-10-01

    In this work, we present a case study to explore the challenges associated with finding novel molecules for a receptor that has been studied in depth and has a wealth of chemical information available. Specifically, we apply a previously described protocol that incorporates explicit water molecules in the ligand binding site to prospectively screen over 2.5 million drug-like and lead-like compounds from the commercially available eMolecules database in search of novel binders to the adenosine A 2A receptor (A 2A AR). A total of seventy-one compounds were selected for purchase and biochemical assaying based on high ligand efficiency and high novelty (Tanimoto coefficient ≤0.25 to any A 2A AR tested compound). These molecules were then tested for their affinity to the adenosine A 2A receptor in a radioligand binding assay. We identified two hits that fulfilled the criterion of ~50 % radioligand displacement at a concentration of 10 μM. Next we selected an additional eight novel molecules that were predicted to make a bidentate interaction with Asn253 6.55 , a key interacting residue in the binding pocket of the A 2A AR. None of these eight molecules were found to be active. Based on these results we discuss the advantages of structure-based methods and the challenges associated with finding chemically novel molecules for well-explored targets.

  8. GW627368X ((N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl} benzene sulphonamide): a novel, potent and selective prostanoid EP4 receptor antagonist

    PubMed Central

    Wilson, Richard J; Giblin, Gerard M P; Roomans, Susan; Rhodes, Sharron A; Cartwright, Kerri-Ann; Shield, Vanessa J; Brown, Jason; Wise, Alan; Chowdhury, Jannatara; Pritchard, Sara; Coote, Jim; Noel, Lloyd S; Kenakin, Terry; Burns-Kurtis, Cynthia L; Morrison, Valerie; Gray, David W; Giles, Heather

    2006-01-01

    N-{2-[4-(4,9-diethoxy-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl)phenyl]acetyl}benzene sulphonamide (GW627368X) is a novel, potent and selective competitive antagonist of prostanoid EP4 receptors with additional human TP receptor affinity. At recombinant human prostanoid EP4 receptors expressed in HEK293 cells, GW627368X produced parallel rightward shifts of PGE2 concentration–effect (E/[A]) curves resulting in an affinity (pKb) estimate of 7.9±0.4 and a Schild slpoe not significantly different from unity. The affinity was independent of the agonist used. In rings of phenylephrine precontracted piglet saphenous vein, GW627368X (30–300 nM) produced parallel rightward displacement of PGE2 E/[A] curves (pKb=9.2±0.2; slope=1). GW627368X appears to bind to human prostanoid TP receptors but not the TP receptors of other species. In human washed platelets, GW627368X (10 μM) produced 100% inhibition of U-46619 (EC100)-induced aggregation (approximate pA2 ∼7.0). However, in rings of rabbit and piglet saphenous vein and of guinea-pig aorta GW627368X (10 μM) did not displace U-46619 E/[A] curves indicating an affinity of <5.0 for rabbit and guinea-pig prostanoid TP receptors. In functional assays GW627368X is devoid of both agonism and antagonist affinity for prostanoid CRTH2, EP2, EP3, IP and FP receptors. At prostanoid EP1 receptors, GW627368X was an antagonist with a pA2 of 6.0, and at prostanoid IP receptors the compound increased the maximum effect of iloprost by 55%. At rabbit prostanoid EP2 receptors the pA2 of GW627368X was <5.0. In competition radioligand bioassays, GW627368X had affinity for human prostanoid EP4 and TP receptors (pKi=7.0±0.2 (n=10) and 6.8 (n=2), respectively). Affinity for all other human prostanoid receptors was <5.3. GW627368X will be a valuable tool to explore the role of the prostanoid EP4 receptor in many physiological and pathological settings. PMID:16604093

  9. Haemodynamic effects of a selective adenosine A2A receptor agonist, CGS 21680, in chronic heart failure in anaesthetized rats.

    PubMed

    Nekooeian, A A; Tabrizchi, R

    1998-10-01

    1. Recently we demonstrated that the administration of an A2A adenosine receptor agonist, CGS 21680, to anaesthetized rats with acute heart failure (1 h post-coronary artery ligation) resulted in an increase in cardiac output. In the present investigation, the effects of CGS 21680 on cardiac output, vascular resistance, heart rate, blood pressure and mean circulatory filling pressure (Pmcf) were investigated in anaesthetized rats with chronic heart failure (8 weeks post-coronary artery ligation). 2. Experiments were conducted in five groups (n = 6) of animals: sham-operated vehicle-treated (0.9% NaCl; 0.037 mL kg(-1) min(-1)) animals in which the occluder was placed but not pulled to ligate the coronary artery; coronary artery-ligated vehicle-treated animals; and coronary artery-ligated CGS 21680-treated (0.1. 0.3 or 1.0 microg kg(-1) min(-1)) animals. 3. Baseline blood pressure, cardiac output and rate of rise in left ventricular pressure (+dP/dt) were significantly reduced in animals with coronary artery ligation when compared to sham-operated animals. Coronary artery ligation resulted in a significant increase in left ventricular end-diastolic pressure, Pmcf and venous resistance when compared to sham-operated animals. 4. Administration of CGS 21680 at 0.3 and 1.0 microg kg(-1) min(-1) significantly (n = 6; P<0.05) increased cardiac output by 19+/-4% and 39+/-5%, and heart rate by 14+/-2% and 15+/-1%, respectively, when compared to vehicle treatment in coronary artery-ligated animals. Administration of CGS 21680 also significantly reduced blood pressure and arterial resistance when compared to coronary artery-ligated vehicle-treated animals. Infusion of CGS 21680 also significantly reduced venous resistance when compared to vehicle-treated coronary artery-ligated animals. 5. The results show that heart failure is characterized by reduced cardiac output, and increased left ventricular end-diastolic pressure, venous resistance and Pmcf. Acute treatment with CGS

  10. Haemodynamic effects of a selective adenosine A2A receptor agonist, CGS 21680, in chronic heart failure in anaesthetized rats

    PubMed Central

    Nekooeian, Ali A; Tabrizchi, Reza

    1998-01-01

    Recently we demonstrated that the administration of an A2A adenosine receptor agonist, CGS 21680, to anaesthetized rats with acute heart failure (1 h post-coronary artery ligation) resulted in an increase in cardiac output. In the present investigation, the effects of CGS 21680 on cardiac output, vascular resistance, heart rate, blood pressure and mean circulatory filling pressure (Pmcf) were investigated in anaesthetized rats with chronic heart failure (8 weeks post-coronary artery ligation).Experiments were conducted in five groups (n=6) of animals: sham-operated vehicle-treated (0.9% NaCl; 0.037 mL kg−1 min−1) animals in which the occluder was placed but not pulled to ligate the coronary artery; coronary artery-ligated vehicle-treated animals; and coronary artery-ligated CGS 21680-treated (0.1, 0.3 or 1.0 μg kg−1 min−1) animals.Baseline blood pressure, cardiac output and rate of rise in left ventricular pressure (+dP/dt) were significantly reduced in animals with coronary artery ligation when compared to sham-operated animals. Coronary artery ligation resulted in a significant increase in left ventricular end-diastolic pressure, Pmcf and venous resistance when compared to sham-operated animals.Administration of CGS 21680 at 0.3 and 1.0 μg kg−1 min−1 significantly (n=6; P<0.05) increased cardiac output by 19±4% and 39±5%, and heart rate by 14±2% and 15±1%, respectively, when compared to vehicle treatment in coronary artery-ligated animals. Administration of CGS 21680 also significantly reduced blood pressure and arterial resistance when compared to coronary artery-ligated vehicle-treated animals. Infusion of CGS 21680 also significantly reduced venous resistance when compared to vehicle-treated coronary artery-ligated animals.The results show that heart failure is characterized by reduced cardiac output, and increased left ventricular end-diastolic pressure, venous resistance and Pmcf. Acute treatment with CGS 21680 in

  11. Oropharyngeal Squamous Cell Carcinoma Treated With Radiotherapy or Radiochemotherapy: Prognostic Role of TP53 and HPV Status

    SciTech Connect

    Fallai, Carlo, E-mail: carlo.fallai@istitutotumori.mi.i; Perrone, Federica; Licitra, Lisa

    Purpose: To study the prognostic value of the TP53 mutation and human papilloma virus (HPV) status in oropharyngeal squamous cell carcinoma (OPSCC). Methods and materials: The TP53 mutation and HPV status were analyzed in 78 cases of locoregionally advanced OPSCC. The possible correlation of these factors with locoregiownal control, relapse-free survival, disease-specific survival, and overall survival (OS) was also investigated. Results: Of these 78 cases, 22 had disruptive and 22 had non-disruptive (silent) TP53 mutations; the remaining 34 cases had wild-type (WT) TP53. HPV 16 DNA was found in 9 cases (11%), but all HPV-positive (HPV+) cases carried a functionalmore » p53 protein, except for 1 case that had a silent mutation. HPV+ patients fared better than HPV-negative (HPV-) patients in terms of all survival parameters, with highly statistically significant differences between the groups. Specifically, no distant metastases were observed in the HPV+ patients, whereas they occurred in 17% of the HPV- patients. However, no difference was observed between the WT TP53 and mutation group, even when this was analyzed in terms of disruptive and non-disruptive mutations. Regardless, treatment with chemotherapy nearly doubled the 5-year OS rates, both in the mutation (42% vs. 22%) and WT (30 vs. 16%) group, but only the mutation group showed improvement in all survival parameters. In addition, the second tumor-free 5-year survival rate was 72% in HPV- cases, but no second tumors were observed in HPV+ and WT p53 cases. Conclusions: Patients with HPV+ OPSCC have an excellent prognosis after radiochemotherapy, but cisplatin-based chemotherapy may not confer a satisfactory outcome, especially in WT cases, thereby justifying the additional or alternative use of taxanes and epidermal growth factor receptors inhibitors. Uncommon distant metastases and second tumors in the HPV+ group may be cause for clinicians to review the follow-up policies in these patients.« less

  12. Increased expression of matrix metalloproteinases mediates thromboxane A2-induced invasion in lung cancer cells.

    PubMed

    Li, Xiuling; Tai, Hsin-Hsiung

    2012-07-01

    Thromboxane A(2) receptor (TP) has been shown to play an important role in multiple aspects of cancer development including regulation of tumor growth, survival and metastasis. Here we report that TP mediates cancer cell invasion by inducing expression of matrix metalloproteinases (MMPs). TP agonist, I-BOP, significantly elevated MMP-1, MMP-3, MMP-9 and MMP-10 mRNA levels in A549 human lung adenocarcinoma cells overexpressing TPα or TPβ. The secretion of MMP-1 and MMP-9 in conditioned media was determined using Western blot analysis and zymographic assay. Signaling pathways of I-BOP-induced MMP-1 expression were examined in further detail as a model system for MMPs induction. Signaling molecules involved in I-BOP-induced MMP-1 expression were identified by using specific inhibitors including small interfering (si)-RNAs of signaling molecules and promoter reporter assay. The results indicate that I-BOP-induced MMP-1 expression is mediated by protein kinase C (PKC), extracellular signal-regulated kinase (ERK)-activator protein-1(AP-1) and ERK-CCAAT/enhancer-binding protein β (C/EBPβ) pathways. I-BOP-induced cellular invasiveness of A549 cells expressing TPα or TPβ was determined by invasion assay. GM6001, a general inhibitor of MMPs, decreased basal and I-BOP-induced cell invasion. Knockdown of MMP-1 and MMP-9 by their respective siRNA partially reduced I-BOP-stimulated cell invasion suggesting that other MMPs induced by I-BOP were also involved. Our studies establish the relationship between TP and MMPs in cancer cell invasion and suggest that the thromboxane A(2) (TXA(2))-TP signaling is a potential therapeutic target for cancer invasion and metastasis.

  13. Activation of bradykinin B2 receptor induced the inflammatory responses of cytosolic phospholipase A2 after the early traumatic brain injury.

    PubMed

    Chao, Honglu; Liu, Yinlong; Lin, Chao; Xu, Xiupeng; Li, Zheng; Bao, Zhongyuan; Fan, Liang; Tao, Chao; Zhao, Lin; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-06-09

    Phospholipase A 2 is a known aggravator of inflammation and deteriorates neurological outcomes after traumatic brain injury (TBI), however the exact inflammatory mechanisms remain unknown. This study investigated the role of bradykinin and its receptor, which are known initial mediators within inflammation activation, as well as the mechanisms of the cytosolic phospholipase A 2 (cPLA 2 )-related inflammatory responses after TBI. We found that cPLA 2 and bradykinin B2 receptor were upregulated after a TBI. Rats treated with the bradykinin B2 receptor inhibitor LF 16-0687 exhibited significantly less cPLA 2 expression and related inflammatory responses in the brain cortex after sustaining a controlled cortical impact (CCI) injury. Both the cPLA 2 inhibitor and the LF16-0687 improved CCI rat outcomes by decreasing neuron death and reducing brain edema. The following TBI model utilized both primary astrocytes and primary neurons in order to gain further understanding of the inflammation mechanisms of the B2 bradykinin receptor and the cPLA 2 in the central nervous system. There was a stronger reaction from the astrocytes as well as a protective effect of LF16-0687 after the stretch injury and bradykinin treatment. The protein kinase C pathway was thought to be involved in the B2 bradykinin receptor as well as the cPLA 2 -related inflammatory responses. Rottlerin, a Protein Kinase C (PKC) δ inhibitor, decreased the activity of the cPLA 2 activity post-injury, and LF16-0687 suppressed both the PKC pathway and the cPLA 2 activity within the astrocytes. These results indicated that the bradykinin B2 receptor-mediated pathway is involved in the cPLA 2 -related inflammatory response from the PKC pathway. Copyright © 2018. Published by Elsevier B.V.

  14. Evaluation of antidepressant-like and anxiolytic-like activity of purinedione-derivatives with affinity for adenosine A2A receptors in mice.

    PubMed

    Dziubina, Anna; Szmyd, Karina; Zygmunt, Małgorzata; Sapa, Jacek; Dudek, Magdalena; Filipek, Barbara; Drabczyńska, Anna; Załuski, Michał; Pytka, Karolina; Kieć-Kononowicz, Katarzyna

    2016-12-01

    It has recently been suggested that the adenosine A 2A receptor plays a role in several animal models of depression. Additionally, A 2A antagonists have reversed behavioral deficits and exhibited a profile similar to classical antidepressants. In the present study, imidazo- and pyrimido[2,1-f]purinedione derivatives (KD 66, KD 167, KD 206) with affinity to A 2A receptors but poor A 1 affinity were evaluated for their antidepressant- and anxiolytic-like activity. The activity of these derivatives was tested using a tail suspension and forced swim test, two widely-used behavioral paradigms for the evaluation of antidepressant-like activity. In turn, the anxiolytic activity was evaluated using the four-plate test. The results showed the antidepressant-like activity of pyrimido- and imidazopurinedione derivatives (i.e. KD 66, KD 167 and KD 206) in acute and chronic behavioral tests in mice. KD 66 revealed an anxiolytic-like effect, while KD 167 increased anxiety behaviors. KD 206 had no effect on anxiety. Furthermore, none of the tested compounds increased locomotor activity. Available data support the proposition that the examined compounds with adenosine A 2A receptor affinity may be an interesting target for the development of antidepressant and/or anxiolytic agents. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  15. Identification of novel thiazolo[5,4-d]pyrimidine derivatives as human A1 and A2A adenosine receptor antagonists/inverse agonists.

    PubMed

    Varano, Flavia; Catarzi, Daniela; Falsini, Matteo; Vincenzi, Fabrizio; Pasquini, Silvia; Varani, Katia; Colotta, Vittoria

    2018-07-23

    In this study a new set of thiazolo[5,4-d]pyrimidine derivatives was synthesized. These derivatives bear different substituents at positions 2 and 5 of the thiazolopyrimidine core while maintaining a free amino group at position-7. The new compounds were tested for their affinity and potency at human (h) A 1 , A 2A , A 2B and A 3 adenosine receptors expressed in CHO cells. The results reveal that the higher affinity of these new set of thiazolopyrimidines is toward the hA 1 and hA 2A adenosine receptors subtypes and is tuned by the substitution pattern at both the 2 and 5 positions of the thiazolopyrimidine nucleus. Functional studies evidenced that the compounds behaved as dual A 1 /A 2A antagonists/inverse agonists. Compound 3, bearing a 5-((2-methoxyphenyl) methylamino) group and a phenyl moiety at position 2, displayed the highest affinity (hA 1 K i  = 10.2 nM; hA 2A K i  = 4.72 nM) and behaved as a potent A 1 /A 2A antagonist/inverse agonist (hA 1 IC 50  = 13.4 nM; hA 2A IC 50  = 5.34 nM). Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Effect of the adenosine A2A receptor antagonist MSX-3 on motivational disruptions of maternal behavior induced by dopamine antagonism in the early postpartum rat.

    PubMed

    Pereira, Mariana; Farrar, Andrew M; Hockemeyer, Jörg; Müller, Christa E; Salamone, John D; Morrell, Joan I

    2011-01-01

    Mesolimbic dopamine (DA), particularly in the nucleus accumbens, importantly regulates activational aspects of maternal responsiveness. DA antagonism and accumbens DA depletions interfere with early postpartum maternal motivation by selectively affecting most forms of active maternal behaviors, while leaving nursing behavior relatively intact. Considerable evidence indicates that there is a functional interaction between DA D2 and adenosine A(2A) receptors in striatal areas, including the nucleus accumbens. This study was conducted to determine if adenosine A(2A) receptor antagonism could reverse the effects of DA receptor antagonism on early postpartum maternal behavior. The adenosine A(2A) receptor antagonist MSX-3 (0.25-2.0 mg/kg, IP) was investigated for its ability to reverse the effects of the DA D2 receptor antagonist haloperidol (0.1 mg/kg, IP) on the maternal behavior of early postpartum female rats. Haloperidol severely impaired the expression of active maternal components, including retrieval and grouping the pups at the nest site, pup licking, and nest building. Co-administration of MSX-3 (0.25-2.0 mg/kg, IP) with haloperidol produced a dose-related attenuation of the haloperidol-induced behavioral deficits in early postpartum females. Doses of MSX-3 that effectively reversed the effects of haloperidol (0.5, 1.0 mg/kg), when administered in the absence of haloperidol, did not affect maternal responding or locomotor activity. Adenosine and DA systems interact to regulate early postpartum maternal responsiveness. This research may potentially contribute to the development of strategies for treatments of psychiatric disorders during the postpartum period, with particular emphasis in maintaining or restoring the mother-infant relationship.

  17. Effect of the adenosine A2A receptor antagonist MSX-3 on motivational disruptions of maternal behavior induced by dopamine antagonism in the early postpartum rat

    PubMed Central

    Farrar, Andrew M.; Hockemeyer, Jörg; Müller, Christa E.; Salamone, John D.; Morrell, Joan I.

    2011-01-01

    Rationale Mesolimbic dopamine (DA), particularly in the nucleus accumbens, importantly regulates activational aspects of maternal responsiveness. DA antagonism and accumbens DA depletions interfere with early postpartum maternal motivation by selectively affecting most forms of active maternal behaviors, while leaving nursing behavior relatively intact. Considerable evidence indicates that there is a functional interaction between DA D2 and adenosine A2A receptors in striatal areas, including the nucleus accumbens. Objective This study was conducted to determine if adenosine A2A receptor antagonism could reverse the effects of DA receptor antagonism on early postpartum maternal behavior. Methods The adenosine A2A receptor antagonist MSX-3 (0.25–2.0 mg/kg, IP) was investigated for its ability to reverse the effects of the DA D2 receptor antagonist haloperidol (0.1 mg/kg, IP) on the maternal behavior of early postpartum female rats. Results Haloperidol severely impaired the expression of active maternal components, including retrieval and grouping the pups at the nest site, pup licking, and nest building. Co-administration of MSX-3 (0.25–2.0 mg/kg, IP) with haloperidol produced a dose-related attenuation of the haloperidol-induced behavioral deficits in early postpartum females. Doses of MSX-3 that effectively reversed the effects of haloperidol (0.5, 1.0 mg/kg), when administered in the absence of haloperidol, did not affect maternal responding or locomotor activity. Conclusions Adenosine and DA systems interact to regulate early postpartum maternal responsiveness. This research may potentially contribute to the development of strategies for treatments of psychiatric disorders during the postpartum period, with particular emphasis in maintaining or restoring the mother–infant relationship. PMID:20848086

  18. Alpha 2-adrenergic receptor stimulation of phospholipase A2 and of adenylate cyclase in transfected Chinese hamster ovary cells is mediated by different mechanisms

    SciTech Connect

    Jones, S.B.; Halenda, S.P.; Bylund, D.B.

    1991-02-01

    The effect of alpha 2-adrenergic receptor activation on adenylate cyclase activity in Chinese hamster ovary cells stably transfected with the alpha 2A-adrenergic receptor gene is biphasic. At lower concentrations of epinephrine forskolin-stimulated cyclic AMP production is inhibited, but at higher concentrations the inhibition is reversed. Both of these effects are blocked by the alpha 2 antagonist yohimbine but not by the alpha 1 antagonist prazosin. Pretreatment with pertussis toxin attenuates inhibition at lower concentrations of epinephrine and greatly potentiates forskolin-stimulated cyclic AMP production at higher concentrations of epinephrine. alpha 2-Adrenergic receptor stimulation also causes arachidonic acid mobilization, presumably via phospholipasemore » A2. This effect is blocked by yohimbine, quinacrine, removal of extracellular Ca2+, and pretreatment with pertussis toxin. Quinacrine and removal of extracellular Ca2+, in contrast, have no effect on the enhanced forskolin-stimulated cyclic AMP production. Thus, it appears that the alpha 2-adrenergic receptor in these cells can simultaneously activate distinct signal transduction systems; inhibition of adenylate cyclase and stimulation of phospholipase A2, both via G1, and potentiation of cyclic AMP production by a different (pertussis toxin-insensitive) mechanism.« less

  19. Chimeric Antigen Receptor-Modified T Cells Redirected to EphA2 for the Immunotherapy of Non-Small Cell Lung Cancer.

    PubMed

    Li, Ning; Liu, Shaohui; Sun, Mingjiao; Chen, Wei; Xu, Xiaogang; Zeng, Zhu; Tang, Yemin; Dong, Yongquan; Chang, Alex H; Zhao, Qiong

    2018-02-01

    Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is overexpressed in more than 90% of non-small cell lung cancer (NSCLC) but not significantly in normal lung tissue. It is therefore an important tumor antigen target for chimeric antigen receptors (CAR)-T-based therapy in NSCLC. Here, we developed a specific CAR targeted to EphA2, and the anti-tumor effects of this CAR were investigated. A second generation CAR with co-stimulatory receptor 4-1BB targeted to EphA2 was developed. The functionality of EphA2-specific T cells in vitro was tested with flow cytometry and real-time cell electronic sensing system assays. The effect in vivo was evaluated in xenograft SCID Beige mouse model of EphA2 positive NSCLC. These EphA2-specifc T cells can cause tumor cell lysis by producing the cytokines IFN-γ when cocultured with EphA2-positive targets, and the cytotoxicity effects was specific in vitro. In vivo, the tumor signals of mice treated with EphA2-specifc T cells presented the tendency of decrease, and was much lower than the mice treated with non-transduced T cells. The anti-tumor effects of this CAR-T technology in vivo and vitro had been confirmed. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive NSCLC. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Colonic motor dysfunctions in a mouse model of high-fat diet-induced obesity: an involvement of A2B adenosine receptors.

    PubMed

    Antonioli, Luca; Pellegrini, Carolina; Fornai, Matteo; Tirotta, Erika; Gentile, Daniela; Benvenuti, Laura; Giron, Maria Cecilia; Caputi, Valentina; Marsilio, Ilaria; Orso, Genny; Bernardini, Nunzia; Segnani, Cristina; Ippolito, Chiara; Csóka, Balázs; Németh, Zoltán H; Haskó, György; Scarpignato, Carmelo; Blandizzi, Corrado; Colucci, Rocchina

    2017-12-01

    Adenosine A 2B receptors (A 2B R) regulate several enteric functions. However, their implication in the pathophysiology of intestinal dysmotility associated with high-fat diet (HFD)-induced obesity has not been elucidated. We investigated the expression of A 2B R in mouse colon and their role in the mechanisms underlying the development of enteric dysmotility associated with obesity. Wild-type C57BL/6J mice were fed with HFD (60% kcal from fat) or normocaloric diet (NCD; 18% kcal from fat) for 8 weeks. Colonic A 2B R localization was examined by immunofluorescence. The role of A 2B R in the control of colonic motility was examined in functional experiments on longitudinal muscle preparations (LMPs). In NCD mice, A 2B R were predominantly located in myenteric neurons; in HFD animals, their expression increased throughout the neuromuscular layer. Functionally, the A 2B R antagonist MRS1754 enhanced electrically induced NK 1 -mediated tachykininergic contractions in LMPs from HFD mice, while it was less effective in tissues from NCD mice. The A 2B receptor agonist BAY 60-6583 decreased colonic tachykininergic contractions in LMPs, with higher efficacy in preparations from obese mice. Both A 2B R ligands did not affect contractions elicited by exogenous substance P. Obesity is related with a condition of colonic inflammation, leading to an increase of A 2B R expression. A 2B R, modulating the activity of excitatory tachykininergic nerves, participate to the enteric dysmotility associated with obesity.

