Sample records for a2a receptor a2ar

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925.

    PubMed

    Rafehi, Muhammad; Burbiel, Joachim C; Attah, Isaac Y; Abdelrahman, Aliaa; Müller, Christa E

    2017-03-01

    The G q protein-coupled, ATP- and UTP-activated P2Y 2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y 2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y 2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

  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. Glycine Potentiates AMPA Receptor Function through Metabotropic Activation of GluN2A-Containing NMDA Receptors

    PubMed Central

    Li, Li-Jun; Hu, Rong; Lujan, Brendan; Chen, Juan; Zhang, Jian-Jian; Nakano, Yasuko; Cui, Tian-Yuan; Liao, Ming-Xia; Chen, Jin-Cao; Man, Heng-Ye; Feng, Hua; Wan, Qi

    2016-01-01

    NMDA receptors are Ca2+-permeable ion channels. The activation of NMDA receptors requires agonist glutamate and co-agonist glycine. Recent evidence indicates that NMDA receptor also has metabotropic function. Here we report that in cultured mouse hippocampal neurons, glycine increases AMPA receptor-mediated currents independent of the channel activity of NMDA receptors and the activation of glycine receptors. The potentiation of AMPA receptor function by glycine is antagonized by the inhibition of ERK1/2. In the hippocampal neurons and in the HEK293 cells transfected with different combinations of NMDA receptors, glycine preferentially acts on GluN2A-containing NMDA receptors (GluN2ARs), but not GluN2B-containing NMDA receptors (GluN2BRs), to enhance ERK1/2 phosphorylation independent of the channel activity of GluN2ARs. Without requiring the channel activity of GluN2ARs, glycine increases AMPA receptor-mediated currents through GluN2ARs. Thus, these results reveal a metabotropic function of GluN2ARs in mediating glycine-induced potentiation of AMPA receptor function via ERK1/2 activation. PMID:27807405

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. Common α2A and α2C adrenergic receptor polymorphisms do not affect plasma membrane trafficking.

    PubMed

    Hurt, Carl M; Sorensen, Matt W; Angelotti, Timothy

    2014-06-01

    Various naturally occurring polymorphic forms of human G protein-coupled receptors (GPCRs) have been identified and linked to diverse pathological diseases, including receptors for vasopressin type 2 (nephrogenic diabetes insipidus) and gonadotropin releasing hormone (hypogonadotropic hypogonadism). In most cases, polymorphic amino acid mutations disrupt protein folding, altering receptor function as well as plasma membrane expression. Other pathological GPCR variants have been found that do not alter receptor function, but instead affect only plasma membrane trafficking (e.g., delta opiate and histamine type 1 receptors). Thus, altered membrane trafficking with retained receptor function may be another mechanism causing polymorphic GPCR dysfunction. Two common human α2A and α2C adrenergic receptor (AR) variants have been identified (α2A N251K and α2C Δ322-325 ARs), but pharmacological analysis of ligand binding and second messenger signaling has not consistently demonstrated altered receptor function. However, possible alterations in plasma membrane trafficking have not been investigated. We utilized a systematic approach previously developed for the study of GPCR trafficking motifs and accessory proteins to assess whether these α2 AR variants affected intracellular trafficking or plasma membrane expression. By combining immunofluorescent microscopy, glycosidic processing analysis, and quantitative fluorescent-activated cell sorting (FACS), we demonstrate that neither variant receptor had altered intracellular localization, glycosylation, nor plasma membrane expression compared to wild-type α2 ARs. Therefore, pathopharmacological properties of α2A N251K and α2C Δ322-325 ARs do not appear to be due to altered receptor pharmacology or plasma membrane trafficking, but may involve interactions with other intracellular signaling cascades or proteins.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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

  14. 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

  15. 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.

  16. The role of serotonin 5-HT2A receptors in memory and cognition

    PubMed Central

    Zhang, Gongliang; Stackman, Robert W.

    2015-01-01

    Serotonin 5-HT2A receptors (5-HT2ARs) are widely distributed in the central nervous system, especially in brain region essential for learning and cognition. In addition to endogenous 5-HT, several hallucinogens, antipsychotics, and antidepressants function by targeting 5-HT2ARs. Preclinical studies show that 5-HT2AR antagonists have antipsychotic and antidepressant properties, whereas agonist ligands possess cognition-enhancing and hallucinogenic properties. Abnormal 5-HT2AR activity is associated with a number of psychiatric disorders and conditions, including depression, schizophrenia, and drug addiction. In addition to its traditional activity as a G protein-coupled receptor (GPCR), recent studies have defined novel operations of 5-HT2ARs. Here we review progress in the (1) receptor anatomy and biology: distribution, signaling, polymerization and allosteric modulation; and (2) receptor functions: learning and memory, hallucination and spatial cognition, and mental disorders. Based on the recent progress in basic research on the 5-HT2AR, it appears that post-training 5-HT2AR activation enhances non-spatial memory consolidation, while pre-training 5-HT2AR activation facilitates fear extinction. Further, the potential influence that 5-HT2AR-elicited visual hallucinations may have on visual cue (i.e., landmark) guided spatial cognition is discussed. We conclude that the development of selective 5-HT2AR modulators to target distinct signaling pathways and neural circuits represents a new possibility for treating emotional, neuropsychiatric, and neurodegenerative disorders. PMID:26500553

  17. Targeting the adenosine 2A receptor enhances chimeric antigen receptor T cell efficacy

    PubMed Central

    Beavis, Paul A.; Henderson, Melissa A.; Giuffrida, Lauren; Mills, Jane K.; Sek, Kevin; Cross, Ryan S.; Davenport, Alexander J.; John, Liza B.; Mardiana, Sherly; Slaney, Clare Y.; Johnstone, Ricky W.; Trapani, Joseph A.; Stagg, John; Loi, Sherene; Kats, Lev; Gyorki, David; Kershaw, Michael H.; Darcy, Phillip K.

    2017-01-01

    Chimeric antigen receptor (CAR) T cells have been highly successful in treating hematological malignancies, including acute and chronic lymphoblastic leukemia. However, treatment of solid tumors using CAR T cells has been largely unsuccessful to date, partly because of tumor-induced immunosuppressive mechanisms, including adenosine production. Previous studies have shown that adenosine generated by tumor cells potently inhibits endogenous antitumor T cell responses through activation of adenosine 2A receptors (A2ARs). Herein, we have observed that CAR activation resulted in increased A2AR expression and suppression of both murine and human CAR T cells. This was reversible using either A2AR antagonists or genetic targeting of A2AR using shRNA. In 2 syngeneic HER2+ self-antigen tumor models, we found that either genetic or pharmacological targeting of the A2AR profoundly increased CAR T cell efficacy, particularly when combined with PD-1 blockade. Mechanistically, this was associated with increased cytokine production of CD8+ CAR T cells and increased activation of both CD8+ and CD4+ CAR T cells. Given the known clinical relevance of the CD73/adenosine pathway in several solid tumor types, and the initiation of phase I trials for A2AR antagonists in oncology, this approach has high translational potential to enhance CAR T cell efficacy in several cancer types. PMID:28165340

  18. 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.

  19. Lgr4 promotes prostate tumorigenesis through the Jmjd2a/AR signaling pathway.

    PubMed

    Zhang, Jianwei; Li, Qi; Zhang, Shaojin; Xu, Quanquan; Wang, Tianen

    2016-11-15

    Lgr4 (leucine-rich repeat domain containing G protein-coupled receptor 4) is implicated in the transcriptional regulation of multiple histone demethylases in the progression of diverse cancers, but there are few reports concerning the molecular mechanism by which Lgr4 regulates histone demethylase activation in prostate cancer (PCa) progression. As Jmjd2a is a histone demethylase, in the current study, we investigated the relationship between interaction Lgr4 with Jmjd 2a and Jmjd2a/androgen receptor (AR) signaling pathway in PCa progression. Firstly, Lgr4 was overexpressed by transfecting pcDNA3.1(+)/Lgr4 plasmids into PCa (LNCaP and PC-3) cell lines. Next, we found that Lgr4 overexpression promoted Jmjd2a mRNA expression, reduced cell apoptosis and arrested cell cycle in the S phase, these effects were reversed by Jmjd2a silencing. Moreover, Lgr4 overexpression markedly elevated AR levels and its interaction with Jmjd2a, which was tested by co-immunoprecipitation and luciferase reporter assays. Furthermore, interaction AR with PSA promoter (containing an AR response element) was obviously improved by Lgr4 overexpression, and PSA silencing reduced Lgr4-induced cell apoptosis and cell cycle arrest in PCa cells. Taken together, Lgr4 may be a novel tumor marker providing new mechanistic insights into PCa progression. Lgr4 activates Jmjd2a/AR signaling pathway to promote interaction AR with PSA promoter, causing reduction of PCa apoptosis and cell cycle arrest. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. 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

  1. Hallucinogenic 5-HT2AR agonists LSD and DOI enhance dopamine D2R protomer recognition and signaling of D2-5-HT2A heteroreceptor complexes.