  1. Functional role of striatal A2A, D2, and mGlu5 receptor interactions in regulating striatopallidal GABA neuronal transmission.

    PubMed

    Beggiato, Sarah; Tomasini, Maria Cristina; Borelli, Andrea Celeste; Borroto-Escuela, Dasiel Oscar; Fuxe, Kjell; Antonelli, Tiziana; Tanganelli, Sergio; Ferraro, Luca

    2016-07-01

    In this study, the functional role of individual striatal receptors for adenosine (A2AR), dopamine (D2R), and the metabotropic glutamate receptor mGlu5R in regulating rat basal ganglia activity was characterized in vivo using dual-probe microdialysis in freely moving rats. In particular, intrastriatal perfusion with the D2R agonist quinpirole (10 μM, 60 min) decreased ipsilateral pallidal GABA and glutamate levels, whereas intrastriatal CGS21680 (A2AR agonist; 1 μM, 60 min) was ineffective on either pallidal GABA and glutamate levels or the quinpirole-induced effects. Intrastriatal perfusion with the mGlu5R agonist (RS)-2-chloro-5-hydroxyphenylglycine (600 μM, 60 min), by itself ineffective on pallidal GABA and glutamate levels, partially counteracted the effects of quinpirole. When combined with CGS21680 (1 μM, 60 min), (RS)-2-chloro-5-hydroxyphenylglycine (CHPG; 600 μM, 60 min) fully counteracted the quinpirole (10 μM, 60 min)-induced reduction in ipsilateral pallidal GABA and glutamate levels. These effects were fully counteracted by local perfusion with the mGlu5R antagonist MPEP (300 μM) or the A2AR antagonist ZM 241385 (100 nM). These results suggest that A2ARs and mGlu5Rs interact synergistically in modulating the D2R-mediated control of striatopallidal GABA neurons. Using dual-probe microdialysis, we characterized the functional role of striatal adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R), and metabotropic glutamate receptor 5 (mGluR5) interactions in regulating rat basal ganglia activity. The results suggest the possible usefulness of using an A2AR antagonist and mGluR5 antagonist combination in the treatment of Parkinson's disease to increase the inhibitory D2 signaling on striatopallidal GABA neurons. © 2016 International Society for Neurochemistry.

  2. TP53 mutations in primary breast carcinomas from white and African-Brazilian patients.

    PubMed

    Nagai, Maria Aparecida; Schaer Barbosa, Helenemarie; Zago, Marco Antonio; Araújo Silva, Wilson; Nishimoto, Inês Nobuko; Salaorni, Sibeli; Guerreiro Costa, Lívia Nery Franco; Silva Araújo, Marcos; Caldas Oliveira, Ana Gabriela; Mourâo Neto, Mário; Brentani, Maria Mitzi

    2003-07-01

    We have attempted to determine the incidence, nature and clinical significance of TP53 mutation in a group of white (242 cases) and African-Brazilian (52 cases) patients with breast cancer. The interethnic admixture as estimated by STR markers showed that white subjects displayed 67.9+/-0.4%, 25.0+/-1.7% and 7.0%+/-1.6% and the black populations had 34.4+/-1.9%, 56.2+/-1.9 and 9.4+/-2.2% respectively of European, African and Amerindian genes. Clinical parameters such as age, lymph node status and steroid receptors were similar in both groups. African-Brazilian patients presented more advanced lesions. Mutation screening was performed using polymerase chain reaction-single strand conformation analysis followed by sequencing. Compared to whites (13.6%), a relatively high frequency of TP53 mutation was found in blacks (32.7%) (p=0.001). African-Brazilian women have a larger proportion of mutations in exons 5 and 7, whereas white women have more mutations in exon 8. Mutations within exon 4 were found only in tumors of white patients. The spectra of TP53 mutations show that A:T-->G:C nucleotide transversion and G:C-->C:G transition were more common in African-Brazilian women whereas G:C-->T:A transversion occurs very frequently in whites. A high prevalence of G:C-->A:T nucleotide transitions and deletions was detected in both groups. No association was found between p53 gene mutation and tumor or clinical parameters independently of the ethnic group. With a median follow-up of 35.6 months for whites and 43.4 months for the blacks, no differences in overall survival were found. If white patients were stratified according to the type and location of TP53 mutations, patients with mutations affecting amino acids directly involved in DNA or Zn binding displayed a poor prognosis. The pattern of mutations found in our population seems to reflect a base line pattern observed in populations with similar ethnic profile with some modifications, which might be derived from specific

  3. Structural investigation of a C-terminal EphA2 receptor mutant: Does mutation affect the structure and interaction properties of the Sam domain?

    PubMed

    Mercurio, Flavia A; Costantini, Susan; Di Natale, Concetta; Pirone, Luciano; Guariniello, Stefano; Scognamiglio, Pasqualina L; Marasco, Daniela; Pedone, Emilia M; Leone, Marilisa

    2017-09-01

    Ephrin A2 receptor (EphA2) plays a key role in cancer, it is up-regulated in several types of tumors and the process of ligand-induced receptor endocytosis, followed by degradation, is considered as a potential path to diminish tumor malignancy. Protein modulators of this mechanism are recruited at the cytosolic Sterile alpha motif (Sam) domain of EphA2 (EphA2-Sam) through heterotypic Sam-Sam associations. These interactions engage the C-terminal helix of EphA2 and close loop regions (the so called End Helix side). In addition, several studies report on destabilizing mutations in EphA2 related to cataract formation and located in/or close to the Sam domain. Herein, we analyzed from a structural point of view, one of these mutants characterized by the insertion of a novel 39 residue long polypeptide at the C-terminus of EphA2-Sam. A 3D structural model was built by computational methods and revealed partial disorder in the acquired C-terminal tail and a few residues participating in an α-helix and two short β-strands. We investigated by CD and NMR studies the conformational properties in solution of two peptides encompassing the whole C-terminal tail and its predicted helical region, respectively. NMR binding experiments demonstrated that these peptides do not interact relevantly with either EphA2-Sam or its interactor Ship2-Sam. Molecular dynamics (MD) simulations further indicated that the EphA2 mutant could be represented only through a conformational ensemble and that the C-terminal tail should not largely wrap the EphA2-Sam End-Helix interface and affect binding to other Sam domains. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Blockade of adenosine A2A receptors recovers early deficits of memory and plasticity in the triple transgenic mouse model of Alzheimer's disease.

    PubMed

    Silva, António C; Lemos, Cristina; Gonçalves, Francisco Q; Pliássova, Anna V; Machado, Nuno J; Silva, Henrique B; Canas, Paula M; Cunha, Rodrigo A; Lopes, João Pedro; Agostinho, Paula

    2018-05-31

    Alzheimer's disease (AD) begins with a deficit of synaptic function and adenosine A 2A receptors (A 2A R) are mostly located in synapses controlling synaptic plasticity. The over-activation of adenosine A 2A receptors (A 2A R) causes memory deficits and the blockade of A 2A R prevents memory damage in AD models. We now enquired if this prophylactic role of A 2A R might be extended to a therapeutic potential. We used the triple transgenic model of AD (3xTg-AD) and defined that the onset of memory dysfunction occurred at 4 months of age in the absence of locomotor or emotional alterations. At the onset of memory deficits, 3xTg mice displayed a decreased density of markers of excitatory synapses (10.6 ± 3.8% decrease of vGluT1) without neuronal or glial overt damage and an increase of synaptic A 2A R in the hippocampus (130 ± 22%). After the onset of memory deficits in 3xTg-AD mice, a three weeks treatment with the selective A 2A R antagonist normalized the up-regulation of hippocampal A 2A R and restored hippocampal-dependent reference memory, as well as the decrease of hippocampal synaptic plasticity (60.0 ± 3.7% decrease of long-term potentiation amplitude) and the decrease of global (syntaxin-I) and glutamatergic synaptic markers (vGluT1). These findings show a therapeutic-like ability of A 2A R antagonists to recover synaptic and memory dysfunction in early AD. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. A genetic variation in the adenosine A2A receptor gene (ADORA2A) contributes to individual sensitivity to caffeine effects on sleep.

    PubMed

    Rétey, J V; Adam, M; Khatami, R; Luhmann, U F O; Jung, H H; Berger, W; Landolt, H-P

    2007-05-01

    Caffeine is the most widely used stimulant in Western countries. Some people voluntarily reduce caffeine consumption because it impairs the quality of their sleep. Studies in mice revealed that the disruption of sleep after caffeine is mediated by blockade of adenosine A2A receptors. Here we show in humans that (1) habitual caffeine consumption is associated with reduced sleep quality in self-rated caffeine-sensitive individuals, but not in caffeine-insensitive individuals; (2) the distribution of distinct c.1083T>C genotypes of the adenosine A2A receptor gene (ADORA2A) differs between caffeine-sensitive and -insensitive adults; and (3) the ADORA2A c.1083T>C genotype determines how closely the caffeine-induced changes in brain electrical activity during sleep resemble the alterations observed in patients with insomnia. These data demonstrate a role of adenosine A2A receptors for sleep in humans, and suggest that a common variation in ADORA2A contributes to subjective and objective responses to caffeine on sleep.

  6. EphA2 receptor activation with ephrin-A1 ligand restores cetuximab efficacy in NRAS-mutant colorectal cancer cells.

    PubMed

    Cuyàs, Elisabet; Queralt, Bernardo; Martin-Castillo, Begoña; Bosch-Barrera, Joaquim; Menendez, Javier A

    2017-07-01

    Patients with wild-type KRAS metastatic colorectal cancer (mCRC) that harbors NRAS activating mutations do not benefit from anti-EGFR therapies. Very little is known about oncogenic NRAS signaling driving mCRC unresponsiveness to the EGFR-directed antibody cetuximab. Using a system of paired NRAS-mutant and wild-type isogenic mCRC cell lines to explore signaling pathways engaged by the common oncogenic NRAS Q61K variant upon challenge with cetuximab, we uncovered an unexpected mechanism of resistance to cetuximab involving dysregulation of the ephrin-A1/EphA2 signaling axis. Parental NRAS+/+ cells, but not NRASQ61K/+ cells, activated the ephrin receptor ephA1 in response to cetuximab treatment. Moreover, whereas cetuximab treatment significantly downregulated EPHA2 gene expression in NRAS+/+ cells, EPHA2 expression in NRASQ61K/+ cells was refractory to cetuximab. Remarkably, pharmacologically mimicked ephrin-A1 engagement to ephA2 converted NRAS-mutant into RAS wild-type mCRC cells in terms of cetuximab efficacy. Accordingly, activation of the ephA2 receptor by bioactive recombinant human ephrin-A1/Fc-fusion protein suppressed the cetuximab-unresponsive hyperactivation of MAPK and AKT and fully restored cetuximab activity in NRAS-mutant colorectal cells. Collectively, these findings reveal that the clinical benefit of cetuximab in mCRC might necessarily involve the suppression of the ligandless oncogenic signaling of the ephA2 receptor. Hence, ligand-dependent tumor suppressor signaling using therapeutic ephA2 agonists might offer new therapeutic opportunities to clinically widen the use of cetuximab in NRAS-mutated and/or ephA2-dependent mCRC tumors.

  7. Effects of a Proprietary Standardized Orthosiphon stamineus Ethanolic Leaf Extract on Enhancing Memory in Sprague Dawley Rats Possibly via Blockade of Adenosine A2A Receptors

    PubMed Central

    Choudhary, Yogendra; Choudhary, Vandana Kotak; Bommu, Praveen; Wong, Hoi Jin

    2015-01-01

    The aim of the study was to explore a propriety standardized ethanolic extract from leaves of Orthosiphon stamineus Benth in improving impairments in short-term social memory in vivo, possibly via blockade of adenosine A2A receptors (A2AR). The ethanolic extract of O. stamineus leaves showed significant in vitro binding activity of A2AR with 74% inhibition at 150 μg/ml and significant A2AR antagonist activity with 98% inhibition at 300 μg/mL. A significant adenosine A1 receptor (A1R) antagonist activity with 100% inhibition was observed at 300 μg/mL. Its effect on learning and memory was assessed via social recognition task using Sprague Dawley rats whereby the ethanolic extract of O. stamineus showed significant (p < 0.001) change in recognition index (RI) at 300 mg/kg and 600 mg/kg p.o and 120 mg/kg i.p., respectively, compared to the vehicle control. In comparison, the ethanolic extract of Polygonum minus aerial parts showed small change in inflexion; however, it remained insignificant in RI at 200 mg/kg p.o. Our findings suggest that the ethanolic extract of O. stamineus leaves improves memory by reversing age-related deficits in short-term social memory and the possible involvement of adenosine A1 and adenosine A2A as a target bioactivity site in the restoration of memory. PMID:26649059

  8. Cognitive impairments associated with alterations in synaptic proteins induced by the genetic loss of adenosine A2A receptors in mice.

    PubMed

    Moscoso-Castro, Maria; López-Cano, Marc; Gracia-Rubio, Irene; Ciruela, Francisco; Valverde, Olga

    2017-11-01

    The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A 2A adenosine receptor subtype (A 2A R) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A 2A R in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A 2A R constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A 2A R knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Two-Photon/Laser-Induced Fluorescence (TP/LIF) sensor

    NASA Technical Reports Server (NTRS)

    Bradshaw, John D.

    1994-01-01

    The Two-Photon/Laser-Induced Fluorescence (TP/LIF) technique is based on the stepwise excitation of the OH transitions, X(exp 2)II, v(exp '') = 0 yields X(exp 2)II, v(exp '') = 1 (lambda = 2.9 microns) and X(exp 2)II v(exp '') = 1 yields A(exp2)Sigma, v' = 0 (lambda = 345 nm) with background free fluorescence monitoring of the A(exp 2)Sigma, v' = 0 yields X(exp 2)II, v(exp '') = 0 transition near 309 nm. This technique has awaited the advent of a suitable mid-infrared (2.9 microns) laser source. Turnable mid-IR lasers now exist that are capable of meeting the specifications required of a high sensitivity TP/LIF OH sensor.

  10. The role of adenosine A1 and A2A receptors in the caffeine effect on MDMA-induced DA and 5-HT release in the mouse striatum.

    PubMed

    Górska, A M; Gołembiowska, K

    2015-04-01

    3,4-Methylenedioxymethamphetamine (MDMA, "ecstasy") popular as a designer drug is often used with caffeine to gain a stronger stimulant effect. MDMA induces 5-HT and DA release by interaction with monoamine transporters. Co-administration of caffeine and MDMA may aggravate MDMA-induced toxic effects on DA and 5-HT terminals. In the present study, we determined whether caffeine influences DA and 5-HT release induced by MDMA. We also tried to find out if adenosine A1 and A2A receptors play a role in the effect of caffeine by investigating the effect of the selective adenosine A1 and A2A receptor antagonists, DPCPX and KW 6002 on DA and 5-HT release induced by MDMA. Mice were treated with caffeine (10 mg/kg) and MDMA (20 or 40 mg/kg) alone or in combination. DA and 5-HT release in the mouse striatum was measured using in vivo microdialysis. Caffeine exacerbated the effect of MDMA on DA and 5-HT release. DPCPX or KW 6002 co-administered with MDMA had similar influence as caffeine, but KW 6002 was more potent than caffeine or DPCPX. To exclude the contribution of MAO inhibition by caffeine in the caffeine effect on MDMA-induced increase in DA and 5-HT, we also tested the effect of the nonxanthine adenosine receptor antagonist CGS 15943A lacking properties of MAO activity modification. Our findings indicate that adenosine A1 and A2A receptor blockade may account for the caffeine-induced exacerbation of the MDMA effect on DA and 5-HT release and may aggravate MDMA toxicity.

  11. Blockade of adenosine A2A receptor enhances CD8+ T cells response and decreases regulatory T cells in head and neck squamous cell carcinoma.

    PubMed

    Ma, Si-Rui; Deng, Wei-Wei; Liu, Jian-Feng; Mao, Liang; Yu, Guang-Tao; Bu, Lin-Lin; Kulkarni, Ashok B; Zhang, Wen-Feng; Sun, Zhi-Jun

    2017-06-07

    Cancer immunotherapy offers a promising approach in cancer treatment. The adenosine A2A receptor (A2AR) could protect cancerous tissues from immune clearance via inhibiting T cells response. To date, the role of A2AR in head and neck squamous cell carcinoma (HNSCC) has not been investigated. Here, we sought to explore the expression and immunotherapeutic value of A2AR blockade in HNSCC. The expression of A2AR was evaluated by immunostaining in 43 normal mucosae, 48 dysplasia and 165 primary HNSCC tissues. The immunotherapeutic value of A2AR blockade was assessed in vivo in genetically defined immunocompetent HNSCC mouse model. Immunostaining of HNSCC tissue samples revealed that increased expression of A2AR on tumor infiltrating immune cells correlated with advanced pathological grade, larger tumor size and positive lymph node status. Elevated A2AR expression was also detected in recurrent HNSCC and HNSCC tissues with induction chemotherapy. The expression of A2AR was found to be significantly correlated with HIF-1α, CD73, CD8 and Foxp3. Furthermore, the increased population of CD4 + Foxp3 + regulatory T cells (Tregs), which partially expressed A2AR, was observed in an immunocompetent mouse model that spontaneously develops HNSCC. Pharmacological blockade of A2AR by SCH58261 delayed the tumor growth in the HNSCC mouse model. Meanwhile, A2AR blockade significantly reduced the population of CD4 + Foxp3 + Tregs and enhanced the anti-tumor response of CD8 + T cells. These results offer a preclinical proof for the administration of A2AR inhibitor on prophylactic experimental therapy of HNSCC and suggest that A2AR blockade can be a potential novel strategy for HNSCC immunotherapy.

  12. Bilateral wilms tumor with TP53-related anaplasia.

    PubMed

    Popov, Sergey D; Vujanic, Gordan M; Sebire, Neil J; Chagtai, Tasnim; Williams, Richard; Vaidya, Sucheta; Pritchard-Jones, Kathy

    2013-01-01

    Wilms tumor (WT) with diffuse anaplasia has an unfavorable prognosis and is often (>70%) associated with mutations in the TP53 gene. Although most WTs are unilateral, 5-10% are bilateral, and they are almost always present with nephrogenic rests. The latter are considered a precursor of WT. Two cases of bilateral WTs with nephroblastomatosis, in which anaplastic changes were detected over a period of time, were analyzed using clinical, radiological, histopathological, and molecular-genetic data. TP53 was analyzed by direct sequencing of its full coding sequence and intron-exon boundaries in 11 fragments. DNA was extracted from paraffin-embedded or frozen specimens. High-resolution genomic copy number profiling was carried out by UCL Genomics on the Affymetrix Human Mapping 250K Nsp or Genome-Wide Human SNP Array 6.0 platform. Both cases demonstrated a strong association between the appearance of anaplastic clones and TP53 mutations. Synchronous ganglioneuroma was diagnosed in one case. Our cases are unique as they represent a long disease history and demonstrate the difficulties in managing rare cases of bilateral WT with anaplasia. These cases also emphasize the practical importance of modern molecular-genetic techniques and their clinical application. Moreover, they highlight the issue of the adequate sampling needed in order to gather comprehensive, efficient, and sufficient information about genetic events in a single tumor.

  13. Study of the Antimicrobial Activity of Tilapia Piscidin 3 (TP3) and TP4 and Their Effects on Immune Functions in Hybrid Tilapia (Oreochromis spp.)

    PubMed Central

    Pan, Chieh-Yu; Tsai, Tsung-Yu; Su, Bor-Chyuan; Hui, Cho-Fat; Chen, Jyh-Yih

    2017-01-01

    To address the growing concern over antibiotic-resistant microbial infections in aquatic animals, we tested several promising alternative agents that have emerged as new drug candidates. Specifically, the tilapia piscidins are a group of peptides that possess antimicrobial, wound-healing, and antitumor functions. In this study, we focused on tilapia piscidin 3 (TP3) and TP4, which are peptides derived from Oreochromis niloticus, and investigated their inhibition of acute bacterial infections by infecting hybrid tilapia (Oreochromis spp.) with Vibrio vulnificus and evaluating the protective effects of pre-treating, co-treating, and post-treating fish with TP3 and TP4. In vivo experiments showed that co-treatment with V. vulnificus and TP3 (20 μg/fish) or TP4 (20 μg/fish) achieved 95.3% and 88.9% survival rates, respectively, after seven days. When we co-injected TP3 or TP4 and V. vulnificus into tilapia and then re-challenged the fish with V. vulnificus after 28 days, the tilapia exhibited survival rates of 35.6% and 42.2%, respectively. Pre-treatment with TP3 (30 μg/fish) or TP4 (20 μg/fish) for 30 minutes prior to V. vulnificus infection resulted in high survival rates of 28.9% and 37.8%, respectively, while post-treatment with TP3 (20 μg/fish or 30 μg/fish) or TP4 (20 μg/fish) 30 minutes after V. vulnificus infection yielded high survival rates of 33.3% and 48.9%. In summary, pre-treating, co-treating, and post-treating fish with TP3 or TP4 all effectively decreased the number of V. vulnificus bacteria and promoted significantly lower mortality rates in tilapia. The minimum inhibitory concentrations (MICs) of TP3 and TP4 that were effective for treating fish infected with V. vulnificus were 7.8 and 62.5 μg/ml, respectively, whereas the MICs of kanamycin and ampicillin were 31.2 and 3.91 μg/ml. The antimicrobial activity of these peptides was confirmed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM), both of which showed

  14. Blockade of 5-HT2A/2C-type receptors impairs learning in female rats in the course of estrous cycle.

    PubMed

    Fedotova, Yu O; Ordyan, N E

    2010-12-01

    We studied the effects of chronic administration (14 days) of agonist of 5-HT2B/2C serotonin receptors m-CPP (0.5 mg/kg subcutaneously) and agonist of 5-HT2A/2C serotonin receptors ketanserin (0.1 mg/kg intraperitoneally) on conditioned reactions in female rats in different phases of the estrous cycle. Passive avoidance (PA) paradigm and Morris water maze were used as behavioral tests. Chronic administration of m-CPP did not affect PA retrieval during the proestrus and estrus phases, but improved the dynamics of spatial learning in Morris water maze in comparison with control rats. Chronic administration of ketanserin uniformly impaired processes of spatial and nonspatial learning in female rats irrespective to the phase of the estrous cycle. A modulating role of 5-HT2A/2C and 5-HT2B/2C serotonin receptors in process of learning in female rats during the key phases of the estrous cycle was demonstrated.