    PubMed

    Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Narvaez, Manuel; Oflijan, Julia; Agnati, Luigi F; Fuxe, Kjell

    2014-01-03

    Dopamine D2LR-serotonin 5-HT2AR heteromers were demonstrated in HEK293 cells after cotransfection of the two receptors and shown to have bidirectional receptor-receptor interactions. In the current study the existence of D2L-5-HT2A heteroreceptor complexes was demonstrated also in discrete regions of the ventral and dorsal striatum with in situ proximity ligation assays (PLA). The hallucinogenic 5-HT2AR agonists LSD and DOI but not the standard 5-HT2AR agonist TCB2 and 5-HT significantly increased the density of D2like antagonist (3)H-raclopride binding sites and significantly reduced the pKiH values of the high affinity D2R agonist binding sites in (3)H-raclopride/DA competition experiments. Similar results were obtained in HEK293 cells and in ventral striatum. The effects of the hallucinogenic 5-HT2AR agonists on D2R density and affinity were blocked by the 5-HT2A antagonist ketanserin. In a forskolin-induced CRE-luciferase reporter gene assay using cotransfected but not D2R singly transfected HEK293 cells DOI and LSD but not TCB2 significantly enhanced the D2LR agonist quinpirole induced inhibition of CRE-luciferase activity. Haloperidol blocked the effects of both quinpirole alone and the enhancing actions of DOI and LSD while ketanserin only blocked the enhancing actions of DOI and LSD. The mechanism for the allosteric enhancement of the D2R protomer recognition and signalling observed is likely mediated by a biased agonist action of the hallucinogenic 5-HT2AR agonists at the orthosteric site of the 5-HT2AR protomer. This mechanism may contribute to the psychotic actions of LSD and DOI and the D2-5-HT2A heteroreceptor complex may thus be a target for the psychotic actions of hallunicogenic 5-HT2A agonists. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. 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

  3. 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.

  4. Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury

    PubMed Central

    Li, Li; Huang, Liping; Ye, Hong; Song, Steven P.; Bajwa, Amandeep; Lee, Sang Ju; Moser, Emily K.; Jaworska, Katarzyna; Kinsey, Gilbert R.; Day, Yuan J.; Linden, Joel; Lobo, Peter I.; Rosin, Diane L.; Okusa, Mark D.

    2012-01-01

    DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A2AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A2AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A2AR agonists. In addition, administration of DCs treated ex vivo with an A2AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A2AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A2AR–induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI. PMID:23093781

  5. 3-(Fur-2-yl)-10-(2-phenylethyl)-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one, a novel adenosine receptor antagonist with A(2A)-mediated neuroprotective effects.

    PubMed

    Scatena, Alessia; Fornai, Francesco; Trincavelli, Maria Letizia; Taliani, Sabrina; Daniele, Simona; Pugliesi, Isabella; Cosconati, Sandro; Martini, Claudia; Da Settimo, Federico

    2011-09-21

    In this study, compound FTBI (3-(2-furyl)-10-(2-phenylethyl)[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one) was selected from a small library of triazinobenzimidazole derivatives as a potent A(2A) adenosine receptor (AR) antagonist and tested for its neuroprotective effects against two different kinds of dopaminergic neurotoxins, 1-methyl-4-phenylpyridinium (MPP+) and methamphetamine (METH), in rat PC12 and in human neuroblastoma SH-SY5Y cell lines. FTBI, in a concentration range corresponding to its affinity for A(2A) AR subtype, significantly increased the number of viable PC12 cells after their exposure to METH and, to a similar extent, to MPP+, as demonstrated in both trypan blue exclusion assay and in cytological staining. These neuroprotective effects were also observed with a classical A(2A) AR antagonist, ZM241385, and appeared to be completely counteracted by the AR agonist, NECA, supporting A(2A) ARs are directly involved in FTBI-mediated effects. Similarly, in human SH-SY5Y cells, FTBI was able to prevent cell toxicity induced by MPP+ and METH, showing that this A(2A) AR antagonist has a neuroprotective effect independently by the specific cell model. Altogether these results demonstrate that the A(2A) AR blockade mediates cell protection against neurotoxicity induced by dopaminergic neurotoxins in dopamine containing cells, supporting the potential use of A(2A) AR antagonists in dopaminergic degenerative diseases including Parkinson's disease.

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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.

  13. Optical characteristics of a RF DBD plasma jet in various {Ar}/ {O}_{2}Ar/O2 mixtures

    NASA Astrophysics Data System (ADS)

    Falahat, A.; Ganjovi, A.; Taraz, M.; Ravari, M. N. Rostami; Shahedi, A.

    2018-02-01

    In this paper, using the optical emission spectroscopy (OES) technique, the optical characteristics of a radiofrequency (RF) plasma jet are examined. The Ar/O2 mixture is taken as the operational gas and, the Ar percentage in the Ar/O2 mixture is varied from 70% to 95%. Using the optical emission spectrum analysis of the RF plasma jet, the excitation temperature is determined based on the Boltzmann plot method. The electron density in the plasma medium of the RF plasma jet is obtained by the Stark broadening of the hydrogen Balmer H_{β }. It is mostly seen that, the radiation intensity of Ar 4p→ 4s transitions at higher argon contributions in Ar/O2 mixture is higher. It is found that, at higher Ar percentages, the emission intensities from atomic oxygen (O) are higher and, the line intensities from the argon atoms and ions including O atoms linearly increase. It is observed that the quenching of Ar^{*} with O2 results in higher O species with respect to O2 molecules. In addition, at higher percentages of Ar in the Ar/O2 mixture, while the excitation temperature is decreased, the electron density is increased.

  14. 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

  15. Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists

    PubMed Central

    Moreno, José L.; Holloway, Terrell; Albizu, Laura; Sealfon, Stuart C.; González-Maeso, Javier

    2011-01-01

    Hallucinogenic drugs, including mescaline, psilocybin and lysergic acid diethylamide (LSD), act at serotonin 5-HT2A receptors (5-HT2ARs). Metabotropic glutamate receptor 2/3 (mGluR2/3) ligands show efficacy in modulating the responses induced by activation of 5-HT2ARs. The formation of a 5-HT2AR-mGluR2 complex suggests a functional interaction that affects the hallucinogen-regulated cellular signaling pathways. Here, we tested the cellular and behavioral effects of hallucinogenic 5-HT2AR agonists in mGluR2 knockout (mGluR2-KO) mice. Mice were intraperitoneally injected with the hallucinogens DOI (2 mg/kg) and LSD (0.24 mg/kg), or vehicle. Head-twitch behavioral response, expression of c-fos, which is induced by all 5-HT2AR agonists, and expression of egr-2, which is hallucinogen-specific, were determined in wild type and mGluR2-KO mice. [3H]Ketanserin binding displacement curves by DOI were performed in mouse frontal cortex membrane preparations. Head twitch behavior was abolished in mGluR2-KO mice. The high-affinity binding site of DOI was undetected in mGluR2-KO mice. The hallucinogen DOI induced c-fos in both wild type and mGluR2-KO mice. However, the induction of egr-2 by DOI was eliminated in mGlu2-KO mice. These findings suggest that the 5-HT2AR-mGluR2 complex is necessary for the neuropsychological responses induced by hallucinogens. PMID:21276828

  16. 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

  17. Glycine triggers a non-ionotropic activity of GluN2A-containing NMDA receptors to confer neuroprotection.

    PubMed

    Hu, Rong; Chen, Juan; Lujan, Brendan; Lei, Ruixue; Zhang, Mi; Wang, Zefen; Liao, Mingxia; Li, Zhiqiang; Wan, Yu; Liu, Fang; Feng, Hua; Wan, Qi

    2016-10-03

    Ionotropic activation of NMDA receptors (NMDARs) requires agonist glutamate and co-agonist glycine. Here we show that glycine enhances the activation of cell survival-promoting kinase Akt in cultured cortical neurons in which both the channel activity of NMDARs and the glycine receptors are pre-inhibited. The effect of glycine is reduced by shRNA-mediated knockdown of GluN2A subunit-containing NMDARs (GluN2ARs), suggesting that a non-ionotropic activity of GluN2ARs mediates glycine-induced Akt activation. In support of this finding, glycine enhances Akt activation in HEK293 cells over-expressing GluN2ARs. The effect of glycine on Akt activation is sensitive to the antagonist of glycine-GluN1 binding site. As a functional consequence, glycine protects against excitotoxicity-induced neuronal death through the non-ionotropic activity of GluN2ARs and the neuroprotective effect is attenuated by Akt inhibition. Thus, this study reveals an unexpected role of glycine in eliciting a non-ionotropic activity of GluN2ARs to confer neuroprotection via Akt activation.

  18. Glycine triggers a non-ionotropic activity of GluN2A-containing NMDA receptors to confer neuroprotection

    PubMed Central

    Hu, Rong; Chen, Juan; Lujan, Brendan; Lei, Ruixue; Zhang, Mi; Wang, Zefen; Liao, Mingxia; Li, Zhiqiang; Wan, Yu; Liu, Fang; Feng, Hua; Wan, Qi

    2016-01-01

    Ionotropic activation of NMDA receptors (NMDARs) requires agonist glutamate and co-agonist glycine. Here we show that glycine enhances the activation of cell survival-promoting kinase Akt in cultured cortical neurons in which both the channel activity of NMDARs and the glycine receptors are pre-inhibited. The effect of glycine is reduced by shRNA-mediated knockdown of GluN2A subunit-containing NMDARs (GluN2ARs), suggesting that a non-ionotropic activity of GluN2ARs mediates glycine-induced Akt activation. In support of this finding, glycine enhances Akt activation in HEK293 cells over-expressing GluN2ARs. The effect of glycine on Akt activation is sensitive to the antagonist of glycine-GluN1 binding site. As a functional consequence, glycine protects against excitotoxicity-induced neuronal death through the non-ionotropic activity of GluN2ARs and the neuroprotective effect is attenuated by Akt inhibition. Thus, this study reveals an unexpected role of glycine in eliciting a non-ionotropic activity of GluN2ARs to confer neuroprotection via Akt activation. PMID:27694970

  19. Cross-talk from β-Adrenergic Receptors Modulates α2A-Adrenergic Receptor Endocytosis in Sympathetic Neurons via Protein Kinase A and Spinophilin*

    PubMed Central

    Cottingham, Christopher; Lu, Roujian; Jiao, Kai; Wang, Qin

    2013-01-01

    Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs. PMID:23965992

  20. Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.

    PubMed

    Moreno, José L; Holloway, Terrell; Albizu, Laura; Sealfon, Stuart C; González-Maeso, Javier

    2011-04-15

    Hallucinogenic drugs, including mescaline, psilocybin and lysergic acid diethylamide (LSD), act at serotonin 5-HT2A receptors (5-HT2ARs). Metabotropic glutamate receptor 2/3 (mGluR2/3) ligands show efficacy in modulating the responses induced by activation of 5-HT2ARs. The formation of a 5-HT2AR-mGluR2 complex suggests a functional interaction that affects the hallucinogen-regulated cellular signaling pathways. Here, we tested the cellular and behavioral effects of hallucinogenic 5-HT2AR agonists in mGluR2 knockout (mGluR2-KO) mice. Mice were intraperitoneally injected with the hallucinogens DOI (2 mg/kg) and LSD (0.24 mg/kg), or vehicle. Head-twitch behavioral response, expression of c-fos, which is induced by all 5-HT2AR agonists, and expression of egr-2, which is hallucinogen-specific, were determined in wild type and mGluR2-KO mice. [(3)H]Ketanserin binding displacement curves by DOI were performed in mouse frontal cortex membrane preparations. Head twitch behavior was abolished in mGluR2-KO mice. The high-affinity binding site of DOI was undetected in mGluR2-KO mice. The hallucinogen DOI induced c-fos in both wild type and mGluR2-KO mice. However, the induction of egr-2 by DOI was eliminated in mGlu2-KO mice. These findings suggest that the 5-HT2AR-mGluR2 complex is necessary for the neuropsychological responses induced by hallucinogens. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  1. 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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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

  3. 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

  4. 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

  5. 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

  6. β2-Adrenergic receptor activation mobilizes intracellular calcium via a non-canonical cAMP-independent signaling pathway.

    PubMed

    Galaz-Montoya, Monica; Wright, Sara J; Rodriguez, Gustavo J; Lichtarge, Olivier; Wensel, Theodore G

    2017-06-16

    Beta adrenergic receptorsARs) are G-protein-coupled receptors essential for physiological responses to the hormones/neurotransmitters epinephrine and norepinephrine which are found in the nervous system and throughout the body. They are the targets of numerous widely used drugs, especially in the case of the most extensively studied βAR, β 2 AR, whose ligands are used for asthma and cardiovascular disease. βARs signal through Gα s G-proteins and via activation of adenylyl cyclase and cAMP-dependent protein kinase, but some alternative downstream pathways have also been proposed that could be important for understanding normal physiological functioning of βAR signaling and its disruption in disease. Using fluorescence-based Ca 2+ flux assays combined with pharmacology and gene knock-out methods, we discovered a previously unrecognized endogenous pathway in HEK-293 cells whereby β 2 AR activation leads to robust Ca 2+ mobilization from intracellular stores via activation of phospholipase C and opening of inositol trisphosphate (InsP 3 ) receptors. This pathway did not involve cAMP, Gα s , or Gα i or the participation of the other members of the canonical β 2 AR signaling cascade and, therefore, constitutes a novel signaling mechanism for this receptor. This newly uncovered mechanism for Ca 2+ mobilization by β 2 AR has broad implications for adrenergic signaling, cross-talk with other signaling pathways, and the effects of βAR-directed drugs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. 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.

  16. Analysis of various types of single-polypeptide-chain (sc) heterodimeric A{sub 2A}R/D{sub 2}R complexes and their allosteric receptor–receptor interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamiya, Toshio, E-mail: kamiya@z2.keio.jp; Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526; Cell Biology Laboratory, School of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-Osaka, Osaka 577-8502

    Highlights: • Various scA{sub 2A}R/D{sub 2}R constructs, with spacers between the two receptors, were created. • Using whole cell binding assay, constructs were examined for their binding activity. • Although the apparent ratio of A{sub 2A}R to D{sub 2}R binding sites should be 1, neither was 1. • Counter agonist-independent binding cooperativity occurred in context of scA{sub 2A}R/D{sub 2}R. - Abstract: Adenosine A{sub 2A} receptor (A{sub 2A}R) heteromerizes with dopamine D{sub 2} receptor (D{sub 2}R). However, these class A G protein-coupled receptor (GPCR) dimers are not fully formed, but depend on the equilibrium between monomer and dimer. In order tomore » stimulate the heteromerization, we have previously shown a successful design for a fusion receptor, single-polypeptide-chain (sc) heterodimeric A{sub 2A}R/D{sub 2}R complex. Here, using whole cell binding assay, six more different scA{sub 2A}R/D{sub 2}R constructs were examined. Not only in scA{sub 2A}R/D{sub 2}R ‘liberated’ with longer spacers between the two receptors, which confer the same configuration as the prototype, the A{sub 2A}R-odr4TM-D{sub 2L}R, but differ in size (Forms 1–3), but also in scA{sub 2A}R/D{sub 2L}R (Form 6) fused with a transmembrane (TM) of another type II TM protein, instead of odr4TM, neither of their fixed stoichiometry (the apparent ratios of A{sub 2A}R to D{sub 2}R binding sites) was 1, suggesting their compact folding. This suggests that type II TM, either odr4 or another, facilitates the equilibrial process of the dimer formation between A{sub 2A}R and D{sub 2L}R, resulting in the higher-order oligomer formation from monomer of scA{sub 2A}R/D{sub 2L}R itself. Also, in the reverse type scA{sub 2A}R/D{sub 2L}R, i.e., the D{sub 2L}R-odr4TM-A{sub 2A}R, counter agonist-independent binding cooperativity (cooperative folding) was found to occur (Forms 4 and 5). In this way, the scA{sub 2A}R/D{sub 2L}R system has unveiled the cellular phenomenon as a snapshot of the

  17. 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.

  18. Heterodimerization with beta2-adrenergic receptors promotes surface expression and functional activity of alpha1D-adrenergic receptors.

    PubMed

    Uberti, Michelle A; Hague, Chris; Oller, Heide; Minneman, Kenneth P; Hall, Randy A

    2005-04-01

    The alpha1D-adrenergic receptor (alpha1D-AR) is a G protein-coupled receptor (GPCR) that is poorly trafficked to the cell surface and largely nonfunctional when heterologously expressed by itself in a variety of cell types. We screened a library of approximately 30 other group I GPCRs in a quantitative luminometer assay for the ability to promote alpha1D-AR cell surface expression. Strikingly, these screens revealed only two receptors capable of inducing robust increases in the amount of alpha1D-AR at the cell surface: alpha1B-AR and beta2-AR. Confocal imaging confirmed that coexpression with beta2-AR resulted in translocation of alpha1D-AR from intracellular sites to the plasma membrane. Additionally, coimmunoprecipitation studies demonstrated that alpha1D-AR and beta2-AR specifically interact to form heterodimers when coexpressed in HEK-293 cells. Ligand binding studies revealed an increase in total alpha1D-AR binding sites upon coexpression with beta2-AR, but no apparent effect on the pharmacological properties of the receptors. In functional studies, coexpression with beta2-AR significantly enhanced the coupling of alpha1D-AR to norepinephrine-stimulated Ca2+ mobilization. Heterodimerization of beta2-AR with alpha1D-AR also conferred the ability of alpha1D-AR to cointernalize upon beta2-AR agonist stimulation, revealing a novel mechanism by which these different adrenergic receptor subtypes may regulate each other's activity. These findings demonstrate that the selective association of alpha1D-AR with other receptors is crucial for receptor surface expression and function and also shed light on a novel mechanism of cross talk between alpha1- and beta2-ARs that is mediated through heterodimerization and cross-internalization.