  15. Alteration in contractile G-protein coupled receptor expression by moist snus and nicotine in rat cerebral arteries

    SciTech Connect

    Sandhu, Hardip, E-mail: sandhu.hardip@gmail.com; Xu Cangbao; Edvinsson, Lars

    The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET{sub B}, serotonin 5-HT{sub 1B}, and thromboxane A{sub 2} TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression ofmore » GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24 h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25 ng nicotine/ml). A high dose (250 ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c, 5-HT{sub 1B} receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET{sub B}, 5-HT{sub 1B}, and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25 ng nicotine/ml) lowered the 5-HT{sub 1B} receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250 ng nicotine/ml) increased the ET{sub B} receptor-mediated contraction. The combined 5-HT{sub 1B} and 5-HT{sub 2A} receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS

  16. Suppression of PLCβ2 by Endotoxin Plays a Role in the Adenosine A2A Receptor-Mediated Switch of Macrophages from an Inflammatory to an Angiogenic Phenotype

    PubMed Central

    Grinberg, Stan; Hasko, Gyorgy; Wu, Dianqing; Leibovich, Samuel Joseph

    2009-01-01

    Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A2A receptor (A2AR) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A2ARs by TLR agonists, down-regulation of tumor necrosis factor α (TNFα) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)β1 and β2 expression in macrophages by de-stabilizing their mRNAs. The PLCβ inhibitor U73122 down-regulates TNFα expression by macrophages, and in the presence of A2AR agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A2AR agonists. Selective down-regulation of PLCβ2, but not PLCβ1, using small-interfering RNA resulted in increased VEGF expression in response to A2AR agonists, but did not suppress TNFα expression. Macrophages from PLCβ2−/− mice also expressed increased VEGF in response to A2AR agonists. LPS-mediated suppression of PLCβ1 and β2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 μg/mouse, i.p.) suppressed PLCβ1 and β2 expression in spleen, liver, and lung of wild-type but not MyD88−/− mice. These studies indicate that LPS suppresses PLCβ1 and β2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCβ2 plays an important role in switching M1 macrophages into an M2-like state. PMID:19850892

  17. Cross-talk between the Tissue Factor/coagulation factor VIIa complex and the tyrosine kinase receptor EphA2 in cancer.

    PubMed

    Eriksson, Oskar; Thulin, Åsa; Asplund, Anna; Hegde, Geeta; Navani, Sanjay; Siegbahn, Agneta

    2016-05-31

    Tissue Factor (TF) forms a proteolytically active complex together with coagulation factor VIIa (FVIIa) and functions as the trigger of blood coagulation or alternatively activates cell signaling. We recently described that EphA2 of the Eph tyrosine kinase receptor family is cleaved directly by the TF/FVIIa complex. The aim of the present study was to further characterize the cross-talk between TF/FVIIa and EphA2 using in vitro model systems and human cancer specimens. Cleavage and phosphorylation of EphA2 was studied by Western blot. Subcellular localization of TF and EphA2 was investigated by a proximity ligation assay and confocal microscopy. Phalloidin staining of the actin cytoskeleton was used to study cell rounding and retraction fiber formation. Expression of TF and EphA2 in human colorectal cancer specimens was examined by immunohistochemistry. TF and EphA2 co-localized constitutively in MDA-MB-231 cells, and addition of FVIIa resulted in cleavage of EphA2 by a PAR2-independent mechanism. Overexpression of TF in U251 glioblastoma cells lead to co-localization with EphA2 at the leading edge and FVIIa-dependent cleavage of EphA2. FVIIa potentiated ephrin-A1-induced cell rounding and retraction fiber formation in MDA-MB-231 cells through a RhoA/ROCK-dependent pathway that did not require PAR2-activation. TF and EphA2 were expressed in colorectal cancer specimens, and were significantly correlated. These results suggest that TF/FVIIa-EphA2 cross-talk might potentiate ligand-dependent EphA2 signaling in human cancers, and provide initial evidence that it is possible for this interaction to occur in vivo.

  18. Ethanol and Caffeine Effects on Social Interaction and Recognition in Mice: Involvement of Adenosine A2A and A1 Receptors.

    PubMed

    López-Cruz, Laura; San-Miguel, Noemí; Bayarri, Pilar; Baqi, Younis; Müller, Christa E; Salamone, John D; Correa, Mercé

    2016-01-01

    Ethanol and caffeine are frequently consumed in combination and have opposite effects on the adenosine system: ethanol metabolism leads to an increase in adenosine levels, while caffeine is a non-selective adenosine A 1 /A 2A receptor antagonist. These receptors are highly expressed in striatum and olfactory tubercle, brain areas involved in exploration and social interaction in rodents. Ethanol modulates social interaction processes, but the role of adenosine in social behavior is still poorly understood. The present work was undertaken to study the impact of ethanol, caffeine and their combination on social behavior, and to explore the involvement of A 1 and A 2A receptors on those actions. Male CD1 mice were evaluated in a social interaction three-chamber paradigm, for preference of conspecific vs. object, and also for long-term recognition memory of familiar vs. novel conspecific. Ethanol showed a biphasic effect, with low doses (0.25 g/kg) increasing social contact and higher doses (1.0-1.5 g/kg) reducing social interaction. However, no dose changed social preference; mice always spent more time sniffing the conspecific than the object, independently of the ethanol dose. Ethanol, even at doses that did not change social exploration, produced amnestic effects on social recognition the following day. Caffeine reduced social contact (15.0-60.0 mg/kg), and even blocked social preference at higher doses (30.0-60.0 mg/kg). The A 1 antagonist Cyclopentyltheophylline (CPT; 3-9 mg/kg) did not modify social contact or preference on its own, and the A 2A antagonist MSX-3 (1.5-6 mg/kg) increased social interaction at all doses. Ethanol at intermediate doses (0.5-1.0 g/kg) was able to reverse the reduction in social exploration induced by caffeine (15.0-30.0 mg/kg). Although there was no interaction between ethanol and CPT or MSX-3 on social exploration in the first day, MSX-3 blocked the amnestic effects of ethanol observed on the following day. Thus, ethanol impairs the

  19. The Sam-Sam interaction between Ship2 and the EphA2 receptor: design and analysis of peptide inhibitors.

    PubMed

    Mercurio, Flavia Anna; Di Natale, Concetta; Pirone, Luciano; Iannitti, Roberta; Marasco, Daniela; Pedone, Emilia Maria; Palumbo, Rosanna; Leone, Marilisa

    2017-12-12

    The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

  20. EndophilinA2 protects against angiotensin II-induced cardiac hypertrophy by inhibiting angiotensin II type 1 receptor trafficking in neonatal rat cardiomyocytes.

    PubMed

    Liu, Yun; Shen, Huan-Jia; Wang, Xin-Qiu-Yue; Liu, Hai-Qi; Zheng, Ling-Yun; Luo, Jian-Dong

    2018-06-20

    Cardiac hypertrophy is one of the major risk factors for chronic heart failure. The role of endophilinA2 (EndoA2) in clathrin-mediated endocytosis and clathrin-independent endocytosis is well documented. In the present study, we tested the hypothesis that EndoA2 protects against angiotensin II (Ang II)-induced cardiac hypertrophy by mediating intracellular angiotensin II type 1 receptor (AT1-R) trafficking in neonatal rat cardiomyocytes (NRCMs). Cardiac hypertrophy was evaluated by using cell surface area and quantitative RT-PCR (qPCR) analyses. For the first time, we found that EndoA2 attenuated cardiac hypertrophy and fibrosis induced by Ang II. Moreover, EndoA2 inhibited apoptosis induced by excessive endoplasmic reticulum stress (ERS), which accounted for the beneficial effects of EndoA2 on cardiac hypertrophy. We further revealed that there was an interaction between EndoA2 and AT1-R.The expression levels of EndoA2, which inhibits AT1-R transport from the cytoplasm to the membrane, and the interaction between EndoA2 and AT1-R were obviously decreased after Ang II treatment. Furthermore, Ang II inhibited the co-localization of AT1-R with GRP-78, which was reversed by EndoA2 overexpression. In conclusion, our results suggested that EndoA2 plays a role in protecting against cardiac hypertrophy induced by Ang II, possibly by inhibiting AT1-R transport from the cytoplasm to the membrane to suppress signal transduction. © 2018 Wiley Periodicals, Inc.

  1. Environmental Enrichment Increases Glucocorticoid Receptors and Decreases GluA2 and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism?

    PubMed Central

    Zanca, Roseanna M.; Braren, Stephen H.; Maloney, Brigid; Schrott, Lisa M.; Luine, Victoria N.; Serrano, Peter A.

    2015-01-01

    Environmental enrichment (EE) housing paradigms have long been shown beneficial for brain function involving neural growth and activity, learning and memory capacity, and for developing stress resiliency. The expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2, which is important for synaptic plasticity and memory, is increased with corticosterone (CORT), undermining synaptic plasticity and memory. Thus, we determined the effect of EE and stress on modulating GluA2 expression in Sprague-Dawley male rats. Several markers were evaluated which include: plasma CORT, the glucocorticoid receptor (GR), GluA2, and the atypical protein kinase M zeta (PKMζ). For 1 week standard-(ST) or EE-housed animals were treated with one of the following four conditions: (1) no stress; (2) acute stress (forced swim test, FST; on day 7); (3) chronic restraint stress (6 h/day for 7 days); and (4) chronic + acute stress (restraint stress 6 h/day for 7 days + FST on day 7). Hippocampi were collected on day 7. Our results show that EE animals had reduced time immobile on the FST across all conditions. After chronic + acute stress EE animals showed increased GR levels with no change in synaptic GluA2/PKMζ. ST-housed animals showed the reverse pattern with decreased GR levels and a significant increase in synaptic GluA2/PKMζ. These results suggest that EE produces an adaptive response to chronic stress allowing for increased GR levels, which lowers neuronal excitability reducing GluA2/PKMζ trafficking. We discuss this EE adaptive response to stress as a potential underlying mechanism that is protective for retaining synaptic plasticity and memory function. PMID:26617502

  2. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  3. Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism

    PubMed Central

    Mejias, Rebeca; Adamczyk, Abby; Anggono, Victor; Niranjan, Tejasvi; Thomas, Gareth M.; Sharma, Kamal; Skinner, Cindy; Schwartz, Charles E.; Stevenson, Roger E.; Fallin, M. Daniele; Kaufmann, Walter; Pletnikov, Mikhail; Valle, David; Huganir, Richard L.; Wang, Tao

    2011-01-01

    Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) via its PDZ domains 4 to 6 (PDZ4–6). We found an association (P < 0.05) of a SNP within the PDZ4-6 genomic region with autism by genotyping autistic patients (n = 480) and matched controls (n = 480). Parallel sequencing identified five rare missense variants within or near PDZ4–6 only in the autism cohort, resulting in a higher cumulative mutation load (P = 0.032). Two variants correlated with a more severe deficit in reciprocal social interaction in affected sibling pairs from proband families. These variants were associated with altered interactions with GluA2/3 and faster recycling and increased surface distribution of GluA2 in neurons, suggesting gain-of-function because GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout mice exhibited increased sociability and impaired prepulse inhibition. These results support a role for GRIP in social behavior and implicate GRIP1 variants in modulating autistic phenotype. PMID:21383172

  4. Cannabidiol, a non-psychotropic plant-derived cannabinoid, decreases inflammation in a murine model of acute lung injury: role for the adenosine A(2A) receptor.

    PubMed

    Ribeiro, Alison; Ferraz-de-Paula, Viviane; Pinheiro, Milena L; Vitoretti, Luana B; Mariano-Souza, Domenica P; Quinteiro-Filho, Wanderley M; Akamine, Adriana T; Almeida, Vinícius I; Quevedo, João; Dal-Pizzol, Felipe; Hallak, Jaime E; Zuardi, Antônio W; Crippa, José A; Palermo-Neto, João

    2012-03-05

    Acute lung injury is an inflammatory condition for which treatment is mainly supportive because effective therapies have not been developed. Cannabidiol, a non-psychotropic cannabinoid component of marijuana (Cannabis sativa), has potent immunosuppressive and anti-inflammatory properties. Therefore, we investigated the possible anti-inflammatory effect of cannabidiol in a murine model of acute lung injury. Analysis of total inflammatory cells and differential in bronchoalveolar lavage fluid was used to characterize leukocyte migration into the lungs; myeloperoxidase activity of lung tissue and albumin concentration in the bronchoalveolar lavage fluid were analyzed by colorimetric assays; cytokine/chemokine production in the bronchoalveolar lavage fluid was also analyzed by Cytometric Bead Arrays and Enzyme-Linked Immunosorbent Assay (ELISA). A single dose of cannabidiol (20mg/kg) administered prior to the induction of LPS (lipopolysaccharide)-induced acute lung injury decreases leukocyte (specifically neutrophil) migration into the lungs, albumin concentration in the bronchoalveolar lavage fluid, myeloperoxidase activity in the lung tissue, and production of pro-inflammatory cytokines (TNF and IL-6) and chemokines (MCP-1 and MIP-2) 1, 2, and 4days after the induction of LPS-induced acute lung injury. Additionally, adenosine A(2A) receptor is involved in the anti-inflammatory effects of cannabidiol on LPS-induced acute lung injury because ZM241385 (4-(2-[7-Amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol) (a highly selective antagonist of adenosine A(2A) receptor) abrogated all of the anti-inflammatory effects of cannabidiol previously described. Thus, we show that cannabidiol has anti-inflammatory effects in a murine model of acute lung injury and that this effect is most likely associated with an increase in the extracellular adenosine offer and signaling through adenosine A(2A) receptor. Copyright © 2012 Elsevier B.V. All rights

  5. Prolonged Tp-e Interval in Down Syndrome Patients with Congenitally Normal Hearts.

    PubMed

    Kucuk, Mehmet; Karadeniz, Cem; Ozdemir, Rahmi; Meşe, Timur

    2018-03-25

    Heterogeneity of ventricular repolarization has been assessed by using the QT dispersion in Down syndrome (DS) patients with congenitally normal hearts. However, novel repolarization indexes, the Tp-e interval and Tp-e/QT ratio, have not previously been evaluated in these patients. The aim of this study was to evaluate the Tp-e interval and Tp-e/QT ratio in DS patients without congenital heart defects. Twelve-lead surface electrocardiograms of 160 DS patients and 110 age- and sex-matched healthy controls were used to evaluate and compare the Tp-e interval, Tp-e dispersion, and Tp-e/QT ratio. Heart rate, Tp-e interval, Tp-e dispersion, Tp-e/QT and Tp-e/QTc ratios were significantly higher in DS group than in the controls. Myocardial repolarization indexes in DS patients with congenitally normal hearts were found to be prolonged compared to those in normal controls. Further evaluation is warranted to reveal a relationship between prolonged repolarization indexes and arrhythmic events in these patients. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  6. Detection of Heteromers Formed by Cannabinoid CB1, Dopamine D2, and Adenosine A2A G-Protein-Coupled Receptors by Combining Bimolecular Fluorescence Complementation and Bioluminescence Energy Transfer

    PubMed Central

    Navarro, Gemma; Carriba, Paulina; Gandí, Jorge; Ciruela, Francisco; Casadó, Vicent; Cortés, Antoni; Mallol, Josefa; Canela, Enric I.; Lluis, Carmen; Franco, Rafael

    2008-01-01

    Functional interactions in signaling occur between dopamine D2 (D2R) and cannabinoid CB1 (CB1R) receptors, between CB1R and adenosine A2A (A2AR) receptors, and between D2R and A2AR. Furthermore, direct molecular interactions have been reported for the pairs CB1R-D2R, A2AR-D2R, and CB1R-A2AR. Here a combination of bimolecular fluorescence complementation and bioluminescence energy transfer techniques was used to identify the occurrence of D2R-CB1R-A2AR hetero-oligomers in living cells. PMID:18956124

  7. RITA displays anti-tumor activity in medulloblastomas independent of TP53 status.

    PubMed

    Gottlieb, Aline; Althoff, Kristina; Grunewald, Laura; Thor, Theresa; Odersky, Andrea; Schulte, Marc; Deubzer, Hedwig E; Heukamp, Lukas; Eggert, Angelika; Schramm, Alexander; Schulte, Johannes H; Künkele, Annette

    2017-04-25

    Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma.

  8. Synthesis and coordination chemistry of TpC*MI complexes where M=Mg, Ca, Sr, Ba and Zn and TpC*=tris[3-(2-methoxy-1,1-dimethyl)pyrazolyl]hydroborate.

    PubMed

    Chisholm, Malcolm H; Gallucci, Judith C; Yaman, Gulsah

    2009-01-14

    Reactions involving MI2 where M=Mg, Ca, Sr, Ba or Zn and M'TpC* where M'=Na or Tl and TpC*=tris[3-methoxy-1,1-dimethyl)pyrazolyl]hydroborate in tetrahydrofuran are described leading to the isolation and characterization of the complexes TpC*MgI, , TpC*CaI, , TpC*SrI, , TpC*SrI(THF), , TpC*BaI, , TpC*BaI(pz*H), , where pz*H=3-(2-methoxyl-1,1-dimethyl)pyrazole, TpC*BaI.1/2toluene, and TpC*ZnI, . The compounds , , , , and have been characterized by single-crystal X-ray crystallography. Compounds and are isostructural and are salt-like containing kappa6-TpM+ cations and I- anions. In all other structures, the iodide is bound to the metal and TpC* is kappa6 bonded to the group 2 M(2+) ions. Reactions involving TpC*CaI, , and sodium or lithium alkoxides or amides failed to yield the amide or alkoxide calcium TpC* derivative, though related reactions involving TpC*ZnI, , and KOSiMe3 proceeded quantitatively to yield kappa3TpC*ZnOSiMe3, , which was also structurally characterized and shown to have the kappa3-TpC* bound ligand.

  9. Excess adenosine in murine penile erectile tissues contributes to priapism via A2B adenosine receptor signaling

    PubMed Central

    Mi, Tiejuan; Abbasi, Shahrzad; Zhang, Hong; Uray, Karen; Chunn, Janci L.; Xia, Ling Wei; Molina, Jose G.; Weisbrodt, Norman W.; Kellems, Rodney E.; Blackburn, Michael R.; Xia, Yang

    2008-01-01

    Priapism, abnormally prolonged penile erection in the absence of sexual excitation, is associated with ischemia-mediated erectile tissue damage and subsequent erectile dysfunction. It is common among males with sickle cell disease (SCD), and SCD transgenic mice are an accepted model of the disorder. Current strategies to manage priapism suffer from a poor fundamental understanding of the molecular mechanisms underlying the disorder. Here we report that mice lacking adenosine deaminase (ADA), an enzyme necessary for the breakdown of adenosine, displayed unexpected priapic activity. ADA enzyme therapy successfully corrected the priapic activity both in vivo and in vitro, suggesting that it was dependent on elevated adenosine levels. Further genetic and pharmacologic evidence demonstrated that A2B adenosine receptor–mediated (A2BR-mediated) cAMP and cGMP induction was required for elevated adenosine–induced prolonged penile erection. Finally, priapic activity in SCD transgenic mice was also caused by elevated adenosine levels and A2BR activation. Thus, we have shown that excessive adenosine accumulation in the penis contributes to priapism through increased A2BR signaling in both Ada–/– and SCD transgenic mice. These findings provide insight regarding the molecular basis of priapism and suggest that strategies to either reduce adenosine or block A2BR activation may prove beneficial in the treatment of this disorder. PMID:18340377

  10. Adenosine A(2A) receptor antagonists are broad facilitators of antinicotinic neuromuscular blockade monitored either with 2 Hz train-of-four or 50 Hz tetanic stimuli.

    PubMed

    Pereira, Monalisa W; Correia-de-Sá, Paulo; Alves-Do-Prado, Wilson

    2012-10-01

    1. The 2 Hz train-of-four ratio (TOF(ratio)) is used to monitor the degree of patient curarization. Using a rat phrenic nerve-hemidiaphragm preparation, we showed that antinicotinic agents, such as hexamethonium, d-tubocurarine and pancuronium, but not cisatracurium, decreased contractions produced by physiological nerve activity patterns (50 Hz) more efficiently than those caused by 2 Hz trains. Uncertainty about the usefulness of the TOF(ratio) to control safe recovery from curarization prompted us to investigate the muscarinic and adenosine neuromodulation of tetanic (50 Hz) fade induced by antinicotinic agents at concentrations that cause a 25% reduction in the TOF(ratio) (TOF(fade)). 2. Tetanic fade caused by d-tubocurarine (1.1 μmol/L), pancuronium (3 μmol/L) and hexamethonium (5.47 mmol/L) was attenuated by blocking presynaptic inhibitory muscarinic M(2) and adenosine A(1) receptors with methoctramine (1 μmol/L) and 1,3-dipropyl-8-cyclopentylxanthine (2.5 nmol/L), respectively. These compounds enhanced rather than decreased tetanic fade induced by cisatracurium (2.2 μmol/L), but they consistently attenuated cisatracurium-induced TOF(fade). The effect of the M(1) receptor antagonist pirenzepine (10 nmol/L) on fade produced by antinicotinic agents at 50 Hz was opposite to that observed with TOF stimulation. Blockade of adenosine A(2A) receptors with ZM 241385 (10 nmol/L) attenuated TOF(fade) caused by all antinicotinic drugs tested, with the exception of the 'pure' presynaptic nicotinic antagonist hexamethonium. ZM 241385 was the only compound tested in this series that facilitated recovery from tetanic fade produced by cisatracurium. 3. The data suggest that distinct antinicotinic relaxants interfere with fine-tuning neuromuscular adaptations to motor nerve stimulation patterns via activation of presynaptic muscarinic and adenosine receptors. These results support the use of A(2A) receptor antagonists together with atropine to facilitate recovery from

  11. Structure-Activity Relationships of Truncated C2- or C8-Substituted Adenosine Derivatives as Dual Acting A2A and A3 Adenosine Receptor Ligands

    PubMed Central

    Hou, Xiyan; Majik, Mahesh S.; Kim, Kyunglim; Pyee, Yuna; Lee, Yoonji; Alexander, Varughese; Chung, Hwa-Jin; Lee, Hyuk Woo; Chandra, Girish; Lee, Jin Hee; Park, Seul-gi; Choi, Won Jun; Kim, Hea Ok; Phan, Khai; Gao, Zhan-Guo; Jacobson, Kenneth A.; Choi, Sun; Lee, Sang Kook; Jeong, Lak Shin

    2011-01-01

    Truncated N6-substituted-4′-oxo- and 4′-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A2A and A3 adenosine receptor (AR) ligands. The lithiation-mediated stannyl transfer and palladium-catalyzed cross coupling reactions were utilized for functionalization of the C2 position of 6-chloropurine nucleosides. An unsubstituted 6-amino group and a hydrophobic C2 substituent were required for high affinity at the hA2AAR, but hydrophobic C8 substitution abolished binding at the hA2AAR. However, most of synthesized compounds displayed medium to high binding affinity at the hA3AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA2AAR agonists. C2 substitution probed geometrically through hA2AAR-docking, was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA2AAR agonist and hA3AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases. PMID:22142423

  12. Author Correction: EphA2 is an epithelial cell pattern recognition receptor for fungal β-glucans.

    PubMed

    Swidergall, Marc; Solis, Norma V; Lionakis, Michail S; Filler, Scott G

    2018-06-12

    In the version of this Article originally published, the authors described the ANT compound used in their study as 4-(2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid (ANT). The authors now wish to clarify that the ANT compound used was actually a 2,5-dimethylpyrrolyl benzoic acid derivative 1 that has been shown to inhibit not only the enzymatic activity of EphA2, but also several unrelated enzymes 2 . The description of the compound in the Article has now been changed to 4-(2,5-dimethyl-1H-pyrrol-1-yl)-2-hydroxybenzoic acid derivative (ANT) to reflect this.

  13. HPV-negative penile squamous cell carcinoma: disruptive mutations in the TP53 gene are common.

    PubMed

    Kashofer, Karl; Winter, Elke; Halbwedl, Iris; Thueringer, Andrea; Kreiner, Marisa; Sauer, Stefan; Regauer, Sigrid

    2017-07-01

    The majority of penile squamous cell carcinomas is caused by transforming human papilloma virus (HPV) infection. The etiology of HPV-negative cancers is unclear, but TP53 mutations have been implicated. Archival tissues of 108 invasive squamous cell carcinoma from a single pathology institution in a low-incidence area were analyzed for HPV-DNA and p16 ink4a overexpression and for TP53 mutations by ion torrent next-generation sequencing. Library preparation failed in 32/108 squamous cell carcinomas. Institutional review board approval was obtained. Thirty of 76 squamous cell carcinomas (43%; average 63 years) were HPV-negative with 8/33 squamous cell carcinomas being TP53 wild-type (24%; average 63 years). Twenty-five of 33 squamous cell carcinomas (76%; average 65 years) showed 32 different somatic TP53 mutations (23 missense mutations in exons 5-8, 6 nonsense, 1 frameshift and 2 splice-site mutations). Several hotspot mutations were detected multiple times (R175H, R248, R282, and R273). Eighteen of 19 squamous cell carcinomas with TP53 expression in immunohistochemistry had TP53 mutations. Fifty percent of TP53-negative squamous cell carcinomas showed mostly truncating loss-of-function TP53 mutations. Patients without mutations had longer survival (5 years: 86% vs 61%; 10 years: 60% vs 22%), but valid clinically relevant conclusions cannot be drawn due to different tumor stages and heterogeneous treatment of the cases presented in this study. Somatic TP53 mutations are a common feature in HPV-negative penile squamous cell carcinomas and offer an explanation for HPV-independent penile carcinogenesis. About half of HPV-negative penile cancers are driven by oncogenic activation of TP53, while a quarter is induced by loss of TP53 tumor suppressor function. Detection of TP53 mutations should be carried out by sequencing, as immunohistochemical TP53 staining could not identify all squamous cell carcinomas with TP53 mutations.