  19. The alpha(2a)-adrenergic receptor plays a protective role in mouse behavioral models of depression and anxiety.

    PubMed

    Schramm, N L; McDonald, M P; Limbird, L E

    2001-07-01

    The noradrenergic system is involved in the regulation of many physiological and psychological processes, including the modulation of mood. The alpha(2)-adrenergic receptors (alpha(2)-ARs) modulate norepinephrine release, as well as the release of serotonin and other neurotransmitters, and are therefore potential targets for antidepressant and anxiolytic drug development. The current studies were undertaken to examine the role of the alpha(2A) subtype of alpha(2)-AR in mouse behavioral models of depression and anxiety. We have observed that the genetic knock-out of the alpha(2A)-AR makes mice less active in a modified version of Porsolt's forced swim test and insensitive to the antidepressant effects of the tricyclic drug imipramine in this paradigm. Furthermore, alpha(2A)-AR knock-out mice appear more anxious than wild-type C57 Bl/6 mice in the rearing and light-dark models of anxiety after injection stress. These findings suggest that the alpha(2A)-AR may play a protective role in some forms of depression and anxiety and that the antidepressant effects of imipramine may be mediated by the alpha(2A)-AR.

  20. 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

  1. 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

  2. Molecular modeling of interactions of the non-peptide antagonist YM087 with the human vasopressin V1a, V2 receptors and with oxytocin receptors.

    NASA Astrophysics Data System (ADS)

    Giełdoń, Artur; Kaźmierkiewicz, Rajmund; Ślusarz, Rafał; Ciarkowski, Jerzy

    2001-12-01

    The nonapeptide hormones arginine vasopressin (CYFQNCPRG-NH2, AVP) and oxytocin (CYIQNCPLG-NH2, OT), control many essential functions in mammals. Their main activities include the urine concentration (via stimulation of AVP V2 receptors, V2R, in the kidneys), blood pressure regulation (via stimulation of vascular V1a AVP receptors, V1aR), ACTH control (via stimulation of V1b receptors, V1bR, in the pituitary) and labor and lactation control (via stimulation of OT receptors, OTR, in the uterus and nipples, respectively). All four receptor subtypes belong to the GTP-binding (G) protein-coupled receptor (GPCR) family. This work consists of docking of YM087, a potent non-peptide V1aR and V2R - but not OTR - antagonist, into the receptor models based on relatively new theoretical templates of rhodopsin (RD) and opiate receptors, proposed by Mosberg et al. (Univ. of Michigan, Ann Arbor, USA). It is simultaneously demonstrated that this RD template satisfactorily compares with the first historical GPCR structure of bovine rhodopsin (Palczewski et al., 2000) and that homology-modeling of V2R, V1aR and OTR using opiate receptors as templates is rational, based on relatively high (20-60%) sequence homology among the set of 4 neurophyseal and 4 opiate receptors. YM087 was computer-docked to V1aR, V2R and OTR using the AutoDock (Olson et al., Scripps Research Institute, La Jolla, USA) and subsequently relaxed using restrained simulated annealing and molecular dynamics, as implemented in AMBER program (Kollman et al., University of California, San Francisco, USA). From about 80 diverse configurations, sampled for each of the three ligand/receptor systems, 3 best energy-relaxed complexes were selected for mutual comparisons. Similar docking modes were found for the YM087/V1aR and YM087/V2R complexes, diverse from those of the YM087/OTR complexes, in agreement with the molecular affinity data.

  3. 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

  4. A pulsed plasma jet with the various Ar/N2 mixtures

    NASA Astrophysics Data System (ADS)

    Barkhordari, A.; Ganjovi, A.; Mirzaei, I.; Falahat, A.; Rostami Ravari, M. N.

    2017-12-01

    In this paper, using the Optical Emission Spectroscopy technique, the physical properties of a fabricated pulsed DBD plasma jet are studied. Ar/N2 gaseous mixture is taken as operational gas, and Ar contribution in Ar/N2 mixture is varied from 75 to 95%. Through the optical emission spectra analysis of the pulsed DBD plasma jet, the rotational, vibrational and excitation temperatures and density of electrons in plasma medium of the pulsed plasma jet are obtained. It is seen that, at the wavelength of 750.38 nm, the radiation intensity from the Ar 4p → 4 s transition increases at the higher Ar contributions in Ar/N2 mixture. It is found that, for 95% of Ar presence in the mixture, the emission intensities from argon and molecular nitrogen are higher, and the emission line intensities will increase nonlinearly. In addition, it is observed that the quenching of Ar* by N2 results in the higher intensities of N2 excited molecules. Moreover, at the higher percentages of Ar in Ar/N2 mixture, while all the plasma temperatures are increased, the plasma electron density is reduced.

  5. 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

  6. 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

  7. Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

    PubMed Central

    Fancher, Ashley T.; Hua, Yun; Camarco, Daniel P.; Close, David A.; Strock, Christopher J.

    2016-01-01

    Abstract The continued activation of androgen receptor (AR) transcription and elevated expression of AR and transcriptional intermediary factor 2 (TIF2) coactivator observed in prostate cancer (CaP) recurrence and the development of castration-resistant CaP (CRPC) support a screening strategy for small-molecule inhibitors of AR-TIF2 protein–protein interactions (PPIs) to find new drug candidates. Small molecules can elicit tissue selective effects, because the cells of distinct tissues express different levels and cohorts of coregulatory proteins. We reconfigured the AR-TIF2 PPI biosensor (PPIB) assay in the PC-3 CaP cell line to determine whether AR modulators and hits from an AR-TIF2 PPIB screen conducted in U-2 OS cells would behave differently in the CaP cell background. Although we did not observe any significant differences in the compound responses between the assay performed in osteosarcoma and CaP cells, the U-2 OS AR-TIF2 PPIB assay would be more amenable to screening, because both the virus and cell culture demands are lower. We implemented a testing paradigm of counter-screens and secondary hit characterization assays that allowed us to identify and deprioritize hits that inhibited/disrupted AR-TIF2 PPIs and AR transcriptional activation (AR-TA) through antagonism of AR ligand binding or by non-specifically blocking nuclear receptor trafficking. Since AR-TIF2 PPI inhibitor/disruptor molecules act distally to AR ligand binding, they have the potential to modulate AR-TA in a cell-specific manner that is distinct from existing anti-androgen drugs, and to overcome the development of resistance to AR antagonism. We anticipate that the application of this testing paradigm to characterize the hits from an AR-TIF2 PPI high-content screening campaign will enable us to prioritize the AR-TIF2 PPI inhibitor/disruptor leads that have potential to be developed into novel therapeutics for CaP and CRPC. PMID:27606620

  8. Characterization of a novel androgen receptor (AR) coregulator RIPK1 and related chemicals that suppress AR-mediated prostate cancer growth via peptide and chemical screening.

    PubMed

    Hsu, Cheng-Lung; Liu, Jai-Shin; Lin, Ting-Wei; Chang, Ying-Hsu; Kuo, Yung-Chia; Lin, An-Chi; Ting, Huei-Ju; Pang, See-Tong; Lee, Li-Yu; Ma, Wen-Lung; Lin, Chun-Cheng; Wu, Wen-Guey

    2017-09-19

    Using bicalutamide-androgen receptor (AR) DNA binding domain-ligand binding domain as bait, we observed enrichment of FxxFY motif-containing peptides. Protein database searches revealed the presence of receptor-interacting protein kinase 1 (RIPK1) harboring one FxxFY motif. RIPK1 interacted directly with AR and suppressed AR transactivation in a dose-dependent manner. Domain mapping experiments showed that the FxxFY motif in RIPK1 is critical for interactions with AR and the death domain of RIPK1 plays a crucial role in its inhibitory effect on transactivation. In terms of tissue expression, RIPK1 levels were markedly higher in benign prostate hyperplasia and non-cancerous tissue regions relative to the tumor area. With the aid of computer modeling for screening of chemicals targeting activation function 2 (AF-2) of AR, we identified oxadiazole derivatives as good candidates and subsequently generated a small library of these compounds. A number of candidates could effectively suppress AR transactivation and AR-related functions in vitro and in vivo with tolerable toxicity via inhibiting AR-peptide, AR-coregulator and AR N-C interactions. Combination of these chemicals with antiandrogen had an additive suppressive effect on AR transcriptional activity. Our collective findings may pave the way in creating new strategies for the development and design of anti-AR drugs.

  9. N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

    PubMed

    Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

    2017-07-01

    N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

  10. 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.

  11. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors

    PubMed Central

    Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A.; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I.; McCormick, Peter J.; Maldonado, Rafael; Robledo, Patricia

    2015-01-01

    Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties. PMID:26158621

  12. Cognitive Impairment Induced by Delta9-tetrahydrocannabinol Occurs through Heteromers between Cannabinoid CB1 and Serotonin 5-HT2A Receptors.

    PubMed

    Viñals, Xavier; Moreno, Estefanía; Lanfumey, Laurence; Cordomí, Arnau; Pastor, Antoni; de La Torre, Rafael; Gasperini, Paola; Navarro, Gemma; Howell, Lesley A; Pardo, Leonardo; Lluís, Carmen; Canela, Enric I; McCormick, Peter J; Maldonado, Rafael; Robledo, Patricia

    2015-07-01

    Activation of cannabinoid CB1 receptors (CB1R) by delta9-tetrahydrocannabinol (THC) produces a variety of negative effects with major consequences in cannabis users that constitute important drawbacks for the use of cannabinoids as therapeutic agents. For this reason, there is a tremendous medical interest in harnessing the beneficial effects of THC. Behavioral studies carried out in mice lacking 5-HT2A receptors (5-HT2AR) revealed a remarkable 5-HT2AR-dependent dissociation in the beneficial antinociceptive effects of THC and its detrimental amnesic properties. We found that specific effects of THC such as memory deficits, anxiolytic-like effects, and social interaction are under the control of 5-HT2AR, but its acute hypolocomotor, hypothermic, anxiogenic, and antinociceptive effects are not. In biochemical studies, we show that CB1R and 5-HT2AR form heteromers that are expressed and functionally active in specific brain regions involved in memory impairment. Remarkably, our functional data shows that costimulation of both receptors by agonists reduces cell signaling, antagonist binding to one receptor blocks signaling of the interacting receptor, and heteromer formation leads to a switch in G-protein coupling for 5-HT2AR from Gq to Gi proteins. Synthetic peptides with the sequence of transmembrane helices 5 and 6 of CB1R, fused to a cell-penetrating peptide, were able to disrupt receptor heteromerization in vivo, leading to a selective abrogation of memory impairments caused by exposure to THC. These data reveal a novel molecular mechanism for the functional interaction between CB1R and 5-HT2AR mediating cognitive impairment. CB1R-5-HT2AR heteromers are thus good targets to dissociate the cognitive deficits induced by THC from its beneficial antinociceptive properties.

  13. Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex

    PubMed Central

    Béïque, Jean-Claude; Imad, Mays; Mladenovic, Ljiljana; Gingrich, Jay A.; Andrade, Rodrigo

    2007-01-01

    Classic hallucinogens such as lysergic acid diethylamide are thought to elicit their psychotropic actions via serotonin receptors of the 5-hydroxytryptamine 2A subtype (5-HT2AR). One likely site for these effects is the prefrontal cortex (PFC). Previous studies have shown that activation of 5-HT2ARs in this region results in a robust increase in spontaneous glutamatergic synaptic activity, and these results have led to the widely held idea that hallucinogens elicit their effect by modulating synaptic transmission within the PFC. Here, we combine cellular and molecular biological approaches, including single-cell 5-HT2ARs inactivation and 5-HT2AR rescue over a 5-HT2AR knockout genetic background, to distinguish between competing hypotheses accounting for these effects. The results from these experiments do not support the idea that 5-HT2ARs elicit the release of an excitatory retrograde messenger nor that they activate thalamocortical afferents, the two dominant hypotheses. Rather, they suggest that 5-HT2ARs facilitate intrinsic networks within the PFC. Consistent with this idea, we locate a discrete subpopulation of pyramidal cells that is strongly excited by 5-HT2AR activation. PMID:17535909

  14. Synthesis and Evaluation of Phenylxanthine Derivatives as Potential Dual A2AR Antagonists/MAO-B Inhibitors for Parkinson's Disease.

    PubMed

    Wang, Xuebao; Han, Chao; Xu, Yong; Wu, Kaiqi; Chen, Shuangya; Hu, Mangsha; Wang, Luyao; Ye, Yun; Ye, Faqing

    2017-06-17

    The aim of this research was to prove the speculation that phenylxanthine (PX) derivatives possess adenosine A2A receptor (A2AR)-blocking properties and to screening and evaluate these PX derivatives as dual A2AR antagonists/MAO-B inhibitors for Parkinson's disease. To explore this hypothesis, two series of PX derivatives were prepared and their antagonism against A2AR and inhibition against MAO-B were determined in vitro. In order to evaluate further the antiparkinsonian properties, pharmacokinetic and haloperidol-induced catalepsy experiments were carried out in vivo. The PX-D and PX-E analogues acted as potent A2AR antagonists with Ki values ranging from 0.27 to 10 μM, and these analogues displayed relatively mild MAO-B inhibition potencies, with inhibitor dissociation constants (Ki values) ranging from 0.25 to 10 μM. Further, the compounds PX-D-P6 and PX-E-P8 displayed efficacious antiparkinsonian properties in haloperidol-induced catalepsy experiments, verifying that these two compounds were potent A2AR antagonists and MAO-B inhibitors. We conclude that PX-D and PX-E analogues are a promising candidate class of dual-acting compounds for treating Parkinson's disease.

  15. Regulatory mechanism of CCN2 production by serotonin (5-HT) via 5-HT2A and 5-HT2B receptors in chondrocytes.

    PubMed

    Hori, Ayaka; Nishida, Takashi; Takashiba, Shogo; Kubota, Satoshi; Takigawa, Masaharu

    2017-01-01

    Serotonin (5-hydroxytryptamine: 5-HT) is recognized as a neurotransmitter in the central nerve system and as a regulator of systemic blood pressure in the peripheral tissues. Recently, it was reported that 5-HT2 receptors (5-HT2Rs) were expressed in cartilage tissues lacking both vessels and neurons, suggesting possible novel functions of 5-HT during cartilage development and regeneration. Our previous data indicated that CCN family protein 2/connective tissue growth factor (CCN2/CTGF) plays a central role in cartilage development and regeneration. Therefore, the aim of this study was to investigate the effect of 5-HT on the production of CCN2 in chondrocytes. Firstly, we showed that the mRNAs of 5-HT2R subtypes 5-HT2AR and 5-HT2BR, were expressed in a human chondrocytic cell line, HCS-2/8; however, 5-HT2CR mRNA was not detected. In addition, exogenously added 5-HT did not affect the 5-HT2AR and 5-HT2BR expressions. Next, we demonstrated that CCN2 production was increased by treatment with a 5-HT2AR agonist and the combination of 5-HT and 5-HT2BR antagonist. In contrast, treatment with a 5-HT2BR agonist and the combination of 5-HT and 5-HT2AR antagonist decreased CCN2 production. Furthermore, we showed that phosphorylation of Akt and p38 MAPK were increased by treatment with 5-HT2AR agonist, and that phosphorylation of PKCε, PKCζ, ERK1/2 and JNK were increased by treatment with 5-HT2BR agonist. Finally, we found that 5-HT2AR was localized in the growth plate, whereas 5-HT2BR was localized in the articular cartilage. These findings suggest that 5-HT promotes CCN2 production through the 5-HT2AR in growth plates, and that it represses CCN2 production through the 5-HT2BR in articular cartilage for harmonized development of long bones.

  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. 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

  18. Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

    NASA Astrophysics Data System (ADS)

    Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

    2011-02-01

    G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

  19. Chronic cannabis promotes pro-hallucinogenic signaling of 5-HT2A receptors through Akt/mTOR pathway.

    PubMed

    Ibarra-Lecue, Inés; Mollinedo-Gajate, Irene; Meana, J Javier; Callado, Luis F; Diez-Alarcia, Rebeca; Urigüen, Leyre

    2018-04-27

    Long-term use of potent cannabis during adolescence increases the risk of developing schizophrenia later in life, but to date, the mechanisms involved remain unknown. Several findings suggest that the functional selectivity of serotonin 2A receptor (5-HT2AR) through inhibitory G-proteins is involved in the molecular mechanisms responsible for psychotic symptoms. Moreover, this receptor is dysregulated in the frontal cortex of schizophrenia patients. In this context, studies involving cannabis exposure and 5-HT2AR are scarce. Here, we tested in mice the effect of an early chronic Δ 9 -tetrahydrocannabinol (THC) exposure on cortical 5-HT2AR expression, as well as on its in vivo and in vitro functionality. Long-term exposure to THC induced a pro-hallucinogenic molecular conformation of the 5-HT2AR and exacerbated schizophrenia-like responses, such as prepulse inhibition disruption. Supersensitive coupling of 5-HT2AR toward inhibitory Gαi1-, Gαi3-, Gαo-, and Gαz-proteins after chronic THC exposure was observed, without changes in the canonical Gαq/11-protein pathway. In addition, we found that inhibition of Akt/mTOR pathway by rapamycin blocks the changes in 5-HT2AR signaling pattern and the supersensitivity to schizophrenia-like effects induced by chronic THC. The present study provides the first evidence of a mechanistic explanation for the relationship between chronic cannabis exposure in early life and increased risk of developing psychosis-like behaviors in adulthood.