  14. Spatiotemporal brain dynamics of emotional face processing modulations induced by the serotonin 1A/2A receptor agonist psilocybin.

    PubMed

    Bernasconi, Fosco; Schmidt, André; Pokorny, Thomas; Kometer, Michael; Seifritz, Erich; Vollenweider, Franz X

    2014-12-01

    Emotional face processing is critically modulated by the serotonergic system. For instance, emotional face processing is impaired by acute psilocybin administration, a serotonin (5-HT) 1A and 2A receptor agonist. However, the spatiotemporal brain mechanisms underlying these modulations are poorly understood. Here, we investigated the spatiotemporal brain dynamics underlying psilocybin-induced modulations during emotional face processing. Electrical neuroimaging analyses were applied to visual evoked potentials in response to emotional faces, following psilocybin and placebo administration. Our results indicate a first time period of strength (i.e., Global Field Power) modulation over the 168-189 ms poststimulus interval, induced by psilocybin. A second time period of strength modulation was identified over the 211-242 ms poststimulus interval. Source estimations over these 2 time periods further revealed decreased activity in response to both neutral and fearful faces within limbic areas, including amygdala and parahippocampal gyrus, and the right temporal cortex over the 168-189 ms interval, and reduced activity in response to happy faces within limbic and right temporo-occipital brain areas over the 211-242 ms interval. Our results indicate a selective and temporally dissociable effect of psilocybin on the neuronal correlates of emotional face processing, consistent with a modulation of the top-down control. © The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Analytical and clinical comparison of Elecsys syphilis (Roche®) - Architect syphilis TP and reformulated Architect syphilis TP (Abbott®) assay.

    PubMed

    De Keukeleire, Steven; Desmet, Stefanie; Lagrou, Katrien; Oosterlynck, Julie; Verhulst, Manon; Van Besien, Jessica; Saegeman, Veroniek; Reynders, Marijke

    2017-03-01

    The performance of Elecsys Syphilis was compared to Architect Syphilis TP and Reformulated Architect Syphilis TP. The overall sensitivity and specificity were 98.4% and 99.5%, 97.7% and 97.1%, and 99.2% and 99.7% respectively. The assays are comparable and considered adequate for syphilis screening. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine

    PubMed Central

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B.

    2009-01-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O2C) were studied in awake mice lacking one or both of the adenosine A1 or A2A receptors (A1R or A2AR, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A1R knockout (A1RKO) mice but lower in A2ARKO mice and intermediate in A1-A2AR double KO mice. A single dose of an unselective β-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A1Rs had little effect on Temp, whereas deletion of A2ARs increased it in females and decreased it in males. A1-A2ARKO mice had lower Temp than WT mice. LA was unaltered in A1RKO mice and lower in A2ARKO and A1-A2ARKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A2AR. Caffeine ingestion also increased LA in an A2AR-dependent manner in male mice. Caffeine ingestion significantly increased O2C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A1R or A2AR blockade. Selective A2B antagonism had little or no effect. Thus A1R and A2AR influence HR, Temp, LA, and O2C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A2AR plays an important role in the modulation of O2C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A1R and A2AR. PMID:19218506

  17. Physiological roles of A1 and A2A adenosine receptors in regulating heart rate, body temperature, and locomotion as revealed using knockout mice and caffeine.

    PubMed

    Yang, Jiang-Ning; Chen, Jiang-Fan; Fredholm, Bertil B

    2009-04-01

    Heart rate (HR), body temperature (Temp), locomotor activity (LA), and oxygen consumption (O(2)C) were studied in awake mice lacking one or both of the adenosine A(1) or A(2A) receptors (A(1)R or A(2A)R, respectively) using telemetry and respirometry, before and after caffeine administration. All parameters were lower during day than night and higher in females than males. When compared with wild-type (WT) littermates, HR was higher in male A(1)R knockout (A(1)RKO) mice but lower in A(2A)RKO mice and intermediate in A(1)-A(2A)R double KO mice. A single dose of an unselective beta-blocker (timolol; 1 mg/kg) abolished the HR differences between these genotypes. Deletion of A(1)Rs had little effect on Temp, whereas deletion of A(2A)Rs increased it in females and decreased it in males. A(1)-A(2A)RKO mice had lower Temp than WT mice. LA was unaltered in A(1)RKO mice and lower in A(2A)RKO and A(1)-A(2A)RKO mice than in WT mice. Caffeine injection increased LA but only in mice expressing A(2A)R. Caffeine ingestion also increased LA in an A(2A)R-dependent manner in male mice. Caffeine ingestion significantly increased O(2)C in WT mice, but less in the different KO mice. Injection of 30 mg/kg caffeine decreased Temp, especially in KO mice, and hence in a manner unrelated to A(1)R or A(2A)R blockade. Selective A(2B) antagonism had little or no effect. Thus A(1)R and A(2A)R influence HR, Temp, LA, and O(2)C in mice in a sex-dependent manner, indicating effects of endogenous adenosine. The A(2A)R plays an important role in the modulation of O(2)C and LA by acute and chronic caffeine administration. There is also evidence for effects of higher doses of caffeine being independent of both A(1)R and A(2A)R.

  18. [Induction chemotherapy with docetaxel plus cisplatin (TP regimen) followed by concurrent chemoradiotherapy with TP regimen versus cisplatin in treating locally advanced nasopharyngeal carcinoma].

    PubMed

    Xie, Fang-Yun; Zou, Guo-Rong; Hu, Wei-Han; Qi, Shu-Nan; Peng, Miao; Li, Ji-Shi

    2009-03-01

    Clinical trials on docetaxel plus cisplatin (DDP) (TP regimen) in treating nasopharyngeal carcinoma (NPC) are still uncertain due to limited samples. This study was to compare the short-term efficacy and toxicity of induction chemotherapy with TP regimen followed by concurrent chemoradiotherapy with TP regimen versus DDP in treating locally advanced NPC. Fifty-seven patients with stage T3-4N2-3M0 NPC diagnosed pathologically from December 2005 to December 2006 were randomized into TP group (30 patients) and DDP group (27 patients). Both groups received TP regimen as induction chemotherapy with docetaxel (70 mg/m(2)) on Day 1 and DDP (80 mg/m(2)) on Day 2, repeating every 21 days for 2 cycles. For concurrent chemotherapy, TP group were administered docetaxel (60 mg/m(2)) on Day 1 and DDP (80 mg/m(2)) on Day 2; DDP group were administered DDP (80 mg/m(2)) on Day 1. Both schedules were repeated every 21 days for 2 cycles. Linear accelerator was used as radioactive source. Irradiation field was designed with CT-simulation and conventional fractions. The 57 patients received 111 cycles of induction chemotherapy, and 53 of them received 103 cycles of concurrent chemotherapy; four patients ceased induction chemotherapy and three ceased concurrent chemotherapy. All patients completed radiotherapy. The major toxicity of induction chemotherapy was hematologic toxicity; the main toxicities of concurrent chemoradiotherapy were hematologic toxicity and mucositis. The occurrence rates of Grade 3-4 leucopenia and Grade 3-4 neutropenia were significantly higher in TP group than in DDP groups (p <0.05). In concurrent chemoradiotherapy, the application rate of granulocyte colony stimulating factor (G-CSF) was significantly higher in TP group than in DDP group (100% vs. 72.0%, p<0.05). After concurrent chemoradiotherapy, the complete remission (CR) rates of the nasopharynx and regional lymph nodes were 93.3% and 92.9% in TP group, and were 96.3% and 91.3% in DDP group (p>0.05). The

  19. Characterisation of endogenous A2A and A2B receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A2B-selective antagonist PSB 603.

    PubMed

    Goulding, Joelle; May, Lauren T; Hill, Stephen J

    2018-01-01

    Endogenous adenosine A 2B receptors (A 2B AR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the A 2B AR antagonist PSB 603 in HEK 293 cells. The A 2A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A 2A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective A 2A AR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E MAX with no significant effect on EC 50 values. Kinetics analysis of the effect of PSB 603 on the A 2B AR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of A 2B AR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. The TP53 gene polymorphisms and survival of sporadic breast cancer patients.

    PubMed

    Bišof, V; Salihović, M Peričić; Narančić, N Smolej; Skarić-Jurić, T; Jakić-Razumović, J; Janićijević, B; Rudan, P

    2012-06-01

    The TP53 gene polymorphisms, Arg72Pro and PIN3 (+16 bp), can have prognostic and predictive value in different cancers including breast cancer. The aim of the present study is to investigate a potential association between different genotypes of these polymorphisms and clinicopathological variables with survival of breast cancer patients in Croatian population. Ninety-four women with sporadic breast cancer were retrospectively analyzed. Median follow-up period was 67.9 months. The effects of basic clinical and histopathological characteristics of tumor on survival were tested by Cox's proportional hazards regression analysis. The TNM stage was associated with overall survival by Kaplan-Meier analysis, univariate, and multivariate Cox's proportional hazards regression analysis, while grade was associated with survival by Kaplan-Meier analysis and univariate Cox's proportional hazards regression analysis. Different genotypes of the Arg72Pro and PIN3 (+16 bp) polymorphisms had no significant impact on survival in breast cancer patients. However, in subgroup of patients treated with chemotherapy without anthracycline, the A2A2 genotype of the PIN3 (+16 bp) polymorphism was associated with poorer overall survival than other genotypes by Kaplan-Meier analysis (P = 0.048). The TP53 polymorphisms, Arg72Pro and PIN3 (+16 bp), had no impact on survival in unselected sporadic breast cancer patients in Croatian population. However, the results support the role of the A2A2 genotype of the PIN3 (+16 bp) polymorphism as a marker for identification of patients that may benefit from anthracycline-containing chemotherapy.

  1. The nuclear hormone receptor family member NR5A2 controls aspects of multipotent progenitor cell formation and acinar differentiation during pancreatic organogenesis.

    PubMed

    Hale, Michael A; Swift, Galvin H; Hoang, Chinh Q; Deering, Tye G; Masui, Toshi; Lee, Youn-Kyoung; Xue, Jumin; MacDonald, Raymond J

    2014-08-01

    The orphan nuclear receptor NR5A2 is necessary for the stem-like properties of the epiblast of the pre-gastrulation embryo and for cellular and physiological homeostasis of endoderm-derived organs postnatally. Using conditional gene inactivation, we show that Nr5a2 also plays crucial regulatory roles during organogenesis. During the formation of the pancreas, Nr5a2 is necessary for the expansion of the nascent pancreatic epithelium, for the subsequent formation of the multipotent progenitor cell (MPC) population that gives rise to pre-acinar cells and bipotent cells with ductal and islet endocrine potential, and for the formation and differentiation of acinar cells. At birth, the NR5A2-deficient pancreas has defects in all three epithelial tissues: a partial loss of endocrine cells, a disrupted ductal tree and a >90% deficit of acini. The acinar defects are due to a combination of fewer MPCs, deficient allocation of those MPCs to pre-acinar fate, disruption of acinar morphogenesis and incomplete acinar cell differentiation. NR5A2 controls these developmental processes directly as well as through regulatory interactions with other pancreatic transcriptional regulators, including PTF1A, MYC, GATA4, FOXA2, RBPJL and MIST1 (BHLHA15). In particular, Nr5a2 and Ptf1a establish mutually reinforcing regulatory interactions and collaborate to control developmentally regulated pancreatic genes by binding to shared transcriptional regulatory regions. At the final stage of acinar cell development, the absence of NR5A2 affects the expression of Ptf1a and its acinar specific partner Rbpjl, so that the few acinar cells that form do not complete differentiation. Nr5a2 controls several temporally distinct stages of pancreatic development that involve regulatory mechanisms relevant to pancreatic oncogenesis and the maintenance of the exocrine phenotype. © 2014. Published by The Company of Biologists Ltd.

  2. WNT activation by lithium abrogates TP53 mutation associated radiation resistance in medulloblastoma.

    PubMed

    Zhukova, Nataliya; Ramaswamy, Vijay; Remke, Marc; Martin, Dianna C; Castelo-Branco, Pedro; Zhang, Cindy H; Fraser, Michael; Tse, Ken; Poon, Raymond; Shih, David J H; Baskin, Berivan; Ray, Peter N; Bouffet, Eric; Dirks, Peter; von Bueren, Andre O; Pfaff, Elke; Korshunov, Andrey; Jones, David T W; Northcott, Paul A; Kool, Marcel; Pugh, Trevor J; Pomeroy, Scott L; Cho, Yoon-Jae; Pietsch, Torsten; Gessi, Marco; Rutkowski, Stefan; Bognár, Laszlo; Cho, Byung-Kyu; Eberhart, Charles G; Conter, Cecile Faure; Fouladi, Maryam; French, Pim J; Grajkowska, Wieslawa A; Gupta, Nalin; Hauser, Peter; Jabado, Nada; Vasiljevic, Alexandre; Jung, Shin; Kim, Seung-Ki; Klekner, Almos; Kumabe, Toshihiro; Lach, Boleslaw; Leonard, Jeffrey R; Liau, Linda M; Massimi, Luca; Pollack, Ian F; Ra, Young Shin; Rubin, Joshua B; Van Meir, Erwin G; Wang, Kyu-Chang; Weiss, William A; Zitterbart, Karel; Bristow, Robert G; Alman, Benjamin; Hawkins, Cynthia E; Malkin, David; Clifford, Steven C; Pfister, Stefan M; Taylor, Michael D; Tabori, Uri

    2014-12-24

    TP53 mutations confer subgroup specific poor survival for children with medulloblastoma. We hypothesized that WNT activation which is associated with improved survival for such children abrogates TP53 related radioresistance and can be used to sensitize TP53 mutant tumors for radiation. We examined the subgroup-specific role of TP53 mutations in a cohort of 314 patients treated with radiation. TP53 wild-type or mutant human medulloblastoma cell-lines and normal neural stem cells were used to test radioresistance of TP53 mutations and the radiosensitizing effect of WNT activation on tumors and the developing brain. Children with WNT/TP53 mutant medulloblastoma had higher 5-year survival than those with SHH/TP53 mutant tumours (100% and 36.6%±8.7%, respectively (p<0.001)). Introduction of TP53 mutation into medulloblastoma cells induced radioresistance (survival fractions at 2Gy (SF2) of 89%±2% vs. 57.4%±1.8% (p<0.01)). In contrast, β-catenin mutation sensitized TP53 mutant cells to radiation (p<0.05). Lithium, an activator of the WNT pathway, sensitized TP53 mutant medulloblastoma to radiation (SF2 of 43.5%±1.5% in lithium treated cells vs. 56.6±3% (p<0.01)) accompanied by increased number of γH2AX foci. Normal neural stem cells were protected from lithium induced radiation damage (SF2 of 33%±8% for lithium treated cells vs. 27%±3% for untreated controls (p=0.05). Poor survival of patients with TP53 mutant medulloblastoma may be related to radiation resistance. Since constitutive activation of the WNT pathway by lithium sensitizes TP53 mutant medulloblastoma cells and protect normal neural stem cells from radiation, this oral drug may represent an attractive novel therapy for high-risk medulloblastomas.

  3. The receptor tyrosine kinase EphA2 promotes glutamine metabolism in tumors by activating the transcriptional coactivators YAP and TAZ.

    PubMed

    Edwards, Deanna N; Ngwa, Verra M; Wang, Shan; Shiuan, Eileen; Brantley-Sieders, Dana M; Kim, Laura C; Reynolds, Albert B; Chen, Jin

    2017-12-05

    Malignant tumors reprogram cellular metabolism to support cancer cell proliferation and survival. Although most cancers depend on a high rate of aerobic glycolysis, many cancer cells also display addiction to glutamine. Glutamine transporters and glutaminase activity are critical for glutamine metabolism in tumor cells. We found that the receptor tyrosine kinase EphA2 activated the TEAD family transcriptional coactivators YAP and TAZ (YAP/TAZ), likely in a ligand-independent manner, to promote glutamine metabolism in cells and mouse models of HER2-positive breast cancer. Overexpression of EphA2 induced the nuclear accumulation of YAP and TAZ and increased the expression of YAP/TAZ target genes. Inhibition of the GTPase Rho or the kinase ROCK abolished EphA2-dependent YAP/TAZ nuclear localization. Silencing YAP or TAZ substantially reduced the amount of intracellular glutamate through decreased expression of SLC1A5 and GLS , respectively, genes that encode proteins that promote glutamine uptake and metabolism. The regulatory DNA elements of both SLC1A5 and GLS contain TEAD binding sites and were bound by TEAD4 in an EphA2-dependent manner. In patient breast cancer tissues, EphA2 expression positively correlated with that of YAP and TAZ , as well as that of GLS and SLC1A5 Although high expression of EphA2 predicted enhanced metastatic potential and poor patient survival, it also rendered HER2-positive breast cancer cells more sensitive to glutaminase inhibition. The findings define a previously unknown mechanism of EphA2-mediated glutaminolysis through YAP/TAZ activation in HER2-positive breast cancer and identify potential therapeutic targets in patients. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  4. Caffeine Reverts Memory But Not Mood Impairment in a Depression-Prone Mouse Strain with Up-Regulated Adenosine A2A Receptor in Hippocampal Glutamate Synapses.

    PubMed

    Machado, Nuno J; Simões, Ana Patrícia; Silva, Henrique B; Ardais, Ana Paula; Kaster, Manuella P; Garção, Pedro; Rodrigues, Diana I; Pochmann, Daniela; Santos, Ana Isabel; Araújo, Inês M; Porciúncula, Lisiane O; Tomé, Ângelo R; Köfalvi, Attila; Vaugeois, Jean-Marie; Agostinho, Paula; El Yacoubi, Malika; Cunha, Rodrigo A; Gomes, Catarina A

    2017-03-01

    Caffeine prophylactically prevents mood and memory impairments through adenosine A 2A receptor (A 2A R) antagonism. A 2A R antagonists also therapeutically revert mood and memory impairments, but it is not known if caffeine is also therapeutically or only prophylactically effective. Since depression is accompanied by mood and memory alterations, we now explored if chronic (4 weeks) caffeine consumption (0.3 g/L) reverts mood and memory impairment in helpless mice (HM, 12 weeks old), a bred-based model of depression. HM displayed higher immobility in the tail suspension and forced swimming tests, greater anxiety in the elevated plus maze, and poorer memory performance (modified Y-maze and object recognition). HM also had reduced density of synaptic (synaptophysin, SNAP-25), namely, glutamatergic (vGluT1; -22 ± 7 %) and GABAergic (vGAT; -23 ± 8 %) markers in the hippocampus. HM displayed higher A 2A R density (72 ± 6 %) in hippocampal synapses, an enhanced facilitation of hippocampal glutamate release by the A 2A R agonist, CGS21680 (30 nM), and a larger LTP amplitude (54 ± 8 % vs. 21 ± 5 % in controls) that was restored to control levels (30 ± 10 %) by the A 2A R antagonist, SCH58261 (50 nM). Notably, caffeine intake reverted memory deficits and reverted the loss of hippocampal synaptic markers but did not affect helpless or anxiety behavior. These results reinforce the validity of HM as an animal model of depression by showing that they also display reference memory deficits. Furthermore, caffeine intake selectively reverted memory but not mood deficits displayed by HM, which are associated with an increased density and functional impact of hippocampal A 2A R controlling synaptic glutamatergic function.

  5. A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases.

    PubMed

    Abdullah, Nor Linda; Mohd-Zin, Siti W; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M

    2017-01-01

    Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2 tm1Jrui/+ Epha4 rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2 tm1Jrui/+ Epha4 rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent.

  6. A Novel Occulta-Type Spina Bifida Mediated by Murine Double Heterozygotes EphA2 and EphA4 Receptor Tyrosine Kinases

    PubMed Central

    Abdullah, Nor Linda; Mohd-Zin, Siti W.; Ahmad-Annuar, Azlina; Abdul-Aziz, Noraishah M.

    2017-01-01

    Members of the Eph receptor tyrosine kinase have previously been implicated in cranial neural tube development. Failure of neural tube closure leads to the devastating conditions known as anencephaly and spina bifida. EphA2 and EphA4 are expressed at the tips of the closing spinal neural folds prior and during neural tube closure. We investigated the possible role of murine EphA2 and EphA4 during the last step of primary neural tube closure, which is adhesion and fusion. The individual mouse knockouts of EphA2 and EphA4 per se do not exhibit neural tube defects (NTDs). The embryos generated by the crossing of double heterozygotes Epha2tm1Jrui/+Epha4rb-2J/+ displayed NTDs with a wide degree of severity including close exencephaly and close spina bifida (spina bifida occulta). Interestingly, mutants displaying NTDs had skin covering the underlying lesion. The tissue sections revealed the elevated neural folds had not adhered and fused. The phenotypes seen in Epha2tm1Jrui/+Epha4rb-2J/+ double heterozygous embryos suggest both genes play a compensatory role with each other in the adhesion and fusion of the neural tube. In this study, there exists a >50% penetrance of NTDs in the mouse mutants, which genetically have a single allele each of EphA2 and EphA4 absent. PMID:29312933

  7. High coffee intake, but not caffeine, is associated with reduced estrogen receptor negative and postmenopausal breast cancer risk with no effect modification by CYP1A2 genotype.

    PubMed

    Lowcock, Elizabeth C; Cotterchio, Michelle; Anderson, Laura N; Boucher, Beatrice A; El-Sohemy, Ahmed

    2013-01-01

    Associations between caffeine and coffee consumption and breast cancer risk are uncertain, with studies suggesting inverse and null associations. Variation in cytochrome P450 1A2 (CYP1A2), a gene responsible for caffeine metabolism, may modify these associations. Cases (n = 3,062) were recruited through the Ontario Cancer Registry and controls (n = 3,427) through random digit dialing. Logistic regression was used to evaluate associations between breast cancer risk and intakes of 7 caffeine-containing items and total caffeine, and examine whether a genetic variant in CYP1A2 (rs762551) modified these associations. Analyses were stratified by estrogen receptor (ER), menopausal, and smoking status. Generally, coffee and caffeine were not associated with breast cancer risk; however, a significant reduction in risk was observed with the highest category of coffee consumption [≥5 cups per day vs. never, multivariate-adjusted odds ratio (MVOR) = 0.71, 95% confidence interval (CI): 0.51, 0.98]. Variant rs762551 did not modify associations. In stratified analyses, high coffee intake was associated with reduced risk of ER- (MVOR = 0.41, 95% CI: 0.19, 0.92) and postmenopausal breast cancer (MVOR = 0.63, 95% CI: 0.43, 0.94). High coffee consumption, but not total caffeine, may be associated with reduced risk of ER- and postmenopausal breast cancers, independent of CYP1A2 genotype. Further studies are needed to replicate these findings.

  8. Attenuation of nicotine's discriminative stimulus effects in rats and its locomotor activity effects in mice by serotonergic 5-HT2A/2C receptor agonists.

    PubMed

    Batman, Angela M; Munzar, Patrik; Beardsley, Patrick M

    2005-05-01

    Reports have indicated that administration of nicotine inhibits, while withdrawal of chronically administered nicotine augments effects of serotonergic 5HT2A/2C agonists. It was our objective to determine whether 5HT2A/2C agonists can modulate the discriminative stimulus effects of nicotine in rats or its locomotor activity effects in mice. Adult male Sprague-Dawley rats were trained to discriminate 0.3 mg/kg nicotine base from saline in a two-lever, fixed-ratio (FR10), food-reinforced, operant-conditioning task during daily (Monday-Friday) 15-min experimental sessions. After characterizing a dose-response curve for nicotine, we tested the ability of the 5HT(2A/2C) agonists (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCL (DOI; 0.18-1.0 mg/kg) and 1-(4-bromo-2, 5-dimethoxyphenyl)-2-aminopropane (DOB; 0.1-1.0 mg/kg), the 5HT2C agonist 6-chloro-2-(1-piperazinyl)pyrazine hydrochloride (MK 212; 0.1 mg/kg-1.0 mg/kg), and the 5HT1A agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT; 0.01 mg/kg-1.0 mg/kg) to modulate nicotine's discriminative stimulus effects. After finding that DOI was able to attenuate the percentage nicotine lever responding (%NLR), we tested for it to also reverse nicotine's effects on locomotor activity in mice. The 5HT2A/2C agonists-in particular DOI-dose dependently attenuated %NLR. The effects of DOI were reversed by the 5HT2A/2C antagonist ketanserin. MK 212 and 8-OH-DPAT had irregular effects among rats and only reduced %NLR to below 50% levels at doses markedly suppressing responding. DOI also dose dependently blocked nicotine's acute rate-lowering locomotor activity effects. These results indicate that activation of serotonin 5HT2A/2C receptors can blunt the discriminative stimulus and locomotor activity effects of nicotine and presents the possibility that activation of these receptors might also be able to attenuate other effects of nicotine.