  20. Temperature-Sensitive Intracellular Traffic of α2C-Adrenergic Receptor.

    PubMed

    Filipeanu, Catalin M

    2015-01-01

    α(2C)-Adrenergic receptor (α(2C)-AR) is the least characterized adrenergic receptor subtype and still very little is known about the intracellular traffic properties and pathophysiological roles of this receptor. α(2C)-AR has an atypical subcellular localization. At 37 °C, in the vascular smooth muscle cells and in fibroblasts, the receptor is poorly localized at the plasma membrane and accumulates inside the cell. Exposure to lower temperatures stimulates α(2C)-AR transport to the cell surface. This particular intracellular trafficking of α(2C)-AR is significant in the pathology of Raynaud phenomenon. In this brief review, I will present general information on the tissue distribution and cellular localization of α(2C)-AR. Also, I will discuss the mechanisms involved in the receptor transport by focusing on the trafficking motifs and on the molecular chaperones. © 2015 Elsevier Inc. All rights reserved.

  1. Structure-activity relationship studies and pharmacological characterization of N5-heteroarylalkyl-substituted-2-(2-furanyl)thiazolo[5,4-d]pyrimidine-5,7-diamine-based derivatives as inverse agonists at human A2A adenosine receptor.

    PubMed

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

    2018-06-09

    This paper describes the synthesis and characterization of N 5 -(hetero)arylalkyl-substituted-thiazolo [5,4-d]pyrimidine-5,7-diamine derivatives (4-19) as novel human (h) A 2A adenosine receptor (AR) inverse agonists. Competition binding and cyclic AMP assays indicate that the examined compounds behave as hA 2A AR inverse agonists showing binding affinity values in the nanomolar or subnanomolar range. Notably, compounds 4, 5, 6 and 11 showed two affinity values for the hA 2A ARs with the highest (KH) falling in the femtomolar range and the lowest (KL) of the nanomolar order. In addition, in cyclic AMP assays, compounds 4, 5, 6 and 11 exhibited potency (IC 50 ) values in the picomolar range. This study has confirmed that 2-(2-furanyl)thiazolo [5,4-d]pyrimidine-5,7-diamine-based derivatives represent a unique new class of hA 2A AR inverse agonists. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  2. Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

    PubMed

    Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

    2001-02-01

    alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

  3. Baicalein suppresses the androgen receptor (AR)-mediated prostate cancer progression via inhibiting the AR N-C dimerization and AR-coactivators interaction.

    PubMed

    Xu, Defeng; Chen, Qiulu; Liu, Yalin; Wen, Xingqiao

    2017-12-01

    Androgen receptor (AR) plays a critical role in prostate cancer (PCa) development and progression. Androgen deprivation therapy with antiandrogens to reduce androgen biosynthesis or prevent androgens from binding to AR are widely used to suppress AR-mediated PCa growth. However, most of ADT may eventually fail with development of the castration resistance after 12-24 months. Here we found that a natural product baicalein can effectively suppress the PCa progression via targeting the androgen-induced AR transactivation with little effect to AR protein expression. PCa cells including LNCaP, CWR22Rv1, C4-2, PC-3, and DU145, were treated with baicalein and luciferase assay was used to evaluate their effect on the AR transactivation. Cell growth and IC 50 were determined by MTT assay after 48 hrs treatment. RT-PCR was used to evaluate the mRNA levels of AR target genes including PSA, TMPRSS2, and TMEPA1. Western blot was used to determine AR and PSA protein expression. The natural product of baicalein can selectively inhibit AR transactivation with little effect on the other nuclear receptors, including ERα, and GR. At a low concentration, 2.5 μM of baicalein effectively suppresses the growth of AR-positive PCa cells, and has little effect on AR-negative PCa cells. Mechanism dissection suggest that baicalein can suppress AR target genes (PSA, TMPRSS2, and TMEPA1) expression in both androgen responsive LNCaP cells and castration resistant CWR22Rv1 cells, that may involve the inhibiting the AR N/C dimerization and AR-coactivators interaction. Baicalein may be developed as an effective anti-AR therapy via its ability to inhibit AR transactivation and AR-mediated PCa cell growth.

  4. Agonist-Directed Desensitization of the β2-Adrenergic Receptor

    PubMed Central

    Goral, Vasiliy; Jin, Yan; Sun, Haiyan; Ferrie, Ann M.; Wu, Qi; Fang, Ye

    2011-01-01

    The β2-adrenergic receptor2AR) agonists with reduced tachyphylaxis may offer new therapeutic agents with improved tolerance profile. However, receptor desensitization assays are often inferred at the single signaling molecule level, thus ligand-directed desensitization is poorly understood. Here we report a label-free biosensor whole cell assay with microfluidics to determine ligand-directed desensitization of the β2AR. Together with mechanistic deconvolution using small molecule inhibitors, the receptor desensitization and resensitization patterns under the short-term agonist exposure manifested the long-acting agonism of salmeterol, and differentiated the mechanisms of agonist-directed desensitization between a full agonist epinephrine and a partial agonist pindolol. This study reveals the cellular mechanisms of agonist-selective β2AR desensitization at the whole cell level. PMID:21541288

  5. CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells.

    PubMed

    Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

    2018-04-03

    The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination.

  6. 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

  7. Confinement of β1- and β2-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae

    PubMed Central

    Valentine, Cathleen D.; Haggie, Peter M.

    2011-01-01

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β1- and β2AR, are structurally similar but mediate distinct signaling responses. Scaffold protein–mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β1- and β2AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)–domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β2AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β2AR confinement. For both β1- and β2AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β1- or β2AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes. PMID:21680711

  8. Confinement of β(1)- and β(2)-adrenergic receptors in the plasma membrane of cardiomyocyte-like H9c2 cells is mediated by selective interactions with PDZ domain and A-kinase anchoring proteins but not caveolae.

    PubMed

    Valentine, Cathleen D; Haggie, Peter M

    2011-08-15

    The sympathetic nervous system regulates cardiac output by activating adrenergic receptors (ARs) in cardiac myocytes. The predominant cardiac ARs, β(1)- and β(2)AR, are structurally similar but mediate distinct signaling responses. Scaffold protein-mediated compartmentalization of ARs into discrete, multiprotein complexes has been proposed to dictate differential signaling responses. To test the hypothesis that βARs integrate into complexes in live cells, we measured receptor diffusion and interactions by single-particle tracking. Unstimulated β(1)- and β(2)AR were highly confined in the membrane of H9c2 cardiomyocyte-like cells, indicating that receptors are tethered and presumably integrated into protein complexes. Selective disruption of interactions with postsynaptic density protein 95/disks large/zonula occludens-1 (PDZ)-domain proteins and A-kinase anchoring proteins (AKAPs) increased receptor diffusion, indicating that these scaffold proteins participate in receptor confinement. In contrast, modulation of interactions between the putative scaffold caveolae and β(2)AR did not alter receptor dynamics, suggesting that these membrane domains are not involved in β(2)AR confinement. For both β(1)- and β(2)AR, the receptor carboxy-terminus was uniquely responsible for scaffold interactions. Our data formally demonstrate that distinct and stable protein complexes containing β(1)- or β(2)AR are formed in the plasma membrane of cardiomyocyte-like cells and that selective PDZ and AKAP interactions are responsible for the integration of receptors into complexes.

  9. Src regulates sequence-dependent beta-2 adrenergic receptor recycling via cortactin phosphorylation*

    PubMed Central

    Vistein, Rachel; Puthenveedu, Manojkumar A.