  9. Beneficial Role of Erythrocyte Adenosine A2B Receptor-Mediated AMP-Activated Protein Kinase Activation in High-Altitude Hypoxia.

    PubMed

    Liu, Hong; Zhang, Yujin; Wu, Hongyu; D'Alessandro, Angelo; Yegutkin, Gennady G; Song, Anren; Sun, Kaiqi; Li, Jessica; Cheng, Ning-Yuan; Huang, Aji; Edward Wen, Yuan; Weng, Ting Ting; Luo, Fayong; Nemkov, Travis; Sun, Hong; Kellems, Rodney E; Karmouty-Quintana, Harry; Hansen, Kirk C; Zhao, Bihong; Subudhi, Andrew W; Jameson-Van Houten, Sonja; Julian, Colleen G; Lovering, Andrew T; Eltzschig, Holger K; Blackburn, Michael R; Roach, Robert C; Xia, Yang

    2016-08-02

    High altitude is a challenging condition caused by insufficient oxygen supply. Inability to adjust to hypoxia may lead to pulmonary edema, stroke, cardiovascular dysfunction, and even death. Thus, understanding the molecular basis of adaptation to high altitude may reveal novel therapeutics to counteract the detrimental consequences of hypoxia. Using high-throughput, unbiased metabolomic profiling, we report that the metabolic pathway responsible for production of erythrocyte 2,3-bisphosphoglycerate (2,3-BPG), a negative allosteric regulator of hemoglobin-O2 binding affinity, was significantly induced in 21 healthy humans within 2 hours of arrival at 5260 m and further increased after 16 days at 5260 m. This finding led us to discover that plasma adenosine concentrations and soluble CD73 activity rapidly increased at high altitude and were associated with elevated erythrocyte 2,3-BPG levels and O2 releasing capacity. Mouse genetic studies demonstrated that elevated CD73 contributed to hypoxia-induced adenosine accumulation and that elevated adenosine-mediated erythrocyte A2B adenosine receptor activation was beneficial by inducing 2,3-BPG production and triggering O2 release to prevent multiple tissue hypoxia, inflammation, and pulmonary vascular leakage. Mechanistically, we demonstrated that erythrocyte AMP-activated protein kinase was activated in humans at high altitude and that AMP-activated protein kinase is a key protein functioning downstream of the A2B adenosine receptor, phosphorylating and activating BPG mutase and thus inducing 2,3-BPG production and O2 release from erythrocytes. Significantly, preclinical studies demonstrated that activation of AMP-activated protein kinase enhanced BPG mutase activation, 2,3-BPG production, and O2 release capacity in CD73-deficient mice, in erythrocyte-specific A2B adenosine receptor knockouts, and in wild-type mice and in turn reduced tissue hypoxia and inflammation. Together, human and mouse studies reveal novel

  10. The effect of adenosine A(2A) receptor antagonists on hydroxyl radical, dopamine, and glutamate in the striatum of rats with altered function of VMAT2.

    PubMed

    Gołembiowska, Krystyna; Dziubina, Anna

    2012-08-01

    It has been shown that a decreased vesicular monoamine transporter (VMAT2) function and the disruption of dopamine (DA) storage is an early contributor to oxidative damage of dopamine neurons in Parkinson's disease (PD). In our previous study, we demonstrated that adenosine A(2A) receptor antagonists suppressed oxidative stress in 6-hydroxydopamine-treated rats suggesting that this effect may account for neuroprotective properties of drugs. In the present study, rats were injected with reserpine (10 mg/kg sc) and 18 h later the effect of the adenosine A(2A) receptor antagonists 8-(3-chlorostyryl)caffeine (CSC) and 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385) on extracellular DA, glutamate and hydroxyl radical formation was studied in the rat striatum using in vivo microdialysis. By disrupting VMAT2 function, reserpine depleted DA stores, and increased glutamate and hydroxyl radical levels in the rat striatum. CSC (1 mg/kg) but not ZM 241385 (3 mg/kg) increased extracellular DA level and production of hydroxyl radical in reserpinised rats. Both antagonists decreased the reserpine-induced increase in extracellular glutamate. L-3,4-Dihydroxyphenylalanine (L-DOPA) (25 mg/kg) significantly enhanced extracellular DA, had no effect on reserpine-induced hydroxyl radical production and decreased extracellular glutamate concentration. CSC but not ZM 241385 given jointly with L-DOPA increased the effect of L-DOPA on extracellular DA and augmented the reserpine-induced hydroxyl radical production. CSC and ZM 241385 did not influence extracellular glutamate level, which was decreased by L-DOPA. It seems that by decreasing the MAO-dependent DA metabolism rate, CSC raised cytosolic DA and by DA autoxidation, it induced hydroxyl radical overproduction. Thus, the methylxanthine A(2A) receptor antagonists bearing properties of MAO-B inhibitor, like CSC, may cause a risk of oxidative stress resulting from dysfunctional DA storage

  11. DOR/Tp53inp2 and Tp53inp1 constitute a metazoan gene family encoding dual regulators of autophagy and transcription.

    PubMed

    Sancho, Ana; Duran, Jordi; García-España, Antonio; Mauvezin, Caroline; Alemu, Endalkachew A; Lamark, Trond; Macias, Maria J; DeSalle, Rob; Royo, Miriam; Sala, David; Chicote, Javier U; Palacín, Manuel; Johansen, Terje; Zorzano, Antonio

    2012-01-01

    Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28-42; region 2, 66-112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription.

  12. DOR/Tp53inp2 and Tp53inp1 Constitute a Metazoan Gene Family Encoding Dual Regulators of Autophagy and Transcription

    PubMed Central

    Sancho, Ana; Duran, Jordi; García-España, Antonio; Mauvezin, Caroline; Alemu, Endalkachew A.; Lamark, Trond; Macias, Maria J.; DeSalle, Rob; Royo, Miriam; Sala, David; Chicote, Javier U.; Palacín, Manuel; Johansen, Terje; Zorzano, Antonio

    2012-01-01

    Human DOR/TP53INP2 displays a unique bifunctional role as a modulator of autophagy and gene transcription. However, the domains or regions of DOR that participate in those functions have not been identified. Here we have performed structure/function analyses of DOR guided by identification of conserved regions in the DOR gene family by phylogenetic reconstructions. We show that DOR is present in metazoan species. Invertebrates harbor only one gene, DOR/Tp53inp2, and in the common ancestor of vertebrates Tp53inp1 may have arisen by gene duplication. In keeping with these data, we show that human TP53INP1 regulates autophagy and that different DOR/TP53INP2 and TP53INP1 proteins display transcriptional activity. The use of molecular evolutionary information has been instrumental to determine the regions that participate in DOR functions. DOR and TP53INP1 proteins share two highly conserved regions (region 1, aa residues 28–42; region 2, 66–112 in human DOR). Mutation of conserved hydrophobic residues in region 1 of DOR (that are part of a nuclear export signal, NES) reduces transcriptional activity, and blocks nuclear exit and autophagic activity under autophagy-activated conditions. We also identify a functional and conserved LC3-interacting motif (LIR) in region 1 of DOR and TP53INP1 proteins. Mutation of conserved acidic residues in region 2 of DOR reduces transcriptional activity, impairs nuclear exit in response to autophagy activation, and disrupts autophagy. Taken together, our data reveal DOR and TP53INP1 as dual regulators of transcription and autophagy, and identify two conserved regions in the DOR family that concentrate multiple functions crucial for autophagy and transcription. PMID:22470510

  13. TASI/PiTP/ISS Lectures on Moduli and Microphysics

    SciTech Connect

    Silverstein, E

    2004-05-10

    I review basic forces on moduli that lead to their stabilization, for example in the supercritical and KKLT models of de Sitter space in string theory, as well as an AdS{sub 4} x S{sup 3} x S{sup 3} model I include which is not published elsewhere. These forces come from the classical dilaton tadpole in generic dimensionality, internal curvature, fluxes, and branes and orientifolds as well as non-perturbative effects. The resulting (A)dS solutions of string theory make detailed predictions for microphysical entropy, whose leading behavior we exhibit on the Coulomb branch of the system. Finally, I briefly review recent developmentsmore » concerning the role of velocity-dependent effects in the dynamics of moduli. These lecture notes are based on material presented at various stages in the 1999 TASI, 2002 PiTP, 2003 TASI, and 2003 ISS schools.« less

  14. TP53 status and response to chemotherapy in breast cancer.

    PubMed

    Bertheau, Philippe; Espié, Marc; Turpin, Elisabeth; Lehmann, Jacqueline; Plassa, Louis-François; Varna, Mariana; Janin, Anne; de Thé, Hugues

    2008-01-01

    Despite its central role in the control of apoptosis, senescence and cell cycle arrest, the tumor suppressor protein p53 remains an enigma for its possible role in predicting response to chemotherapy in cancer patients. Many studies remained inconclusive, others showed a better response for tumors with normal p53, and some recent studies showed adverse effects of normal p53 for response to treatment. p53 is not only a powerful pro-apoptotic factor in response to drug-induced DNA damages but also a potential inducer of cell cycle arrest, protecting tumor cells from further cytotoxic damages. Our review describes the classical as well as the more recent concepts. In order to draw definite conclusions, future works should use more reliable methods to assess the TP53 status and should address more homogeneous tumor subpopulations treated with homogeneous chemotherapy regimens. Copyright 2008 S. Karger AG, Basel.

  15. Nuclear TP53: An unraveled function as transcriptional repressor of PINK1.

    PubMed

    Checler, Frédéric; Goiran, Thomas; Alves da Costa, Cristine

    2018-05-11

    The tumor suppressor TP53/p53 is a key protein in both neurodegenerative diseases and cancer. Thus, TP53-linked cell death appears exacerbated in several age-related neuropathologies, while TP53 mutation-associated phenotypes indicate a loss of function accounting for approximately half of cancers. Thus, TP53 plays a pivotal role in these phenotypically distinct pathologies, a hypothesis reinforced by recent epidemiological studies suggesting an opposite risk to develop one type of pathology relative to the other. Dysfunctions in mitophagic processes also occur in both types of pathologies and again, TP53 has been proposed as one of the regulators of this cellular process. The consensus view postulates that TP53 exerts both anti- and pro-autophagy functions that are directly driven by a specific subcellular localization. Thus, TP53 positively modulates autophagy via the transcriptional control of several genes while it is acknowledged that its anti-autophagy phenotype is exclusively linked to a transcription-independent cytosolic control of an AMPK-MTOR cascade. Our study indicates that TP53 can also downregulate the specialized autophagy-related mitophagy response via the transcriptional repression of PINK1. This is the first demonstration of an anti-mitophagic control by nuclear TP53.

  16. L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.

    PubMed

    Krintel, Christian; Frydenvang, Karla; Ceravalls de Rabassa, Anna; Kaern, Anne M; Gajhede, Michael; Pickering, Darryl S; Kastrup, Jette S

    2014-05-01

    In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp). © 2014 FEBS.

  17. Dlg-1 Interacts With and Regulates the Activities of Fibroblast Growth Factor Receptors and EphA2 in the Mouse Lens.

    PubMed

    Lee, SungKyoung; Shatadal, Shalini; Griep, Anne E

    2016-02-01

    We previously showed that Discs large-1 (Dlg-1) regulates lens fiber cell structure and the fibroblast growth factor receptor (Fgfr) signaling pathway, a pathway required for fiber cell differentiation. Herein, we investigated the mechanism through which Dlg-1 regulates Fgfr signaling. Immunofluorescence was used to measure levels of Fgfr1, Fgfr2, and activated Fgfr signaling intermediates, pErk and pAkt, in control and Dlg-1-deficient lenses that were haplodeficient for Fgfr1 or Fgfr2. Immunoblotting was used to measure levels of N-cadherin, EphA2, β-catenin, and tyrosine-phosphorylated EphA2, Fgfr1, Fgfr2, and Fgfr3 in cytoskeletal-associated and cytosolic fractions of control and Dlg-1-deficient lenses. Complex formation between Dlg-1, N-cadherin, β-catenin, Fgfr1, Fgfr2, Fgfr3, and EphA2 was assessed by coimmunoprecipitation. Lenses deficient for Dlg-1 and haplodeficient for Fgfr1 or Fgfr2 showed increased levels of Fgfr2 or Fgfr1, respectively. Levels of pErk and pAkt correlated with the level of Fgfr2. N-cadherin was reduced in the cytoskeletal-associated fraction and increased in the cytosolic fraction of Dlg-1-deficient lenses. Dlg-1 complexed with β-catenin, EphA2, Fgfr1, Fgfr2, and Fgfr3. EphA2 complexed with N-cadherin, β-catenin, Fgfr1, Fgfr2, and Fgfr3. Levels of these interactions were altered in Dlg-1-deficient lenses. Loss of Dlg-1 led to changes in Fgfr1, Fgfr2, Fgfr3, and EphA2 levels and to greater changes in the levels of their activation. Dlg-1 complexes with and regulates the activities of EphA2, Fgfr1, Fgfr2, and Fgfr3. As EphA2 contains a Psd95/Dlg/ZO-1 (PDZ) binding motif, whereas Fgfrs do not, we propose that the PDZ protein, Dlg-1, modulates Fgfr signaling through regulation of EphA2.

  18. Unprecedented Therapeutic Potential with a Combination of A2A/NR2B Receptor Antagonists as Observed in the 6-OHDA Lesioned Rat Model of Parkinson's Disease

    PubMed Central

    Michel, Anne; Downey, Patrick; Nicolas, Jean-Marie; Scheller, Dieter

    2014-01-01

    In Parkinson's disease, the long-term use of dopamine replacing agents is associated with the development of motor complications; therefore, there is a need for non-dopaminergic drugs. This study evaluated the potential therapeutic impact of six different NR2B and A2A receptor antagonists given either alone or in combination in unilateral 6-OHDA-lesioned rats without (monotherapy) or with (add-on therapy) the co-administration of L-Dopa: Sch-58261+ Merck 22; Sch-58261+Co-101244; Preladenant + Merck 22; Preladenant + Radiprodil; Tozadenant + Radiprodil; Istradefylline + Co-101244. Animals given monotherapy were assessed on distance traveled and rearing, whereas those given add-on therapy were assessed on contralateral rotations. Three-way mixed ANOVA were conducted to assess the main effect of each drug separately and to determine whether any interaction between two drugs was additive or synergistic. Additional post hoc analyses were conducted to compare the effect of the combination with the effect of the drugs alone. Motor activity improved significantly and was sustained for longer when the drugs were given in combination than when administered separately at the same dose. Similarly, when tested as add-on treatment to L-Dopa, the combinations resulted in higher levels of contralateral rotation in comparison to the single drugs. Of special interest, the activity observed with some combinations could not be described by a simplistic additive effect and involved more subtle synergistic pharmacological interactions. The combined administration of A2A/NR2B-receptor antagonists improved motor behaviour in 6-OHDA rats. Given the proven translatability of this model such a combination may be expected to be effective in improving motor symptoms in patients. PMID:25513815

  19. Antiaggregant and antivasospastic properties of the new thromboxane A2 receptor antagonist sodium 4-[[1-[[[(4-chlorophenyl)sulfonyl]amino]methyl]cyclopentyl] methyl]benzeneacetate.

    PubMed

    Lardy, C; Rousselot, C; Chavernac, G; Depin, J C; Guerrier, D

    1994-11-01

    LCB 2853 (sodium 4-[[1-[[[(4-chlorophenyl)sulfonyl]amino]methyl]cyclopentyl] methyl]benzeneacetate, CAS 141335-11-7) was demonstrated to be a potent thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist in in vitro, ex vivo and in vivo experiments. The specific mechanism of action was studied in [3H]SQ 29548 receptor binding studies (pKi = 7.93) and was shown to be of competitive nature in U 46619-induced platelet aggregation (pA2 = 6.82). TXA2-dependent platelet rich plasma (PRP) aggregation (U 46619, arachidonic acid (AA), collagen, ADP or serotonin second phase) was inhibited in vitro in humans (IC50:0.037-0.65 mumol/l) and different animal species, as well as ex vivo i.v. rat and p.o. guinea-pig AA-induced aggregation (ED50 = 48 and 57 micrograms/kg). The U 46619-induced contractions of aorta, caudal artery and trachea were inhibited in a dose-dependent way (IC50 = 0.07, 0.02 and 0.5 mumol/l respectively). In vivo, both against platelet aggregation and vasoconstriction, LCB 2853 showed an ED50 lower than 1 mg/kg i.v. in rat AA-induced thrombocytopenia or U 46619-induced hypertension (ED50 = 0.25 and 0.16 mg/kg) as well as in AA-induced sudden death in the mouse (ED50 = 0.44 mg/kg). The U 46619-induced bronchoconstriction was blocked after i.v. administration of LCB 2853 (ED50 = 18.4 micrograms/kg). The duration of action observed in different models was 6 h by oral route and between 3 and 5 h by intravenous route. These properties in TXA2-dependent models led to further investigations of the antithrombotic activity of this novel TXA2 antagonist.

  20. The Tp0684 (MglB-2) Lipoprotein of Treponema pallidum: A Glucose-Binding Protein with Divergent Topology.

    PubMed

    Brautigam, Chad A; Deka, Ranjit K; Liu, Wei Z; Norgard, Michael V

    2016-01-01

    Treponema pallidum, the bacterium that causes syphilis, is an obligate human parasite. As such, it must acquire energy, in the form of carbon sources, from the host. There is ample evidence that the principal source of energy for this spirochete is D-glucose acquired from its environment, likely via an ABC transporter. Further, there is genetic evidence of a D-glucose chemotaxis system in T. pallidum. Both of these processes may be dependent on a single lipidated chemoreceptor: Tp0684, also called TpMglB-2 for its sequence homology to MglB of Escherichia coli. To broaden our understanding of this potentially vital protein, we determined a 2.05-Å X-ray crystal structure of a soluble form of the recombinant protein. Like its namesake, TpMglB-2 adopts a bilobed fold that is similar to that of the ligand-binding proteins (LBPs) of other ABC transporters. However, the protein has an unusual, circularly permuted topology. This feature prompted a series of biophysical studies that examined whether the protein's topological distinctiveness affected its putative chemoreceptor functions. Differential scanning fluorimetry and isothermal titration calorimetry were used to confirm that the protein bound D-glucose in a cleft between its two lobes. Additionally, analytical ultracentrifugation was employed to reveal that D-glucose binding is accompanied by a significant conformational change. TpMglB-2 thus appears to be fully functional in vitro, and given the probable central importance of the protein to T. pallidum's physiology, our results have implications for the viability and pathogenicity of this obligate human pathogen.

  1. Advanced Running Performance by Genetic Predisposition in Male Dummerstorf Marathon Mice (DUhTP) Reveals Higher Sterol Regulatory Element-Binding Protein (SREBP) Related mRNA Expression in the Liver and Higher Serum Levels of Progesterone

    PubMed Central

    Brenmoehl, Julia; Walz, Christina; Ponsuksili, Siriluck; Schwerin, Manfred; Fuellen, Georg; Hoeflich, Andreas

    2016-01-01

    Long-term-selected DUhTP mice represent a non-inbred model for inborn physical high-performance without previous training. Abundance of hepatic mRNA in 70-day male DUhTP and control mice was analyzed using the Affymetrix mouse array 430A 2.0. Differential expression analysis with PLIER corrected data was performed using AltAnalyze. Searching for over-representation in biochemical pathways revealed cholesterol metabolism being most prominently affected in DUhTP compared to unselected control mice. Furthermore, pathway analysis by AltAnalyze plus PathVisio indicated significant induction of glycolysis, fatty acid synthesis and cholesterol biosynthesis in the liver of DUhTP mice versus unselected control mice. In contrast, gluconeogenesis was partially inactivated as judged from the analysis of hepatic mRNA transcript abundance in DUhTP mice. Analysis of mRNA transcripts related to steroid hormone metabolism inferred elevated synthesis of progesterone and reduced levels of sex steroids. Abundance of steroid delta isomerase-5 mRNA (Hsd3b5, FC 4.97) was increased and steroid 17-alpha-monooxygenase mRNA (Cyp17a1, FC -11.6) was massively diminished in the liver of DUhTP mice. Assessment of steroid profiles by LC-MS revealed increased levels of progesterone and decreased levels of sex steroids in serum from DUhTP mice versus controls. Analysis of hepatic mRNA transcript abundance indicates that sterol regulatory element-binding protein-1 (SREBP-1) may play a major role in metabolic pathway activation in the marathon mouse model DUhTP. Thus, results from bioinformatics modeling of hepatic mRNA transcript abundance correlated with direct steroid analysis by mass spectrometry and further indicated functions of SREBP-1 and steroid hormones for endurance performance in DUhTP mice. PMID:26799318

  2. Interaction between caffeine and polymorphisms of glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) and cytochrome P450 1A2 (CYP1A2) on Parkinson's disease risk.

    PubMed

    Kim, Iris Y; O'Reilly, Éilis J; Hughes, Katherine C; Gao, Xiang; Schwarzschild, Michael A; McCullough, Marjorie L; Hannan, Marian T; Betensky, Rebecca A; Ascherio, Alberto

    2018-03-01

    Caffeine intake has been inversely associated with Parkinson's disease (PD) risk. This relationship may be modified by polymorphisms of glutamate ionotropic receptor NMDA type subunit 2A (GRIN2A) and cytochrome P450 1A2 (CYP1A2), but the results of previous studies have been inconsistent. We examined the interaction of caffeine intake with GRIN2A-rs4998386 and CYP1A2-rs762551 polymorphisms in influencing PD risk among 829 incident cases of PD and 2,754 matched controls selected among participants in the following 3 large prospective ongoing cohorts: the Nurses' Health Study, the Health Professionals' Follow-up Study, and the Cancer Prevention Study II Nutrition Cohort. Matching factors included cohort, birth year, source of DNA, date of DNA collection, and race. Relative risks and 95% confidence intervals were estimated using conditional logistic models. Interactions were tested both on the multiplicative scale and on the additive scale. Overall, caffeine intake was associated with a lower PD risk (adjusted relative risk for highest versus lowest tertile = 0.70; 95% confidence interval, 0.57-0.86; p < .001). In analyses stratified by the GRIN2A-rs4998386 genotype, the multivariable-adjusted relative risk of PD comparing the highest to the lowest tertile of caffeine was 0.69 (95% confidence interval, 0.55-0.88; p < .01) among individuals homozygous for the C allele, and 0.85 (95% confidence interval, 0.55-1.32; p = .47; p RERI  = .43) among carriers for the T allele. Interactions between caffeine and GRIN2A were not significant in either the multiplicative or additive scales. We also did not observe significant interactions for CYP1A2-rs762551 and incident PD risk. Our findings do not support the hypothesis of an interaction between the GRIN2A-rs4998386 or CYP1A2-rs762551 polymorphism and caffeine intake in determining PD risk. © 2018 International Parkinson and Movement Disorder Society. © 2018 International Parkinson and Movement Disorder

  3. Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

    PubMed

    Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

    2016-06-23

    The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors

  4. [Evaluation of the Performance of Two Kinds of Anti-TP Enzyme-Linked Immunosorbent Assay].

    PubMed

    Gao, Nan; Huang, Li-Qin; Wang, Rui; Jia, Jun-Jie; Wu, Shuo; Zhang, Jing; Ge, Hong-Wei

    2018-06-01

    To evaluate the accuracy and precision of 2 kinds of anti-treponema pallidum (anti-TP) ELISA reagents in our laboratory for detecting the anti-TP in voluntary blood donors, so as to provide the data support for use of ELISA reagents after introduction of chemiluminescene immunoassay (CLIA). The route detection of anti-TP was performed by using 2 kinds of ELISA reagents, then 546 responsive positive samples detected by anti-TP ELISA were collected, and the infections status of samples confirmed by treponema pallidum particle agglutination (TPPA) test was identified. The confirmed results of responsive samples detected by 2 kinds of anti-TP ELISA reagents were compared, the accuracy of 2 kinds of anti-TP ELISA reagents was analyzed by drawing ROC and comparing area under curve (AUC), and precision of 2 kinds of anti-TP ELISA reagents was compared by statistical analysis of quality control data from 7.1 2016 to 6.30 2017. There were no statistical difference in confirmed positive rate of responsive samples and weak positive samples between 2 kinds of anti-TP ELISA reagents. The responsive samples detected by 2 kinds of anti-TP ELISA reagents accounted for 85.53%(467/546) of all responsive samples, the positive rate confirmed by TPPA test was 82.87%. 44 responsive samples detected by anti-TP ELISA reagent A and 35 responsive samples detected by anti-TP ELISA reagent B were confirmed to be negative by TPPA test. Comparison of AUC showed that the accuracy of 2 kinds of anti-TP ELISA reagents was more high, the difference between 2 reagents was not statistically significant. The coefficient of variation (CV) of anti-TP ELISA reagent A and B was 14.98% and 18.04% respectively, which met the precision requirement of ELISA test. The accuracy and precision of 2 kinds of anti-TP ELISA reagents used in our laboratory are similar, and using any one of anti-TP ELISA reagents all can satisfy the requirements of blood screening.

  5. Characterizing genomic differences of human cancer stratified by the TP53 mutation status.

    PubMed

    Wang, Mengyao; Yang, Chao; Zhang, Xiuqing; Li, Xiangchun

    2018-06-01

    The key roles of the TP53 mutation in cancer have been well established. TP53 is the most frequently mutated gene, and its inactivation is widespread among human cancer types. However, the landscape of genomic alterations in human cancers stratified by the TP53 mutation has not yet been described. We obtained somatic mutation and copy number change data of 6551 regular-mutated samples from the Cancer Genome Atlas (TCGA) and compared significantly mutated genes (SMGs), copy number alterations, mutational signatures and mutational strand asymmetries between cancer samples with and without the TP53 mutation. We identified 126 SMGs, 30 of which were statistically significant in both the TP53 mutant and wild-type groups. Several SMGs, such as VHL, SMAD4 and PTEN, showed a mutation bias towards the TP53 wild-type group, whereas ATRX, IDH1 and RB1 were more prevalent in the TP53 mutant group. Five mutational signatures were extracted from the combined TCGA dataset on which mutational asymmetry analysis was performed, revealing that the TP53 mutant group exhibited substantially greater replication and transcription biases. Furthermore, we found that alterations of multiple genes in a merged mutually exclusive network composed of BRAF, EGFR, PAK1, PIK3CA, PTEN, APC and TERT were related to shortened survival in the TP53 wild-type group. In summary, we characterized the genomic differences and similarities underlying human cancers stratified by the TP53 mutation and identified multi-gene alterations of a merged mutually exclusive network to be a poor prognostic factor for the TP53 wild-type group.

  6. The TP53 gene promoter is not methylated in families suggestive of Li-Fraumeni syndrome with no germline TP53 mutations.

    PubMed

    Finkova, Alena; Vazna, Alzbeta; Hrachovina, Ondrej; Bendova, Sarka; Prochazkova, Kamila; Sedlacek, Zdenek

    2009-08-01

    Germline TP53 mutations are found in only 70% of families with the Li-Fraumeni syndrome (LFS), and with an even lower frequency in families suggestive of LFS but not meeting clinical criteria of the syndrome. Despite intense efforts, to date, no other genes have been associated with the disorder in a significant number of TP53 mutation-negative families. A search for defects in TP53 other than heterozygous missense mutations showed that neither intron variants nor sequence variants in the TP53 promoter are frequent in LFS, and multiexon deletions have been found to be responsible for LFS only in several cases. Another cancer predisposition syndrome, hereditary non-polyposis colon cancer, has been associated with epigenetic silencing of one allele of the MLH1 or MSH2 genes. This prompted us to test the methylation of the TP53 gene promoter in a set of 14 families suggestive of LFS using bisulphite sequencing of three DNA fragments from the 5' region of the gene. We found no detectable methylation at any of the CG dinucleotides tested. Thus, epigenetic silencing of the TP53 promoter is not a frequent cause of the disorder in families suggestive of LFS but with no germline mutations in the coding part of the gene.

  7. Adenosine A2A receptor blockade prevents synaptotoxicity and memory dysfunction caused by beta-amyloid peptides via p38 mitogen-activated protein kinase pathway.

    PubMed

    Canas, Paula M; Porciúncula, Lisiane O; Cunha, Geanne M A; Silva, Carla G; Machado, Nuno J; Oliveira, Jorge M A; Oliveira, Catarina R; Cunha, Rodrigo A

    2009-11-25

    Alzheimer's disease (AD) is characterized by memory impairment, neurochemically by accumulation of beta-amyloid peptide (namely Abeta(1-42)) and morphologically by an initial loss of nerve terminals. Caffeine consumption prevents memory dysfunction in different models, which is mimicked by antagonists of adenosine A(2A) receptors (A(2A)Rs), which are located in synapses. Thus, we now tested whether A(2A)R blockade prevents the early Abeta(1-42)-induced synaptotoxicity and memory dysfunction and what are the underlying signaling pathways. The intracerebral administration of soluble Abeta(1-42) (2 nmol) in rats or mice caused, 2 weeks later, memory impairment (decreased performance in the Y-maze and object recognition tests) and a loss of nerve terminal markers (synaptophysin, SNAP-25) without overt neuronal loss, astrogliosis, or microgliosis. These were prevented by pharmacological blockade [5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine (SCH58261); 0.05 mg . kg(-1) . d(-1), i.p.; for 15 d] in rats, and genetic inactivation of A(2A)Rs in mice. Moreover, these were synaptic events since purified nerve terminals acutely exposed to Abeta(1-42) (500 nm) displayed mitochondrial dysfunction, which was prevented by A(2A)R blockade. SCH58261 (50 nm) also prevented the initial synaptotoxicity (loss of MAP-2, synaptophysin, and SNAP-25 immunoreactivity) and subsequent loss of viability of cultured hippocampal neurons exposed to Abeta(1-42) (500 nm). This A(2A)R-mediated control of neurotoxicity involved the control of Abeta(1-42)-induced p38 phosphorylation and was independent from cAMP/PKA (protein kinase A) pathway. Together, these results show that A(2A)Rs play a crucial role in the development of Abeta-induced synaptotoxicity leading to memory dysfunction through a p38 MAPK (mitogen-activated protein kinase)-dependent pathway and provide a molecular basis for the benefits of caffeine consumption in AD.

  8. Phenotypic approaches to gene mapping in platelet function disorders - identification of new variant of P2Y12, TxA2 and GPVI receptors.

    PubMed

    Watson, S; Daly, M; Dawood, B; Gissen, P; Makris, M; Mundell, S; Wilde, J; Mumford, A

    2010-01-01

    Platelet number or function disorders cause a range of bleeding symptoms from mild to severe. Patients with platelet dysfunction but normal platelet number are the most prevalent and typically have mild bleeding symptoms. The study of this group of patients is particularly difficult because of the lack of a gold-standard test of platelet function and the variable penetrance of the bleeding phenotype among affected individuals. The purpose of this short review is to discuss the way in which this group of patients can be investigated through platelet phenotyping in combination with targeted gene sequencing. This approach has been used recently to identify patients with mutations in key platelet activation receptors, namely those for ADP, collagen and thromboxane A2 (TxA2). One interesting finding from this work is that for some patients, mild bleeding is associated with heterozygous mutations in platelet proteins that are co-inherited with other genetic disorders of haemostasis such as type 1 von Willebrand's disease. Thus, the phenotype of mild bleeding may be multifactorial in some patients and may be considered to be a complex trait.

  9. STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells

    PubMed Central

    Guo, Liang; Lichten, Louis A.; Ryu, Moon-Suhn; Liuzzi, Juan P.; Wang, Fudi; Cousins, Robert J.

    2010-01-01

    The exocrine pancreas plays an important role in endogenous zinc loss by regulating excretion into the intestinal tract and hence influences the dietary zinc requirement. The present experiments show that the zinc transporter ZnT2 (Slc30a2) is localized to the zymogen granules and that dietary zinc restriction in mice decreased the zinc concentration of zymogen granules and ZnT2 expression. Excess zinc given orally increased ZnT2 expression and was associated with increased pancreatic zinc accumulation. Rat AR42J acinar cells when induced into a secretory phenotype, using the glucocorticoid analog dexamethasone (DEX), exhibited increased ZnT2 expression and labile zinc as measured with a fluorophore. DEX administrated to mice also induced ZnT2 expression that accompanied a reduction of the pancreatic zinc content. ZnT2 promoter analyses identified elements required for responsiveness to zinc and DEX. Zinc regulation was traced to a MRE located downstream from the ZnT2 transcription start site. Responsiveness to DEX is produced by two upstream STAT5 binding sites that require the glucocorticoid receptor for activation. ZnT2 knockdown in the AR42J cells using siRNA resulted in increased cytoplasmic zinc and decreased zymogen granule zinc that further demonstrated that ZnT2 may mediate the sequestration of zinc into zymogen granules. We conclude, based upon experiments with intact mice and pancreatic acinar cells in culture, that ZnT2 participates in zinc transport into pancreatic zymogen granules through a glucocorticoid pathway requiring glucocorticoid receptor and STAT5, and zinc-regulated signaling pathways requiring MTF-1. The ZnT2 transporter appears to function in a physiologically responsive manner involving entero-pancreatic zinc trafficking. PMID:20133611

  10. Serotonin (5-HT)2A/2C receptor agonist (2,5-dimethoxy-4-idophenyl)-2-aminopropane hydrochloride (DOI) improves voiding efficiency in the diabetic rat.

    PubMed

    Tu, Hongjian; Cao, Nailong; Gu, Baojun; Si, Jiemin; Chen, Zhong; Andersson, Karl-Erik

    2015-07-01

    To examine the effects of the serotonin (5-HT)2A/2C receptor agonist (2,5-dimethoxy-4-idophenyl)-2-aminopropane hydrochloride (DOI) on micturition in rats with diabetes mellitus (DM). Female Sprague-Dawley rats (n = 16) were divided into two groups: rats with Type 1 DM and age-matched control rats. DM was induced by i.p. injection of streptozotocin (65 mg/kg) and detailed cystometrogram (CMG) studies were performed 8 weeks post-injection in all rats under urethane anaesthesia. The selective 5-HT2A antagonist ketanserin was administered after each DOI dose-response curve was plotted. All drugs were administered i.v. Compared with controls, comprehensive urodynamic studies showed that DM rats had a higher bladder capacity and post-void residual urine volume (PVR), and a markedly lower voiding efficiency. In DM rats, DOI (0.01-0.3 mg/kg) induced significant dose-dependent increases in micturition volume and reductions in PVR, resulting in greater voiding efficiency. CMG measurements showed a dose-dependent increase in high-frequency oscillation (HFO) activity, evidenced by an increased duration of HFOs per voiding. This correlated with the improved voiding efficiency. Ketanserin (0.1 mg/kg) partially or completely reversed the DOI-induced changes. The HFOs observed in the present study seem to correlate with external urethral sphincter bursting activity during voiding. Bladder voiding efficiency was reduced in DM rats. The 5-HT2A receptor agonist can enhance HFO activity and improves voiding efficiency, and so may represent a new strategy to improve voiding efficiency after DM in experimental studies. © 2014 The Authors BJU International © 2014 BJU International Published by John Wiley & Sons Ltd.

  11. Bioinformatic dissecting of TP53 regulation pathway underlying butyrate-induced histone modification in epigenetic regulation

    USDA-ARS?s Scientific Manuscript database

    Butyrate affects cell proliferation, differentiation and motility. Butyrate inhibits histone deacetylase (HDAC) activities and induces cell cycle arrest and apoptosis. TP53 is one of the most active upstream regulators discovered by IPA in our RNA sequencing data set. The TP53 signaling pathway pl...

  12. Interrelationship between TP53 gene deletion, protein expression and chromosome 17 aneusomy in gastric adenocarcinoma

    PubMed Central

    2009-01-01

    Background This study evaluates the existence of numerical alterations of chromosome 17 and TP53 gene deletion in gastric adenocarcinoma. The p53 protein expression was also evaluated, as well as, possible associations with clinicopathological characteristics. Methods Dual-color fluorescence in situ hybridization and immunostaining were performed in twenty gastric cancer samples of individuals from Northern Brazil. Results Deletion of TP53 was found in all samples. TP53 was inactivated mainly by single allelic deletion, varying to 7–39% of cells/case. Aneusomy of chromosome 17 was observed in 85% of cases. Chromosome 17 monosomy and gain were both observed in about half of cases. Cells with gain of chromosome 17 frequently presented TP53 deletion. The frequency of cells with two chr17 and one TP53 signals observed was higher in diffuse than in intestinal-type GC. Immunoreactivity of p53 was found only in intestinal-type samples. The frequency of cells with two chr17 and two TP53 signals found was higher in samples with positive p53 expression than in negative cases in intestinal-type GC. Conclusion We suggest that TP53 deletion and chromosome 17 aneusomy is a common event in GC and other TP53 alterations, as mutation, may be implicated in the distinct carcinogenesis process of diffuse and intestinal types. PMID:19619279

  13. Crystal growth of triphenylphosphine oxide 4-nitrophenol (TP4N) for nonlinear optical (NLO) applications

    NASA Astrophysics Data System (ADS)

    Pandian, Muthu Senthil; Karuppasamy, P.; Kamalesh, T.; Ramasamy, P.; Verma, Sunil

    2018-04-01

    The optically good quality organic single crystals of triphenylphosphine oxide 4-nitrophenol (TP4N) were successfully grown by slow evaporation solution technique (SEST) using methanol as solvent. The lattice parameters of the grown crystal were confirmed by single crystal X-ray diffraction analysis. The optical transmittance, cut-off wavelength and band gap of the TP4N crystal were obtained by UV-Vis NIR spectrum analysis. The photoluminescence studies were carried out to find out the luminesce properties of TP4N single crystal. The photoconductivity studies reveal that the TP4N crystal has negative photoconductive nature. The third order nonlinear susceptibility (χ(3)) of TP4N crystal was evaluated using the Z-scan technique at 640 nm.

  14. 7-Deazapurine containing DNA: efficiency of c7GdTP, c7AdTP and c7IdTP incorporation during PCR-amplification and protection from endodeoxyribonuclease hydrolysis.

    PubMed Central

    Seela, F; Röling, A

    1992-01-01

    The enzymatic synthesis of 7-deazapurine nucleoside containing DNA (501 bp) is performed by PCR-amplification (Taq polymerase) using a pUC18 plasmid DNA as template and the triphosphates of 7-deaza-2'-deoxyguanosine (c7Gd), -adenosine (c7Ad) and -inosine (c7Id). c7GdTP can fully replace dGTP resulting in a completely modified DNA-fragment of defined size and sequence. The other two 7-deazapurine triphosphates (c7AdTP) and (c7IdTP) require the presence of the parent purine 2'-deoxyribonucleotides. In purine/7-deazapurine nucleotide mixtures Taq polymerase prefers purine over 7-deazapurine nucleotides but accepts c7GdTP much better than c7AdTP or c7IdTP. As incorporation of 7-deazapurine nucleotides represents a modification of the major groove of DNA it can be used to probe DNA/protein interaction. Regioselective phosphodiester hydrolysis of the modified DNA-fragments was studied with 28 endodeoxyribonucleases. c7Gd is able to protect the DNA from the phosphodiester hydrolysis in more than 20 cases, only a few enzymes (Mae III, Rsa I, Hind III, Pvu II or Taq I) do still hydrolyze the modified DNA. c7Ad protects DNA less efficiently, as this DNA could only be modified in part. The absence of N-7 as potential binding position or a geometric distortion of the recognition duplex caused by the 7-deazapurine base can account for protection of hydrolysis. Images PMID:1738604

  15. Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

    USGS Publications Warehouse

    Strobl-Mazzulla, P. H.; Lethimonier, C.; Gueguen, M.M.; Karube, M.; Fernandino, J.I.; Yoshizaki, G.; Patino, R.; Strussmann, C.A.; Kah, O.; Somoza, G.M.

    2008-01-01

    Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, ?? and ??, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor ??, but not ??, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate. ?? 2008 Elsevier Inc.

  16. Molecular Docking and Prediction of Pharmacokinetic Properties of Dual Mechanism Drugs that Block MAO-B and Adenosine A2A Receptors for the Treatment of Parkinson's Disease

    PubMed Central

    Azam, Faizul; Madi, Arwa M.; Ali, Hamed I.

    2012-01-01

    Monoamine oxidase B (MAO-B) inhibitory potential of adenosine A2A receptor (AA2AR) antagonists has raised the possibility of designing dual-target–directed drugs that may provide enhanced symptomatic relief and that may also slow the progression of Parkinson's disease (PD) by protecting against further neurodegeneration. To explain the dual inhibition of MAO-B and AA2AR at the molecular level, molecular docking technique was employed. Lamarckian genetic algorithm methodology was used for flexible ligand docking studies. A good correlation (R2= 0.524 and 0.627 for MAO-B and AA2AR, respectively) was established between docking predicted and experimental Ki values, which confirms that the molecular docking approach is reliable to study the mechanism of dual interaction of caffeinyl analogs with MAO-B and AA2AR. Parameters for Lipinski's “Rule-of-Five” were also calculated to estimate the pharmacokinetic properties of dual-target–directed drugs where both MAO-B inhibition and AA2AR antagonism exhibited a positive correlation with calculated LogP having a correlation coefficient R2 of 0.535 and 0.607, respectively. These results provide some beneficial clues in structural modification for designing new inhibitors as dual-target–directed drugs with desired pharmacokinetic properties for the treatment of PD. PMID:23112538

  17. RITA displays anti-tumor activity in medulloblastomas independent of TP53 status

    PubMed Central

    Gottlieb, Aline; Althoff, Kristina; Grunewald, Laura; Thor, Theresa; Odersky, Andrea; Schulte, Marc; Deubzer, Hedwig E.; Heukamp, Lukas; Eggert, Angelika; Schramm, Alexander; Schulte, Johannes H.; Künkele, Annette

    2017-01-01

    Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40–70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma. PMID:28427187

  18. Crystal structure of thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase).

    PubMed

    Guo, Zheng; Wang, Fengbin; Shen, Tiantian; Huang, Jing; Wang, Yuandong; Ji, Chaoneng

    2014-05-01

    Thermostable p-nitrophenylphosphatase from Bacillus Stearothermophilus (Bs-TpNPPase) is involved in the Mg(2+)-dependent hydrolysis of the phosphoenzyme at an optimum reaction temperature of 55°C. Bs-TpNPPase has been cloned and overexpressed in the E.coli M15 strain. Based on the conserved active sites, the protein was suggested to be a member of the haloalkanoate dehalogenase (HAD) superfamily. Two site-specific point mutants of Bs-TpNPPase were prepared by changing the catalytic Asp10 and Thr43 to Ala10 and Ala43, respectively. The activity of the two mutants further confirms Bs-TpNPPase as a member of the HAD superfamily. HAD superfamily can be divided into the four subfamilies and play several biochemical roles such as DNA repair, signal transduction and secondary metabolism. To understand the relationship between structure and thermostability in HAD superfamily, Bs-TpNPPase from Bacillus Stearothermophilus was selected. The X-ray crystal structure of Bs-TpNPPase was determined at 1.5A resolution using the molecular replacement phasing method. The structure of Bs-TpNPPase has been deposited and the PDB code is 4KN8. Compared with Bsp, a mesophilic prokaryotic putative p-nitrophenyl phosphatase from Bacillus Subtilis, Bs- TpNPPase showed highly homology but variations in the level of leucine content, aromatic clusters, cation-Pi and hydrophobic interaction. These differences may affect the thermal stability of the protein. The crystal structure of Bs-TpNPPase described herein may serve as a guide to better understand the mechanism of thermostability and provide insights for further mutation work.

  19. Targeting IL13Ralpha2 activates STAT6-TP63 pathway to suppress breast cancer lung metastasis.

    PubMed

    Papageorgis, Panagiotis; Ozturk, Sait; Lambert, Arthur W; Neophytou, Christiana M; Tzatsos, Alexandros; Wong, Chen K; Thiagalingam, Sam; Constantinou, Andreas I

    2015-07-25

    Basal-like breast cancer (BLBC) is an aggressive subtype often characterized by distant metastasis, poor patient prognosis, and limited treatment options. Therefore, the discovery of alternative targets to restrain its metastatic potential is urgently needed. In this study, we aimed to identify novel genes that drive metastasis of BLBC and to elucidate the underlying mechanisms of action. An unbiased approach using gene expression profiling of a BLBC progression model and in silico leveraging of pre-existing tumor transcriptomes were used to uncover metastasis-promoting genes. Lentiviral-mediated knockdown of interleukin-13 receptor alpha 2 (IL13Ralpha2) coupled with whole-body in vivo bioluminescence imaging was performed to assess its role in regulating breast cancer tumor growth and lung metastasis. Gene expression microarray analysis was followed by in vitro validation and cell migration assays to elucidate the downstream molecular pathways involved in this process. We found that overexpression of the decoy receptor IL13Ralpha2 is significantly enriched in basal compared with luminal primary breast tumors as well as in a subset of metastatic basal-B breast cancer cells. Importantly, breast cancer patients with high-grade tumors and increased IL13Ralpha2 levels had significantly worse prognosis for metastasis-free survival compared with patients with low expression. Depletion of IL13Ralpha2 in metastatic breast cancer cells modestly delayed primary tumor growth but dramatically suppressed lung metastasis in vivo. Furthermore, IL13Ralpha2 silencing was associated with enhanced IL-13-mediated phosphorylation of signal transducer and activator of transcription 6 (STAT6) and impaired migratory ability of metastatic breast cancer cells. Interestingly, genome-wide transcriptional analysis revealed that IL13Ralpha2 knockdown and IL-13 treatment cooperatively upregulated the metastasis suppressor tumor protein 63 (TP63) in a STAT6-dependent manner. These observations

  20. The relationship of TP53 R72P polymorphism to disease outcome and TP53 mutation in myelodysplastic syndromes

    PubMed Central

    McGraw, K L; Zhang, L M; Rollison, D E; Basiorka, A A; Fulp, W; Rawal, B; Jerez, A; Billingsley, D L; Lin, H-Y; Kurtin, S E; Yoder, S; Zhang, Y; Guinta, K; Mallo, M; Solé, F; Calasanz, M J; Cervera, J; Such, E; González, T; Nevill, T J; Haferlach, T; Smith, A E; Kulasekararaj, A; Mufti, G; Karsan, A; Maciejewski, J P; Sokol, L; Epling-Burnette, P K; Wei, S; List, A F

    2015-01-01

    Nonsynonymous TP53 exon 4 single-nucleotide polymorphism (SNP), R72P, is linked to cancer and mutagen susceptibility. R72P associations with specific cancer risk, particularly hematological malignancies, have been conflicting. Myelodysplastic syndrome (MDS) with chromosome 5q deletion is characterized by erythroid hypoplasia arising from lineage-specific p53 accumulation resulting from ribosomal insufficiency. We hypothesized that apoptotically diminished R72P C-allele may influence predisposition to del(5q) MDS. Bone marrow and blood DNA was sequenced from 705 MDS cases (333 del(5q), 372 non-del(5q)) and 157 controls. Genotype distribution did not significantly differ between del(5q) cases (12.6% CC, 38.1% CG, 49.2% GG), non-del(5q) cases (9.7% CC, 44.6% CG, 45.7% GG) and controls (7.6% CC, 37.6% CG, 54.8% GG) (P=0.13). Allele frequency did not differ between non-del(5q) and del(5q) cases (P=0.91) but trended towards increased C-allele frequency comparing non-del(5q) (P=0.08) and del(5q) (P=0.10) cases with controls. Median lenalidomide response duration increased proportionate to C-allele dosage in del(5q) patients (2.2 (CC), 1.3 (CG) and 0.89 years (GG)). Furthermore, C-allele homozygosity in del(5q) was associated with prolonged overall and progression-free survival and non-terminal interstitial deletions that excluded 5q34, whereas G-allele homozygozity was associated with inferior outcome and terminal deletions involving 5q34 (P=0.05). These findings comprise the largest MDS R72P SNP analysis. PMID:25768405

  1. Neutrophil-derived 5′-Adenosine Monophosphate Promotes Endothelial Barrier Function via CD73-mediated Conversion to Adenosine and Endothelial A2B Receptor Activation

    PubMed Central

    Lennon, Paul F.; Taylor, Cormac T.; Stahl, Gregory L.; Colgan, Sean P.