    2014-01-01

    The recycling of internalized signaling receptors, which has direct functional consequences, is subject to multiple sequence and biochemical requirements. Why signaling receptors recycle via a specialized pathway, unlike many other proteins that recycle by bulk, is a fundamental unanswered question. Here we show that these specialized pathways allow selective control of signaling receptor recycling by heterologous signaling. Using assays to visualize receptor recycling in living cells, we show that the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, is regulated by Src family kinases. The target of Src is cortactin, an essential factor for B2AR sorting into specialized recycling microdomains on the endosome. Phosphorylation of a single cortactin residue, Y466, regulates the rate of fission of B2AR recycling vesicles from these microdomains, and, therefore, the rate of delivery of B2AR to the cell surface. Together, our results indicate that actin-stabilized microdomains that mediate signaling receptor recycling can serve as a functional point of convergence for crosstalk between signaling pathways. PMID:25077552

  10. Experimental study of NO2 reduction in N2/Ar and O2/Ar mixtures by pulsed corona discharge.

    PubMed

    Zhu, Xinbo; Zheng, Chenghang; Gao, Xiang; Shen, Xu; Wang, Zhihua; Luo, Zhongyang; Cen, Kefa

    2014-11-01

    Non-thermal plasma technology has been regarded as a promising alternative technology for NOx removal. The understanding of NO2 reduction characteristics is extremely important since NO2 reduction could lower the total NO oxidation rate in the plasma atmosphere. In this study, NO2 reduction was experimentally investigated using a non-thermal plasma reactor driven by a pulsed power supply for different simulated gas compositions and operating parameters. The NO2 reduction was promoted by increasing the specific energy density (SED), and the highest conversion rates were 33.7%, 42.1% and 25.7% for Ar, N2/Ar and O2/Ar, respectively. For a given SED, the NO2 conversion rate had the order N2/Ar>Ar>O2/Ar. The highest energy yield of 3.31g/kWh was obtained in N2/Ar plasma and decreased with increasing SED; the same trends were also found in the other two gas compositions. The conversion rate decreased with increasing initial NO2 concentration. Furthermore, the presence of N2 or O2 led to different reaction pathways for NO2 conversion due to the formation of different dominating reactive radicals. Copyright © 2014. Published by Elsevier B.V.

  11. Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

    PubMed

    Shishkina, G T; Kalinina, T S; Dygalo, N N

    2004-01-01

    Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain

  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. β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

    PubMed Central

    Kohout, Trudy A.; Lin, Fang-Tsyr; Perry, Stephen J.; Conner, David A.; Lefkowitz, Robert J.

    2001-01-01

    The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation. PMID:11171997

  14. β2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

    PubMed

    Sironi, Chiara; Bodega, Francesca; Armilli, Marta; Porta, Cristina; Zocchi, Luciano; Agostoni, Emilio

    2010-09-30

    Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of β2-adrenoreceptors (β2AR). In this research we tried to provide molecular evidence for β2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of β2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed β2AR and GRK2 specific bands. Copyright 2010 Elsevier B.V. All rights reserved.

  15. 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

  16. CNPY2 inhibits MYLIP-mediated AR protein degradation in prostate cancer cells

    PubMed Central

    Ito, Saya; Ueno, Akihisa; Ueda, Takashi; Nakagawa, Hideo; Taniguchi, Hidefumi; Kayukawa, Naruhiro; Fujihara-Iwata, Atsuko; Hongo, Fumiya; Okihara, Koji; Ukimura, Osamu

    2018-01-01

    The androgen receptor (AR) is a ligand-dependent transcription factor that promotes prostate cancer (PC) cell growth through control of target gene expression. This report suggests that Canopy FGF signaling regulator 2 (CNPY2) controls AR protein levels in PC cells. We found that AR was ubiquitinated by an E3 ubiquitin ligase, myosin regulatory light chain interacting protein (MYLIP) and then degraded through the ubiquitin-proteasome pathway. CNPY2 decreased the ubiquitination activity of MYLIP by inhibition of interaction between MYLIP and UBE2D1, an E2 ubiquitin ligase. CNPY2 up-regulated gene expression of AR target genes such as KLK3 gene which encodes the prostate specific antigen (PSA) and promoted cell growth of PC cells. The cell growth inhibition by CNPY2 knockdown was rescued by AR overexpression. Furthermore, positive correlation of expression levels between CNPY2 and AR/AR target genes was observed in tissue samples from human prostate cancer patients. Together, these results suggested that CNPY2 promoted cell growth of PC cells by inhibition of AR protein degradation through MYLIP-mediated AR ubiquitination. PMID:29707137

  17. Crystal Structures of the β2-Adrenergic Receptor

    NASA Astrophysics Data System (ADS)

    Weis, William I.; Rosenbaum, Daniel M.; Rasmussen, Søren G. F.; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Yao, Xiao-Jie; Day, Peter W.; Parnot, Charles; Fung, Juan J.; Ratnala, Venkata R. P.; Kobilka, Brian K.; Cherezov, Vadim; Hanson, Michael A.; Kuhn, Peter; Stevens, Raymond C.; Edwards, Patricia C.; Schertler, Gebhard F. X.; Burghammer, Manfred; Sanishvili, Ruslan; Fischetti, Robert F.; Masood, Asna; Rohrer, Daniel K.

    G protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome, and are responsible for the majority of signal transduction events involving hormones and neuro-transmitters across the cell membrane. GPCRs that bind to diffusible ligands have low natural abundance, are relatively unstable in detergents, and display basal G protein activation even in the absence of ligands. To overcome these problems two approaches were taken to obtain crystal structures of the β2-adrenergic receptor2AR), a well-characterized GPCR that binds cate-cholamine hormones. The receptor was bound to the partial inverse agonist carazolol and co-crystallized with a Fab made to a three-dimensional epitope formed by the third intracellular loop (ICL3), or by replacement of ICL3 with T4 lysozyme. Small crystals were obtained in lipid bicelles (β2AR-Fab) or lipidic cubic phase (β2AR-T4 lysozyme), and diffraction data were obtained using microfocus technology. The structures provide insights into the basal activity of the receptor, the structural features that enable binding of diffusible ligands, and the coupling between ligand binding and G-protein activation.

  18. Altered Sympathetic-to-Immune Cell Signaling via β 2-Adrenergic Receptors in Adjuvant Arthritis

    PubMed Central

    Bellinger, Denise L.; Schaller, Jill A.; Osredkar, Tracy

    2013-01-01

    Adjuvant-induced arthritic (AA) differentially affects norepinephrine concentrations in immune organs, and in vivo β-adrenergic receptor (β-AR) agonist treatment distinctly regulates ex vivo cytokine profiles in different immune organs. We examined the contribution of altered β-AR functioning in AA to understand these disparate findings. Twenty-one or 28 days after disease induction, we examined β 2-AR expression in spleen and draining lymph nodes (DLNs) for the arthritic limbs using radioligand binding and western blots and splenocyte β-AR-stimulated cAMP production using enzyme-linked immunoassay (EIA). During severe disease, β-AR agonists failed to induce splenocyte cAMP production, and β-AR affinity and density declined, indicating receptor desensitization and downregulation. Splenocyte β 2-AR phosphorylation (pβ 2-AR) by protein kinase A (pβ 2-ARPKA) decreased in severe disease, and pβ 2-AR by G protein-coupled receptor kinases (pβ 2-ARGRK) increased in chronic disease. Conversely, in DLN cells, pβ 2-ARPKA rose during severe disease, but fell during chronic disease, and pβ 2-ARGRK increased during both disease stages. A similar pβ 2-AR pattern in DLN cells with the mycobacterial cell wall component of complete Freund's adjuvant suggests that pattern recognition receptors (i.e., toll-like receptors) are important for DLN pβ 2-AR patterns. Collectively, our findings indicate lymphoid organ- and disease stage-specific sympathetic dysregulation, possibly explaining immune compartment-specific differences in β 2-AR-mediated regulation of cytokine production in AA and rheumatoid arthritis. PMID:24194774

  19. Molecular Determinants of the Human α2C-Adrenergic Receptor Temperature-Sensitive Intracellular Traffic

    PubMed Central

    Pullikuth, Ashok K.; Guidry, Jessie J.

    2015-01-01

    The human α2C-adrenergic receptor2C-AR) is localized intracellularly at physiologic temperature. Decreasing the environmental temperature strongly stimulates the receptor transport to the cell surface. In contrast, rat and mouse α2C-AR plasma membrane levels are less sensitive to decrease in temperature, whereas the opossum α2C-AR cell surface levels are not changed in these conditions. Structural analysis demonstrated that human α2C-AR has a high number of arginine residues in the third intracellular loop and in the C-terminus, organized as putative RXR motifs. Although these motifs do not affect the receptor subcellular localization at 37°C, deletion of the arginine clusters significantly enhanced receptor plasma membrane levels at reduced temperature. We found that this exaggerated transport of the human receptor is mediated by two functional arginine clusters, one in the third intracellular loop and one in the C-terminus. This effect is mediated by interactions with COPI vesicles, but not by 14-3-3 proteins. In rat α2C-AR, the arginine cluster from the third intracellular loop is shifted to the left due to three missing residues. Reinsertion of these residues in the rat α2C-AR restored the same temperature sensitivity as in the human receptor. Proteomic and coimmunoprecipitation experiments identified pontin as a molecule having stronger interactions with human α2C-AR compared with rat α2C-AR. Inhibition of pontin activity enhanced human receptor plasma membrane levels and signaling at 37°C. Our results demonstrate that human α2C-AR has a unique temperature-sensitive traffic pattern within the G protein–coupled receptor class due to interactions with different molecular chaperones, mediated in part by strict spatial localization of specific arginine residues. PMID:25680754

  20. Adenosine A1 receptors link to smooth muscle contraction via CYP4a, PKC-α, and ERK1/2

    PubMed Central

    Kunduri, SS; Mustafa, SJ; Ponnoth, DS; Dick, GM; Nayeem, MA

    2013-01-01

    Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms aren’t thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). 20-HETE can activate protein kinase C-α (PKC-α) which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist CCPA was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished CCPA-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway. PMID:23519140

  1. 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.