    1998-01-01

    During episodes of inflammation, polymorphonuclear leukocyte (PMN) transendothelial migration has the potential to disturb vascular barrier function and give rise to intravascular fluid extravasation and edema. However, little is known regarding innate mechanisms that dampen fluid loss during PMN-endothelial interactions. Using an in vitro endothelial paracellular permeability model, we observed a PMN-mediated decrease in endothelial paracellular permeability. A similar decrease was elicited by cell-free supernatants from activated PMN (FMLP 10−6 M), suggesting the presence of a PMN-derived soluble mediator(s). Biophysical and biochemical analysis of PMN supernatants revealed a role for PMN-derived 5′-adenosine monophosphate (AMP) and its metabolite, adenosine, in modulation of endothelial paracellular permeability. Supernatants from activated PMN contained micromolar concentrations of bioactive 5′-AMP and adenosine. Furthermore, exposure of endothelial monolayers to authentic 5′-AMP and adenosine increased endothelial barrier function more than twofold in both human umbilical vein endothelial cells and human microvascular endothelial cells. 5′-AMP bioactivity required endothelial CD73-mediated conversion of 5′-AMP to adenosine via its 5′-ectonucleotidase activity. Decreased endothelial paracellular permeability occurred through adenosine A2B receptor activation and was accompanied by a parallel increase in intracellular cAMP. We conclude that activated PMN release soluble mediators, such as 5′-AMP and adenosine, that promote endothelial barrier function. During inflammation, this pathway may limit potentially deleterious increases in endothelial paracellular permeability and could serve as a basic mechanism of endothelial resealing during PMN transendothelial migration. PMID:9782120

  2. Human soluble phospholipase A2 receptor is an inhibitor of the integrin-mediated cell migratory response to collagen-I.

    PubMed

    Watanabe, Kazunori; Watanabe, Kazuhiro; Watanabe, Yosuke; Fujioka, Daisuke; Nakamura, Takamitsu; Nakamura, Kazuto; Obata, Jun-Ei; Kugiyama, Kiyotaka

    2018-05-23

    Murine membrane-bound phospholipase A 2 receptor 1 (PLA 2 R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA 2 R. This study examined whether human soluble PLA 2 R may have biological functions and whether soluble PLA 2 R may exist in human plasma. Here, we showed that human recombinant soluble PLA 2 R (rsPLA 2 R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin β1 on the cell surface of HEK293 cells. As a result, rsPLA 2 R suppressed integrin β1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA 2 R released from HEK293 cells transfected with a construct encoding human soluble PLA 2 R. rsPLA 2 R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA 2 R with collagen-I. In addition, rsPLA 2 R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound full-length PLA 2 R. Immunoprecipitation and Western blotting showed the existence of immuno-reactive PLA 2 R in human plasma. In conclusion, human recombinant soluble PLA 2 R inhibits integrin β1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immuno-reactive PLA 2 R in human plasma has the same properties as rsPLA 2 R.

  3. Clinical features, course and prognosis of idiopathic membranous nephropathy depending on the presence of antibodies against M-type phospholipase A2 receptor.

    PubMed

    Jatem Escalante, Elías; Segarra Medrano, Alfons; Carnicer Cáceres, Clara; Martín-Gómez, M Adoración; Salcedo Allende, María Teresa; Ostos Roldan, Helena; Agraz Pamplona, Irene

    2015-01-01

    In membranous nephropathy, the presence of antibodies against M-type phospholipase A2 receptor is considered highly specific for idiopathic forms. However, no specific association to a particular clinical profile has been found for such antibodies. To assess potential differences in initial clinical profile, course and prognosis of idiopathic membranous nephropathy depending on the presence of anti-PLA2R antibodies. Eighty-five patients with idiopathic membranous nephropathy were included (55 anti-PLA2R-positive and 30 anti-PLA2R-negative). Clinical, biochemical and pathological variables were recorded at the time of diagnosis. Frequency of spontaneous remission, incidence of response to first-line therapy, frequency and number of recurrences, survival of renal function free from renal replacement therapy, survival of renal function free from chronic renal insufficiency and frequency of occurrence of malignant, infectious or autoimmune diseases during follow-up were recorded. At the time of diagnosis, anti-PLA2R-negative patients were significantly older and had a higher frequency of spontaneous remission. No differences were noted in the response to first-line treatment, frequency and number of recurrences, survival of renal function free from renal replacement therapy, or survival of renal function free from chronic renal insufficiency. Anti-PLA2R-negative patients with idiopathic membranous nephropathy were older and experienced spontaneous remission more often than anti-PLA2R-positive patients. No differences in terms of treatment response, recurrences, and final prognosis were observed between both groups of patients. Copyright © 2015 The Authors. Published by Elsevier España, S.L.U. All rights reserved.

  4. The potent inhibition of vapiprost, a novel thromboxane A2 receptor antagonist, on the secondary aggregation and ATP release of human platelets.

    PubMed

    Horie, S; Yamada, M; Satoh, M; Noritake, S; Hiraishi, S; Kizaki, K; Kurusu, O; Nakahara, T; Ishii, H; Kazama, M

    1997-06-01

    The inhibitory effects of vapiprost hydrochloride (vapiprost), a novel thromboxane A2 receptor antagonist, on platelet aggregation and ATP release were studied using platelet rich plasma (PRP) of humans, guinea pigs, rabbits and rats. In in vitro experiments with human platelet, vapiprost inhibited the aggregation and ATP release stimulated with U-46619, collagen or arachidonic acid (AA) at an IC50 of less than 2.1 x 10(-8) M. Vapiprost did not inhibit the primary aggregation or ATP release of human platelets stimulated with adenosine 5'-diphosphate (ADP), epinephrine (Epi) or platelet activating factor (PAF), but inhibited the secondary aggregation stimulated with those agonists at an IC50 of less than 1.3 x 10(-7) M. The sensitivity of platelets in various species of animals to vapiprost was in the following order: human > or = guinea pigs > rats > rabbits. In ex vivo experiments with guinea pigs which received a single oral dose of vapiprost, the agent demonstrated strong inhibition of ATP release from platelets stimulated with U-46619, collagen or AA at an ID50 of less than 25.8 micrograms/kg. These inhibitory effects were observed within 30 min and sustained for 24 h at a single dosage of 5 mg/kg of vapiprost. In AA-induced pulmonary infarction models of mice, the sudden death rates decreased significantly with the oral administration of 10 mg/kg or more of vapiprost. These results indicate that vapiprost effectively inhibits the secondary aggregation and ATP release of human platelets stimulated with various agonists, and that guinea pig and human platelets are similar in response to vapiprost. Furthermore, it was demonstrated in ex vivo experiments with guinea pigs that the inhibitory action of vapiprost appears rapidly and lasts for long periods.

  5. AmeriFlux CA-TP2 Ontario - Turkey Point 1989 Plantation White Pine

    DOE Data Explorer

    Arain, M. Altaf [McMaster University

    2016-01-01

    This is the AmeriFlux version of the carbon flux data for the site CA-TP2 Ontario - Turkey Point 1989 Plantation White Pine. Site Description - Plantation established in 1989 over sandy agriculture land

  6. AmeriFlux CA-TP3 Ontario - Turkey Point 1974 Plantation White Pine

    DOE Data Explorer

    Arain, M. Altaf [McMaster University

    2016-01-01

    This is the AmeriFlux version of the carbon flux data for the site CA-TP3 Ontario - Turkey Point 1974 Plantation White Pine. Site Description - White pine plantation established in 1974 over sandy abandoned land

  7. AmeriFlux CA-TP4 Ontario - Turkey Point 1939 Plantation White Pine

    DOE Data Explorer

    Arain, M. Altaf [McMaster University

    2016-01-01

    This is the AmeriFlux version of the carbon flux data for the site CA-TP4 Ontario - Turkey Point 1939 Plantation White Pine. Site Description - White pine plantation established in 1939 over sandy abandoned land

  8. The molecular mechanism of flop-selectivity and subsite recognition for an AMPA receptor allosteric modulator: Structures of GluA2 and GluA3 complexed with PEPA

    PubMed Central

    Ahmed, Ahmed H.; Ptak, Christopher P.; Oswald, Robert E.

    2011-01-01

    Glutamate receptors are important potential drug targets for cognitive enhancement and the treatment of schizophrenia in part because they are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system. One approach to the application of therapeutic agents to the AMPA subtype of glutamate receptors is the use of allosteric modulators, which promote dimerization by binding to a dimer interface thereby reducing desensitization and deactivation. AMPA receptors exist in two alternatively spliced variants (flip and flop) that differ in desensitization and receptor activation profiles. Most of the structural information on modulators of the AMPA receptor target the flip subtype. We report here the crystal structure of the flop-selective allosteric modulator, PEPA, bound to the binding domains of the GluA2 and GluA3 flop isoforms of AMPA receptors. Specific hydrogen bonding patterns can explain the preference for the flop isoform. This includes a bidentate hydrogen bonding pattern between PEPA and N754 of the flop isoforms of GluA2 and GluA3 (the corresponding position in the flip isoform is S754). Comparison with other allosteric modulators provides a framework for the development of new allosteric modulators with preferences for either the flip or flop isoforms. In addition to interactions with N/S754, specific interactions of the sulfonamide with conserved residues in the binding site are characteristics of a number of allosteric modulators. These, in combination, with variable interactions with five subsites on the binding surface lead to different stoichiometries, orientations within the binding pockets, and functional outcomes. PMID:20199107

  9. Somatic and Germline TP53 Alterations in Second Malignant Neoplasms from Pediatric Cancer Survivors.

    PubMed

    Sherborne, Amy L; Lavergne, Vincent; Yu, Katharine; Lee, Leah; Davidson, Philip R; Mazor, Tali; Smirnoff, Ivan V; Horvai, Andrew E; Loh, Mignon; DuBois, Steven G; Goldsby, Robert E; Neglia, Joseph P; Hammond, Sue; Robison, Leslie L; Wustrack, Rosanna; Costello, Joseph F; Nakamura, Alice O; Shannon, Kevin M; Bhatia, Smita; Nakamura, Jean L

    2017-04-01

    Purpose: Second malignant neoplasms (SMNs) are severe late complications that occur in pediatric cancer survivors exposed to radiotherapy and other genotoxic treatments. To characterize the mutational landscape of treatment-induced sarcomas and to identify candidate SMN-predisposing variants, we analyzed germline and SMN samples from pediatric cancer survivors. Experimental Design: We performed whole-exome sequencing (WES) and RNA sequencing on radiation-induced sarcomas arising from two pediatric cancer survivors. To assess the frequency of germline TP53 variants in SMNs, Sanger sequencing was performed to analyze germline TP53 in 37 pediatric cancer survivors from the Childhood Cancer Survivor Study (CCSS) without any history of a familial cancer predisposition syndrome but known to have developed SMNs. Results: WES revealed TP53 mutations involving p53's DNA-binding domain in both index cases, one of which was also present in the germline. The germline and somatic TP53- mutant variants were enriched in the transcriptomes for both sarcomas. Analysis of TP53- coding exons in germline specimens from the CCSS survivor cohort identified a G215C variant encoding an R72P amino acid substitution in 6 patients and a synonymous SNP A639G in 4 others, resulting in 10 of 37 evaluable patients (27%) harboring a germline TP53 variant. Conclusions: Currently, germline TP53 is not routinely assessed in patients with pediatric cancer. These data support the concept that identifying germline TP53 variants at the time a primary cancer is diagnosed may identify patients at high risk for SMN development, who could benefit from modified therapeutic strategies and/or intensive posttreatment monitoring. Clin Cancer Res; 23(7); 1852-61. ©2016 AACR . ©2016 American Association for Cancer Research.

  10. Somatic and germline TP53 alterations in second malignant neoplasms from pediatric cancer survivors

    PubMed Central

    Sherborne, Amy L.; Lavergne, Vincent; Yu, Katharine; Lee, Leah; Davidson, Philip R.; Mazor, Tali; Smirnoff, Ivan; Horvai, Andrew; Loh, Mignon; DuBois, Steven G.; Goldsby, Robert E.; Neglia, Joseph; Hammond, Sue; Robison, Leslie L.; Wustrack, Rosanna; Costello, Joseph; Nakamura, Alice O.; Shannon, Kevin; Bhatia, Smita; Nakamura, Jean L.

    2016-01-01

    Purpose Second malignant neoplasms (SMNs) are severe late complications that occur in pediatric cancer survivors exposed to radiotherapy and other genotoxic treatments. To characterize the mutational landscape of treatment-induced sarcomas and to identify candidate SMN-predisposing variants we analyzed germline and SMN samples from pediatric cancer survivors. Experimental Design We performed whole exome sequencing (WES) and RNA sequencing on radiation-induced sarcomas arising from two pediatric cancer survivors. To assess the frequency of germline TP53 variants in SMNs, Sanger sequencing was performed to analyze germline TP53 in thirty-seven pediatric cancer survivors from the Childhood Cancer Survivor Study (CCSS) without history of a familial cancer predisposition syndrome but known to have developed SMNs. Results WES revealed TP53 mutations involving p53’s DNA binding domain in both index cases, one of which was also present in the germline. The germline and somatic TP53 mutant variants were enriched in the transcriptomes for both sarcomas. Analysis of TP53 coding exons in germline specimens from the CCSS survivor cohort identified a G215C variant encoding an R72P amino acid substitution in six patients and a synonymous single nucleotide polymorphism A639G in four others, resulting in ten out of 37 evaluable patients (27%) harboring a germline TP53 variant. Conclusions Currently, germline TP53 is not routinely assessed in pediatric cancer patients. These data support the concept that identifying germline TP53 variants at the time a primary cancer is diagnosed may identify patients at high risk for SMN development, who could benefit from modified therapeutic strategies and/or intensive post-treatment monitoring. PMID:27683180

  11. Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor

    PubMed Central

    Honjo, Eijiro; Tamada, Taro; Maeda, Yoshitake; Koshiba, Takumi; Matsukura, Yasuko; Okamoto, Tomoyuki; Ishibashi, Matsujiro; Tokunaga, Masao; Kuroki, Ryota

    2005-01-01

    The granulocyte-colony stimulating factor (GCSF) receptor receives signals for regulating the maturation, proliferation and differentiation of the precursor cells of neutrophilic granulocytes. The signalling complex composed of two GCSFs (GCSF, 19 kDa) and two GCSF receptors (GCSFR, 34 kDa) consisting of an Ig-like domain and a cytokine-receptor homologous (CRH) domain was crystallized. A crystal of the complex was grown in 1.0 M sodium formate and 0.1 M sodium acetate pH 4.6 and belongs to space group P41212 (or its enantiomorph P43212), with unit-cell parameters a = b = 110.1, c = 331.8 Å. Unfortunately, this crystal form did not diffract beyond 5 Å resolution. Since the heterogeneity of GCSF receptor appeared to prevent the growth of good-quality crystals, the GCSF receptor was fractionated by anion-exchange chromatography. Crystals of the GCSF–fractionated GCSF receptor complex were grown as a new crystal form in 0.2 M ammonium phosphate. This new crystal form diffracted to beyond 3.0 Å resolution and belonged to space group P3121 (or its enantiomorph P3221), with unit-cell parameters a = b = 134.8, c = 105.7 Å. PMID:16511159

  12. The Structure of the Metal Transporter Tp34 and its Affinity for Divalent Metal Ions

    NASA Astrophysics Data System (ADS)

    Knutsen, Gregory; Deka, Ranjit; Brautigam, Chad; Tomchick, Diana; Machius, Mischa; Norgard, Michael

    2007-10-01

    Tp34 is periplasmic membrane protein of the nonculitvatable spirochete Treponema pallidum, the pathogen of syphillis. It was proposed that Tp34 is a divalent metal transporter, but the identity of the preferred metal ion(s) was unclear. In this study we investigated the ability of divalent metal ions to induce rTp34 dimerization using hydrodynamic techniques and determine the crystal structure of metal bound forms. Using analytical ultracentrifugation sedimentation velocity experiments, we determined that cobalt is superior to nickel at inducing the dimerization of rTp34. rTp34 was crystallized and selected crystals were incubated at a pH 7.5 with CuSO4 and NiSO4. Diffraction experiments were conducted and the processed electron density maps showed that copper was bound to the major metal binding site as well as to three additional minor binding sites. By contrast nickel was only bound to the major metal binding site in one monomer and to three additional minor sites. These results along with previous findings support evidence of Tp34 being involved with metal transport and/or iron utilization.

  13. Classification of TP53 Mutations and HPV Predict Survival in Advanced Larynx Cancer

    PubMed Central

    Scheel, Adam; Bellile, Emily; McHugh, Jonathan B.; Walline, Heather M.; Prince, Mark E.; Urba, Susan; Wolf, Gregory T.; Eisbruch, Avraham; Worden, Francis; Carey, Thomas E.; Bradford, Carol

    2016-01-01

    OBJECTIVE Assess TP53 functional mutations in the context of other biomarkers in advanced larynx cancer. STUDY DESIGN Prospective analysis of pretreatment tumor TP53, HPV, Bcl-xL and cyclin D1 status in stage III and IV larynx cancer patients in a clinical trial. METHODS TP53 exons 4-9 from 58 tumors were sequenced. Mutations were grouped using three classifications based on their expected function. Each functional group was analyzed for response to induction chemotherapy, time to surgery, survival, HPV status, p16INK4a, Bcl-xl and cyclin D1 expression. RESULTS TP53 Mutations were found in 22/58 (37.9%) patients with advanced larynx cancer, including missense mutations in 13/58 (22.4%) patients, nonsense mutations in 4/58 (6.9%), and deletions in 5/58 (8.6%). High risk HPV was found in 20/52 (38.5%) tumors. A classification based on crystal Evolutionary Action score of p53 (EAp53) distinguished missense mutations with high risk for decreased survival from low risk mutations (p=0.0315). A model including this TP53 classification, HPV status, cyclin D1 and Bcl-xL staining significantly predicts survival (p=0.0017). CONCLUSION EAp53 functional classification of TP53 mutants and biomarkers predict survival in advanced larynx cancer. PMID:27345657

  14. Classification of TP53 mutations and HPV predict survival in advanced larynx cancer.

    PubMed

    Scheel, Adam; Bellile, Emily; McHugh, Jonathan B; Walline, Heather M; Prince, Mark E; Urba, Susan; Wolf, Gregory T; Eisbruch, Avraham; Worden, Francis; Carey, Thomas E; Bradford, Carol

    2016-09-01

    Assess tumor suppressor p53 (TP53) functional mutations in the context of other biomarkers in advanced larynx cancer. Prospective analysis of pretreatment tumor TP53, human papillomavirus (HPV), Bcl-xL, and cyclin D1 status in stage III and IV larynx cancer patients in a clinical trial. TP53 exons 4 through 9 from 58 tumors were sequenced. Mutations were grouped using three classifications based on their expected function. Each functional group was analyzed for response to induction chemotherapy, time to surgery, survival, HPV status, p16INK4a, Bcl-xl, and cyclin D1 expression. TP53 mutations were found in 22 of 58 (37.9%) patients with advanced larynx cancer, including missense mutations in 13 of 58 (22.4%) patients, nonsense mutations in four of 58 (6.9%), and deletions in five of 58 (8.6%). High-risk HPV was found in 20 of 52 (38.5%) tumors. A classification based on Evolutionary Action score of p53 (EAp53) distinguished missense mutations with high risk for decreased survival from low-risk mutations (P = 0.0315). A model including this TP53 classification, HPV status, cyclin D1, and Bcl-xL staining significantly predicts survival (P = 0.0017). EAp53 functional classification of TP53 mutants and biomarkers predict survival in advanced larynx cancer. NA. Laryngoscope, 126:E292-E299, 2016. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

  15. A Valuable Tool in Predicting Poor Outcome due to Sepsis in Pediatric Intensive Care Unit: Tp-e/QT Ratio.

    PubMed

    Ozdemir, Rahmi; Isguder, Rana; Kucuk, Mehmet; Karadeniz, Cem; Ceylan, Gokhan; Katipoglu, Nagehan; Yilmazer, Murat Muhtar; Yozgat, Yilmaz; Mese, Timur; Agin, Hasan

    2016-10-01

    To assess the feasibility of 12-lead electrocardiographic (ECG) measures such as P wave dispersion (PWd), QT interval, QT dispersion (QTd), Tp-e interval, Tp-e/QT and Tp-e/QTc ratio in predicting poor outcome in patients diagnosed with sepsis in pediatric intensive care unit (PICU). Ninety-three patients diagnosed with sepsis, severe sepsis or septic shock and 103 age- and sex-matched healthy children were enrolled into the study. PWd, QT interval, QTd, Tp-e interval and Tp-e/QT, Tp-e/QTc ratios were obtained from a 12-lead electrocardiogram. PWd, QTd, Tp-e interval and Tp-e/QT, Tp-e/QTc ratios were significantly higher in septic patients compared with the controls. During the study period, 41 patients had died. In multivariate logistic regression analyses, only Tp-e/QT ratio was found to be an independent predictor of mortality. The ECG measurements can predict the poor outcome in patients with sepsis. The Tp-e/QT ratio may be a valuable tool in predicting mortality for patients with sepsis in the PICU. © The Author [2016]. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Pharmacological stress myocardial perfusion imaging with the potent and selective A(2A) adenosine receptor agonists ATL193 and ATL146e administered by either intravenous infusion or bolus injection.

    PubMed

    Glover, D K; Ruiz, M; Takehana, K; Petruzella, F D; Riou, L M; Rieger, J M; Macdonald, T L; Watson, D D; Linden, J; Beller, G A

    2001-09-04

    Adenosine (Ado) and dipyridamole are alternatives to exercise stress for myocardial perfusion imaging. Though generally safe, side effects frequently occur that cause patient discomfort and sometimes lead to premature termination of the study or require aminophylline administration. Recently, a new class of A(2A) Ado receptor agonists was synthesized. ATL193 and ATL146e are 2-propynylcyclohexyl-5'-N-ethylcarboxamido derivatives of Ado. The study goals were to evaluate the potency and selectivity of these new compounds on recombinant canine Ado receptors and to evaluate their hemodynamic properties in dogs to assess their usefulness as vasodilators for myocardial perfusion imaging. In assays of recombinant canine Ado receptors, ATL-193 and ATL-146e were highly selective for the A(2A) over the A(1) and A(3) receptors and were more potent than MRE-0470 and CGS-21680. In 16 anesthetized dogs, the agonists were administered by infusion (ATL-193; n=7 normal) or bolus injection (ATL-146e; n=9 critical left anterior descending coronary artery stenosis), and hemodynamic responses were compared with those of Ado. Both agonists produced dose-dependent coronary flow (CF) elevation without provoking the hypotension observed with Ado. After an ATL-146e bolus, the CF increase was sustained for several minutes, providing ample time for injection and myocardial uptake of (99m)Tc-sestamibi, and CF returned to baseline within 20 minutes. The CF increase was completely blocked by the selective A(2A) antagonist ZM241385 (3 microgram. kg(-1). min(-1)). ATL-193 and ATL-146e are highly potent and selective Ado A(2A) receptor agonists with excellent potential for use as vasodilators for myocardial perfusion imaging. An important advantage of ATL-146e is the ability to administer it by bolus injection.

  17. Folic acid inhibits COLO-205 colon cancer cell proliferation through activating the FRα/c-SRC/ERK1/2/NFκB/TP53 pathway: in vitro and in vivo studies

    PubMed Central

    Kuo, Chun-Ting; Chang, Chieh; Lee, Wen-Sen

    2015-01-01

    To investigate the molecular mechanism underlying folic acid (FA)-induced anti-colon caner activity, we showed that FA caused G0/G1 arrest in COLO-205. FA activated the proto-oncogene tyrosine-protein kinase Src (c-SRC)-mediated signaling pathway to enhance nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) nuclear translocation and binding onto the tumor protein p53 (TP53) gene promoter, and up-regulated expressions of TP53, cyclin-dependent kinase inhibitor 1A (CDKN1A) and cyclin-dependent kinase inhibitor 1B (CDKN1B). Knock-down of TP53 abolished FA-induced increases in the levels of CDKN1A and CDKN1B protein and G0/G1 arrest in COLO-205. Knock-down of folate receptor alpha (FRα) abolished FA-induced activations in the c-SRC-mediated pathway and increases in the levels of CDKN1A, CDKN1B and TP53 protein. These data suggest that FA inhibited COLO-205 proliferation through activating the FRα/c-SRC/mitogen-activated protein kinase 3/1 (ERK1/2)/NFκB/TP53 pathway-mediated up-regulations of CDKN1A and CDKN1B protein. In vivo studies demonstrated that daily i.p. injections of FA led to profound regression of the COLO-205 tumors and prolong the lifespan. In these tumors, the levels of CDKN1A, CDKN1B and TP53 protein were increased and von willebrand factor (VWF) protein levels were decreased. These findings suggest that FA inhibits COLO-205 colon cancer growth through anti-cancer cell proliferation and anti-angiogenesis. PMID:26056802

  18. Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma.

    PubMed

    Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne-Marie; Brenton, James D

    2016-10-01

    TP53 mutations are ubiquitous in high-grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low-grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged-amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain-of-function (GOF or nonsynonymous), loss-of-function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.

  19. Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma

    PubMed Central

    Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne‐Marie

    2016-01-01

    Abstract TP53 mutations are ubiquitous in high‐grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low‐grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged‐amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain‐of‐function (GOF or nonsynonymous), loss‐of‐function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low‐grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of

  20. Effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output and vascular resistance in acute heart failure in the anaesthetized rat

    PubMed Central

    Nekooeian, Ali A; Tabrizchi, Reza

    1998-01-01

    The effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output, blood pressure, mean circulatory filling pressure (Pmcf), arterial and venous resistances, heart rate and left ventricular end-diastolic pressure were assessed in rats with acute heart failure by means of coronary artery occlusion.Animals (n=6 in each group) were divided into five groups: group I, sham-operated vehicle-treated (0.9% saline; 0.018 mL min−1); groups II-V, subject to coronary artery occlusion and treated with vehicle (0.9% saline; 0.018 ml min−1) and CGS 21680 (0.1, 0.3 and 1.0 μg kg−1 min−1), respectively. Haemodynamic measurements were taken one hour after completion of surgery, ninety minutes after coronary artery occlusion (except in group I), and fifteen minutes after infusion of saline or CGS 21680.Baseline haemodynamic measurements before occlusion were found not to differ significantly between the different groups of animals. However, after occlusion, cardiac output, rate of rise in left ventricular pressure (+dP/dt) and blood pressure were significantly reduced when compared to corresponding values in sham-operated animals. In addition, occlusion of the coronary artery resulted in a significant elevation in venous resistance, Pmcf and left ventricular end-diastolic pressure as compared to corresponding values in sham-operated animals.Infusion with CGS 21680 at the highest dose significantly reduced blood pressure, arterial resistance and left ventricular end-diastolic pressure when compared to occluded vehicle-treated animals (group II). Administration of CGS 21680 at the highest dose also significantly increased cardiac output (28%) and heart rate (10%) in comparison to occluded vehicle-treated animals. In addition, the highest dose of CGS 21680 significantly reduced Pmcf (9%) and venous resistance (62%) in comparison to occluded vehicle-treated animals. Administration of CGS 21680 did not significantly affect +dP/dt when compared

  1. Effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output and vascular resistance in acute heart failure in the anaesthetized rat.

    PubMed

    Nekooeian, A A; Tabrizchi, R

    1998-04-01

    1. The effects of CGS 21680, a selective A2A adenosine receptor agonist, on cardiac output, blood pressure, mean circulatory filling pressure (Pmcf), arterial and venous resistances, heart rate and left ventricular end-diastolic pressure were assessed in rats with acute heart failure by means of coronary artery occlusion. 2. Animals (n=6 in each group) were divided into five groups: group I, sham-operated vehicle-treated (0.9% saline; 0.018 mL min(-1)); groups II-V, subject to coronary artery occlusion and treated with vehicle (0.9% saline; 0.018 ml min(-1)) and CGS 21680 (0.1, 0.3 and 1.0 microg kg(-1) min(-1)), respectively. Haemodynamic measurements were taken one hour after completion of surgery, ninety minutes after coronary artery occlusion (except in group I), and fifteen minutes after infusion of saline or CGS 21680. 3. Baseline haemodynamic measurements before occlusion were found not to differ significantly between the different groups of animals. However, after occlusion, cardiac output, rate of rise in left ventricular pressure (+ dP/dt) and blood pressure were significantly reduced when compared to corresponding values in sham-operated animals. In addition, occlusion of the coronary artery resulted in a significant elevation in venous resistance, Pmcf and left ventricular end-diastolic pressure as compared to corresponding values in sham-operated animals. 4. Infusion with CGS 21680 at the highest dose significantly reduced blood pressure, arterial resistance and left ventricular end-diastolic pressure when compared to occluded vehicle-treated animals (group II). Administration of CGS 21680 at the highest dose also significantly increased cardiac output (28%) and heart rate (10%) in comparison to occluded vehicle-treated animals. In addition, the highest dose of CGS 21680 significantly reduced Pmcf (9%) and venous resistance (62%) in comparison to occluded vehicle-treated animals. Administration of CGS 21680 did not significantly affect +dP/dt when

  2. The role of serotonin-2 (5-HT2) and dopamine receptors in the behavioral actions of the 5-HT2A/2C agonist, DOI, and putative 5-HT2C inverse agonist, SR46349B.

    PubMed

    Scarlota, Laura C; Harvey, John A; Aloyo, Vincent J

    2011-02-01

    Atypical antipsychotic efficacy is often attributed to actions at serotonin-2 (5-HT(2)) and dopamine receptors, indicating a potential benefit of understanding the interplay between these systems. Currently, it is known that 5-HT(2) receptors modulate dopamine release, although the role of specific dopamine receptors in 5-HT(2)-mediated behavior is not well understood. We examined the role of 5-HT(2A), 5-HT(2C), and dopamine (D1 and D2) receptors in the behavioral response to a 5-HT(2A/2C) agonist (DOI) and 5-HT(2A/2C) antagonist (SR46349B). Effects were assessed by measuring rabbit head bobs (previously characterized as 5-HT(2A) receptor-mediated) and body shakes (5-HT(2C)-mediated). As expected, DOI produced head bobs and body shakes, and these DOI-elicited behaviors were attenuated by the SR46349B pretreatment. Unexpectedly, SR46349B also induced head bobs when administered alone. However, SR46349B-elicited head bobs are distinguishable from those produced by DOI since the 5-HT(2A) antagonist, ketanserin, only attenuated DOI-elicited head bobs. Conversely, 5-HT(2C) ligands (SB242084 and SB206553) inhibited SR46349B but not DOI-induced head bobs. Furthermore, when administered alone, SB206553 (a 5-HT(2C) inverse agonist) produced head bobs, indicating the behavior can be either 5-HT(2A) or 5-HT(2C) mediated. Next, it was revealed that D1 and D2 receptors play a role in DOI-elicited head bobs, but only D1 receptors are required for SR46349B-elicited head bobs. 5-HT(2A) receptor agonism and 5-HT(2C) inverse agonism produce the same behavior, likely due to similar downstream actions at D1 receptors. Consequently, 5-HT(2C) agonism or D1 agonism may be effective therapies for disorders, such as schizophrenia, currently being treated with 5-HT(2A) antagonists.

  3. Evaluation of the Recombinant Protein TpF1 of Treponema pallidum for Serodiagnosis of Syphilis

    PubMed Central

    Jiang, Chuanhao; Zhao, Feijun; Xiao, Jinhong; Zeng, Tiebing; Yu, Jian; Ma, Xiaohua; Wu, Haiying

    2013-01-01

    Syphilis is a chronic infection caused by Treponema pallidum subsp. pallidum, and diagnosis with sensitive and specific methods is a challenging process that is important for its prevention and treatment. In the present study, we established a recombinant protein TpF1-based indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and a Western blot assay for human and rabbit sera. The 20-kDa recombinant protein TpF1 was detected by Western blotting performed with sera from rabbits immunized with recombinant TpF1 and infected with the T. pallidum Nichols strain and T. pallidum clinical isolates but was not detected by Western blotting with sera from uninfected rabbits. The sensitivity of the recombinant protein was determined by screening sera from individuals with primary, secondary, latent, and congenital syphilis (n = 82). The specificity of the recombinant protein was determined by screening sera from uninfected controls (n = 30) and individuals with potentially cross-reactive infections, including Lyme disease (n = 30) and leptospirosis (n = 5). The sensitivities of TpF1-based ELISAs were 93.3%, 100%, 100%, and 100% for primary, secondary, latent, and congenital syphilis, respectively, and the specificities were all 100% for sera from uninfected controls and individuals with potentially cross-reactive infections. In Western blot assays, the sensitivities and specificities of TpF1 for human sera were all 100%. The reactivities of TpF1 with syphilitic sera were proportional to the titers of the T. pallidum particle agglutination (TPPA) assay. These data indicate that the recombinant protein TpF1 is a highly immunogenic protein in human and rabbit infections and a promising marker for the screening of syphilis. PMID:23945159

  4. Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary.

    PubMed

    Ahmed, Ahmed Ashour; Etemadmoghadam, Dariush; Temple, Jillian; Lynch, Andy G; Riad, Mohamed; Sharma, Raghwa; Stewart, Colin; Fereday, Sian; Caldas, Carlos; Defazio, Anna; Bowtell, David; Brenton, James D

    2010-05-01

    Numerous studies have tested the association between TP53 mutations in ovarian cancer and prognosis but these have been consistently confounded by limitations in study design, methodology, and/or heterogeneity in the sample cohort. High-grade serous (HGS) carcinoma is the most clinically important histological subtype of ovarian cancer. As these tumours may arise from the ovary, Fallopian tube or peritoneum, they are collectively referred to as high-grade pelvic serous carcinoma (HGPSC). To identify the true prevalence of TP53 mutations in HGPSC, we sequenced exons 2-11 and intron-exon boundaries in tumour DNA from 145 patients. HGPSC cases were defined as having histological grade 2 or 3 and FIGO stage III or IV. Surprisingly, pathogenic TP53 mutations were identified in 96.7% (n = 119/123) of HGPSC cases. Molecular and pathological review of mutation-negative cases showed evidence of p53 dysfunction associated with copy number gain of MDM2 or MDM4, or indicated the exclusion of samples as being low-grade serous tumours or carcinoma of uncertain primary site. Overall, p53 dysfunction rate approached 100% of confirmed HGPSCs. No association between TP53 mutation and progression-free or overall survival was found. From this first comprehensive mapping of TP53 mutation rate in a homogeneous group of HGPSC patients, we conclude that mutant TP53 is a driver mutation in the pathogenesis of HGPSC cancers. Because TP53 mutation is almost invariably present in HGPSC, it is not of substantial prognostic or predictive significance. Copyright (c) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  5. Mutations in TP53 are a prognostic factor in colorectal hepatic metastases undergoing surgical resection.

    PubMed

    Molleví, David G; Serrano, Teresa; Ginestà, Mireia M; Valls, Joan; Torras, Jaume; Navarro, Matilde; Ramos, Emilio; Germà, Josep R; Jaurrieta, Eduardo; Moreno, Víctor; Figueras, Joan; Capellà, Gabriel; Villanueva, Alberto

    2007-06-01

    The aim of this study was to analyze the prognostic value of TP53 mutations in a consecutive series of patients with hepatic metastases (HMs) from colorectal cancer undergoing surgical resection. Ninety-one patients with liver metastases from colorectal carcinoma were included. Mutational analysis of TP53, exons 4-10, was performed by single-strand conformation polymorphism and sequencing. P53 and P21 protein immunostaining was assessed. Multivariate Cox models were adjusted for gender, number of metastasis, resection margin, presence of TP53 mutations and chemotherapy treatment. Forty-six of 91 (50.05%) metastases showed mutations in TP53, observed mainly in exons 5-8, although 14.3% (n = 13) were located in exons 9 and 10. Forty percent (n = 22) were protein-truncating mutations. TP53 status associated with multiple (> or =3) metastases (65.6%, P = 0.033), advanced primary tumor Dukes' stage (P = 0.011) and younger age (<57 years old, P = 0.03). Presence of mutation associated with poor prognosis in univariate (P = 0.017) and multivariate Cox model [hazard ratio (HR) = 1.80, 95% confidence interval (CI) = 1.07-3.06, P = 0.028]. Prognostic value was maintained in patients undergoing radical resection (R0 series, n = 79, P = 0.014). Mutation associated with a worse outcome in chemotherapy-treated patients (HR = 2.54, 95% CI = 1.12-5.75, P = 0.026). The combination of > or =3 metastases and TP53 mutation identified a subset of patients with very poor prognosis (P = 0.009). P53 and P21 protein immunostaining did not show correlation with survival. TP53 mutational status seems to be an important prognostic factor in patients undergoing surgical resection of colorectal cancer HMs.

  6. Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.

    PubMed

    Canela, Laia; Selga, Elisabet; García-Martínez, Juan Manuel; Amaral, Olavo B; Fernández-Dueñas, Víctor; Alberch, Jordi; Canela, Enric I; Franco, Rafael; Noé, Véronique; Lluís, Carme; Ciudad, Carlos J; Ciruela, Francisco

    2012-10-25

    G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the β-synuclein (β-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since β-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that β-Syn might act protectively against α-Syn neuropathology. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. The A2B Adenosine Receptor Modulates the Epithelial– Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells

    PubMed Central

    Giacomelli, Chiara; Daniele, Simona; Romei, Chiara; Tavanti, Laura; Neri, Tommaso; Piano, Ilaria; Celi, Alessandro; Martini, Claudia; Trincavelli, Maria L.

    2018-01-01

    The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different

  8. Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

    PubMed

    Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

    2015-04-29

    Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

  9. The Levels and Distribution of TN, TP and TOC in the South China Sea

    NASA Astrophysics Data System (ADS)

    Wang, H.; Han, D.

    2012-04-01

    The marine biogeochemistries of carbon, nitrogen and phosphorous have come under increased scrutiny because of their close involvement in climate change and coastal eutrophication. The South China Sea is unique in that located in a subtropical zone, and therefore represents an important regime for biogeochemical studies. However, to our knowledge, few data are available for total nitrogen (TN), total phosphorous (TP) and total organic carbon (TOC) in South Sea, China. The present study aims to contribute to the knowledge of their status through investigating the level and distribution of TN, TP and TOC in South China Sea. A total of 108 seawater samples of 11 sites in south sea, china were collected during August 29- September 4, 2006. An automated and simultaneous method for determination of TN and TOC was applied to all seawater samples. The combined system allowed simultaneous determination for TOC and TN in the same sample using a single injection and provided low detection limits and excellent linear ranges for both TOC and TN. The risk of contamination has been remarkably reduced due to the minimal sample manipulation and automated analyses. And quantitative analyses of TP in seawater were accomplished by a typical chemical method. Concentration ranges of TN and TP were 0.06-0.67, and 0.003-0.071 mg/L, respectively, as well as that of TOC were 0.23-2.51mg/L. The values of TN and TP showed that the status of nutrition is relatively better in south china sea than other marine areas. Moreover, the upright change trend of TN concentration level as well as TP and TOC according to the experimental results at the total 11 sites are simultaneity studied. The concentration of TN initial increases with the increasing of the depth, later the value becomes almost constant. In contrast, the concentration of TOC reduces with the increasing of the depth, later the value becomes almost unchangeable. Compared with the trend of TN and TOC, that of TP appears relatively stable

  10. The TP53 dependence of radiation-induced chromosome instability in human lymphoblastoid cells

    NASA Technical Reports Server (NTRS)

    Schwartz, Jeffrey L.; Jordan, Robert; Evans, Helen H.; Lenarczyk, Marek; Liber, Howard

    2003-01-01

    The dose and TP53 dependence for the induction of chromosome instability were examined in cells of three human lymphoblastoid cell lines derived from WIL2 cells: TK6, a TP53-normal cell line, NH32, a TP53-knockout created from TK6, and WTK1, a WIL2-derived cell line that spontaneously developed a TP53 mutation. Cells of each cell line were exposed to (137)Cs gamma rays, and then surviving clones were isolated and expanded in culture for approximately 35 generations before the frequency and characteristics of the instability were analyzed. The presence of dicentric chromosomes, formed by end-to-end fusions, served as a marker of chromosomal instability. Unexposed TK6 cells had low levels of chromosomal instability (0.002 +/- 0.001 dicentrics/cell). Exposure of TK6 cells to doses as low as 5 cGy gamma rays increased chromosome instability levels nearly 10-fold to 0.019 +/- 0.008 dicentrics/cell. There was no further increase in instability levels beyond 5 cGy. In contrast to TK6 cells, unexposed cultures of WTK1 and NH32 cells had much higher levels of chromosome instability of 0.034 +/- 0.007 and 0.041 +/- 0.009, respectively, but showed little if any effect of radiation on levels of chromosome instability. The results suggest that radiation exposure alters the normal TP53-dependent cell cycle checkpoint controls that recognize alterations in telomere structure and activate apoptosis.

  11. Prognostic and predictive value of TP53 mutations in node-positive breast cancer patients treated with anthracycline- or anthracycline/taxane-based adjuvant therapy: results from the BIG 02-98 phase III trial

    PubMed Central

    2012-01-01

    Abstract Introduction Pre-clinical data suggest p53-dependent anthracycline-induced apoptosis and p53-independent taxane activity. However, dedicated clinical research has not defined a predictive role for TP53 gene mutations. The aim of the current study was to retrospectively explore the prognosis and predictive values of TP53 somatic mutations in the BIG 02-98 randomized phase III trial in which women with node-positive breast cancer were treated with adjuvant doxorubicin-based chemotherapy with or without docetaxel. Methods The prognostic and predictive values of TP53 were analyzed in tumor samples by gene sequencing within exons 5 to 8. Patients were classified according to p53 protein status predicted from TP53 gene sequence, as wild-type (no TP53 variation or TP53 variations which are predicted not to modify p53 protein sequence) or mutant (p53 nonsynonymous mutations). Mutations were subcategorized according to missense or truncating mutations. Survival analyses were performed using the Kaplan-Meier method and log-rank test. Cox-regression analysis was used to identify independent predictors of outcome. Results TP53 gene status was determined for 18% (520 of 2887) of the women enrolled in BIG 02-98. TP53 gene variations were found in 17% (90 of 520). Nonsynonymous p53 mutations, found in 16.3% (85 of 520), were associated with older age, ductal morphology, higher grade and hormone-receptor negativity. Of the nonsynonymous mutations, 12.3% (64 of 520) were missense and 3.6% were truncating (19 of 520). Only truncating mutations showed significant independent prognostic value, with an increased recurrence risk compared to patients with non-modified p53 protein (hazard ratio = 3.21, 95% confidence interval = 1.740 to 5.935, P = 0.0002). p53 status had no significant predictive value for response to docetaxel. Conclusions p53 truncating mutations were uncommon but associated with poor prognosis. No significant predictive role for p53 status was detected. Trial

  12. Genetic deletion of GPR52 enhances the locomotor-stimulating effect of an adenosine A2A receptor antagonist in mice: A potential role of GPR52 in the function of striatopallidal neurons.

    PubMed

    Nishiyama, Keiji; Suzuki, Hirobumi; Maruyama, Minoru; Yoshihara, Tomoki; Ohta, Hiroyuki

    2017-09-01

    G protein-coupled receptor 52 (GPR52) is largely co-expressed with dopamine D 2 receptor (DRD2) in the striatum and nucleus accumbens, and this expression pattern is similar to that of adenosine A 2A receptor (ADORA2A). GPR52 has been proposed as a therapeutic target for positive symptoms of schizophrenia, based on observations from pharmacological and transgenic mouse studies. However, the physiological role of GPR52 in dopaminergic functions in the basal ganglia remains unclear. Here, we used GPR52 knockout (KO) mice to examine the role of GPR52 in dopamine receptor-mediated and ADORA2A-mediated locomotor activity and dopamine receptor signaling. High expression of GPR52 protein in the striatum, nucleus accumbens, and lateral globus pallidus of wild type (WT) littermates was confirmed by immunohistochemical analysis. GPR52 KO and WT mice exhibited almost identical locomotor responses to the dopamine releaser methamphetamine and the N-methyl-d-aspartate antagonist MK-801. In contrast, the locomotor response to the ADORA2A antagonist istradefylline was significantly augmented in GPR52 KO mice compared to WT mice. Gene expression analysis revealed that striatal expression of DRD2, but not of dopamine D 1 receptor and ADORA2A, was significantly decreased in GPR52 KO mice. Moreover, a significant reduction in the mRNA expression of enkephalin, a marker of the activity of striatopallidal neurons, was observed in the striatum of GPR52 KO mice, suggesting that GPR52 deletion could enhance DRD2 signaling. Taken together, these results imply the physiological relevance of GPR52 in modulating the function of striatopallidal neurons, possibly by interaction of GPR52 with ADORA2A and DRD2. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A more accurate detection of codon 72 polymorphism and LOH of the TP53 gene.

    PubMed

    Baccouche, Sami; Mabrouk, Imed; Said, Salem; Mosbah, Ali; Jlidi, Rachid; Gargouri, Ali

    2003-01-10

    The polymorphism at codon 72 of the TP53 gene has been extensively studied for its involvement in cancerogenesis and loss of heterozygosity (LOH) detection. Usually, the exon 4 of the TP53 gene is amplified by polymerase chain reaction (PCR) on DNA extracted from blood and tumor tissues, then digested by AccII. In the case of heterozygosity, the comparison of AccII profile from blood and tumor DNA PCR products allowed the identification of a potential LOH in the TP53 locus. This method can be hindered by a partial AccII digestion and/or DNA contamination of non-tumor cells. To circumvent these problems, we have developed a new approach by using the AccII restriction site between exon 4 and exon 6. The PCR amplification of exon 4-6, followed by AccII digestion allowed us to detect without ambiguity any LOH case.

  14. Impaired thromboxane receptor dimerization reduces signaling efficiency: A potential mechanism for reduced platelet function in vivo.

    PubMed

    Capra, Valérie; Mauri, Mario; Guzzi, Francesca; Busnelli, Marta; Accomazzo, Maria Rosa; Gaussem, Pascale; Nisar, Shaista P; Mundell, Stuart J; Parenti, Marco; Rovati, G Enrico

    2017-01-15

    Thromboxane A 2 is a potent mediator of inflammation and platelet aggregation exerting its effects through the activation of a G protein-coupled receptor (GPCR), termed TP. Although the existence of dimers/oligomers in Class A GPCRs is widely accepted, their functional significance still remains controversial. Recently, we have shown that TPα and TPβ homo-/hetero-dimers interact through an interface of residues in transmembrane domain 1 (TM1) whose disruption impairs dimer formation. Here, biochemical and pharmacological characterization of this dimer deficient mutant (DDM) in living cells indicates a significant impairment in its response to agonists. Interestingly, two single loss-of-function TPα variants, namely W29C and N42S recently identified in two heterozygous patients affected by bleeding disorders, match some of the residues mutated in our DDM. These two naturally occurring variants display a reduced potency to TP agonists and are characterized by impaired dimer formation in transfected HEK-293T cells. These findings provide proofs that lack of homo-dimer formation is a crucial process for reduced TPα function in vivo, and might represent one molecular mechanism through which platelet TPα receptor dysfunction affects the patient(s) carrying these mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children's Oncology Group.

    PubMed

    Ooms, Ariadne H A G; Gadd, Samantha; Gerhard, Daniela S; Smith, Malcolm A; Guidry Auvil, Jaime M; Meerzaman, Daoud; Chen, Qing-Rong; Hsu, Chih Hao; Yan, Chunhua; Nguyen, Cu; Hu, Ying; Ma, Yussanne; Zong, Zusheng; Mungall, Andrew J; Moore, Richard A; Marra, Marco A; Huff, Vicki; Dome, Jeffrey S; Chi, Yueh-Yun; Tian, Jing; Geller, James I; Mullighan, Charles G; Ma, Jing; Wheeler, David A; Hampton, Oliver A; Walz, Amy L; van den Heuvel-Eibrink, Marry M; de Krijger, Ronald R; Ross, Nicole; Gastier-Foster, Julie M; Perlman, Elizabeth J

    2016-11-15

    To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs). All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582-91. ©2016 AACR. ©2016 American Association for Cancer Research.

  16. Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children’s Oncology Group

    PubMed Central

    Ooms, Ariadne H.A.G.; Gadd, Samantha; Gerhard, Daniela S.; Smith, Malcolm