  2. TNF-{alpha} upregulates the A{sub 2B} adenosine receptor gene: The role of NAD(P)H oxidase 4

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    St Hilaire, Cynthia; Koupenova, Milka; Carroll, Shannon H.

    2008-10-24

    Proliferation of vascular smooth muscle cells (VSMC), oxidative stress, and elevated inflammatory cytokines are some of the components that contribute to plaque formation in the vasculature. The cytokine tumor necrosis factor-alpha (TNF-{alpha}) is released during vascular injury, and contributes to lesion formation also by affecting VSMC proliferation. Recently, an A{sub 2B} adenosine receptor (A{sub 2B}AR) knockout mouse illustrated that this receptor is a tissue protector, in that it inhibits VSMC proliferation and attenuates the inflammatory response following injury, including the release of TNF-{alpha}. Here, we show a regulatory loop by which TNF-{alpha} upregulates the A{sub 2B}AR in VSMC in vitromore » and in vivo. The effect of this cytokine is mimicked by its known downstream target, NAD(P)H oxidase 4 (Nox4). Nox4 upregulates the A{sub 2B}AR, and Nox inhibitors dampen the effect of TNF-{alpha}. Hence, our study is the first to show that signaling associated with Nox4 is also able to upregulate the tissue protecting A{sub 2B}AR.« less

  3. A dimeric ArC2 compound from Peperomia pellucida.

    PubMed

    Bayma, J D; Arruda, M S; Müller, A H; Arruda, A C; Canto, W C

    2000-12-01

    Pellucidin A, a novel dimeric ArC2 compound, together with dill-apiol have been isolated from the aerial parts of Peperomia pellucida. The structure of pellucidin A was established by 1D and 2D NMR spectroscopy (1H-1H COSY; 1H-13C COSY; DEPT; NOESY and double irradiation) and other spectroscopic techniques. The biogenesis of pellucidin A is also briefly discussed.

  4. 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

  5. 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

  6. Mechanism of allosteric regulation of β2-adrenergic receptor by cholesterol

    PubMed Central

    Manna, Moutusi; Niemelä, Miia; Tynkkynen, Joona; Javanainen, Matti; Kulig, Waldemar; Müller, Daniel J; Rog, Tomasz; Vattulainen, Ilpo

    2016-01-01

    There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human β2-adrenergic receptor2AR) – a prototypical G protein-coupled receptor – is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates β2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5–7 of the receptor. The alternative mechanism, where the β2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of β2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions. DOI: http://dx.doi.org/10.7554/eLife.18432.001 PMID:27897972

  7. Ebselen has lithium-like effects on central 5-HT2A receptor function.

    PubMed

    Antoniadou, I; Kouskou, M; Arsiwala, T; Singh, N; Vasudevan, S R; Fowler, T; Cadirci, E; Churchill, G C; Sharp, T

    2018-02-27

    Lithium's antidepressant action may be mediated by inhibition of inositol monophosphatase (IMPase), a key enzyme in G q protein coupled receptor signalling. Recently, the antioxidant agent ebselen was identified as an IMPase inhibitor. Here we investigated both ebselen and lithium in models of the 5-HT 2A receptor, a G q protein coupled receptor implicated in lithium's actions. 5-HT 2A receptor function was modelled in mice by measuring the behavioural (head-twitches) and cortical immediate early gene (IEG; Arc, c-fos and Erg2 mRNA) responses to 5-HT 2A receptor agonist administration. Ebselen and lithium were administered either acutely or chronically prior to assessment of 5-HT 2A receptor function. Given the SSRI augmenting action of lithium and 5-HT 2A antagonists, ebselen was also tested for this action by co-administration with the SSRI citalopram in microdialysis (extracellular 5-HT) experiments. Acute and repeated administration of ebselen inhibited behavioural and IEG responses to the 5-HT 2A receptor agonist DOI. Repeated lithium also inhibited DOI-evoked behavioural and IEG responses. In comparison, a selective IMPase inhibitor (L-690,330) attenuated the behavioural response to DOI whereas glycogen synthase kinase inhibitor (AR-A014418) did not. Finally, ebselen increased regional brain 5-HT synthesis and enhanced the increase in extracellular 5-HT induced by citalopram. The current data demonstrate lithium-mimetic effects of ebselen in different experimental models of 5-HT 2A receptor function, likely mediated by IMPase inhibition. This evidence of lithium-like neuropharmacological effects of ebselen adds further support for the clinical testing of ebselen in mood disorder, including as an antidepressant augmenting agent. This article is protected by copyright. All rights reserved.

  8. Astrocytic β2-adrenergic receptors mediate hippocampal long-term memory consolidation.

    PubMed

    Gao, Virginia; Suzuki, Akinobu; Magistretti, Pierre J; Lengacher, Sylvain; Pollonini, Gabriella; Steinman, Michael Q; Alberini, Cristina M

    2016-07-26

    Emotionally relevant experiences form strong and long-lasting memories by critically engaging the stress hormone/neurotransmitter noradrenaline, which mediates and modulates the consolidation of these memories. Noradrenaline acts through adrenergic receptors (ARs), of which β2-adrenergic receptorsARs) are of particular importance. The differential anatomical and cellular distribution of βAR subtypes in the brain suggests that they play distinct roles in memory processing, although much about their specific contributions and mechanisms of action remains to be understood. Here we show that astrocytic rather than neuronal β2ARs in the hippocampus play a key role in the consolidation of a fear-based contextual memory. These hippocampal β2ARs, but not β1ARs, are coupled to the training-dependent release of lactate from astrocytes, which is necessary for long-term memory formation and for underlying molecular changes. This key metabolic role of astrocytic β2ARs may represent a novel target mechanism for stress-related psychopathologies and neurodegeneration.

  9. Regulation of androgen receptor and histone deacetylase 1 by Mdm2-mediated ubiquitylation.

    PubMed

    Gaughan, Luke; Logan, Ian R; Neal, David E; Robson, Craig N

    2005-01-01

    The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors and plays a critical role in regulating the expression of genes involved in androgen-dependent and -independent tumour formation. Regulation of the AR is achieved by alternate binding of either histone acetyltransferase (HAT)-containing co-activator proteins, or histone deacetylase 1 (HDAC1). Factors that control AR stability may also constitute an important regulatory mechanism, a notion that has been confirmed with the finding that the AR is a direct target for Mdm2-mediated ubiquitylation and proteolysis. Using chromatin immunoprecipitation (ChIP) and re-ChIP analyses, we show that Mdm2 associates with AR and HDAC1 at the active androgen-responsive PSA promoter in LNCaP prostate cancer cells. Furthermore, we demonstrate that Mdm2-mediated modification of AR and HDAC1 catalyses protein destabilization and attenuates AR sactivity, suggesting that ubiquitylation of the AR and HDAC1 may constitute an additional mechanism for regulating AR function. We also show that HDAC1 and Mdm2 function co-operatively to reduce AR-mediated transcription that is attenuated by the HAT activity of the AR co-activator Tip60, suggesting interplay between acetylation status and receptor ubiquitylation in AR regulation. In all, our data indicates a novel role for Mdm2 in regulating components of the AR transcriptosome.

  10. Divergent Label-free Cell Phenotypic Pharmacology of Ligands at the Overexpressed β2-Adrenergic Receptors

    NASA Astrophysics Data System (ADS)

    Ferrie, Ann M.; Sun, Haiyan; Zaytseva, Natalya; Fang, Ye

    2014-01-01

    We present subclone sensitive cell phenotypic pharmacology of ligands at the β2-adrenergic receptor2-AR) stably expressed in HEK-293 cells. The parental cell line was transfected with green fluorescent protein (GFP)-tagged β2-AR. Four stable subclones were established and used to profile a library of sixty-nine AR ligands. Dynamic mass redistribution (DMR) profiling resulted in a pharmacological activity map suggesting that HEK293 endogenously expresses functional Gi-coupled α2-AR and Gs-coupled β2-AR, and the label-free cell phenotypic activity of AR ligands are subclone dependent. Pathway deconvolution revealed that the DMR of epinephrine is originated mostly from the remodeling of actin microfilaments and adhesion complexes, to less extent from the microtubule networks and receptor trafficking, and certain agonists displayed different efficacy towards the cAMP-Epac pathway. We demonstrate that receptor signaling and ligand pharmacology is sensitive to the receptor expression level, and the organization of the receptor and its signaling circuitry.

  11. 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.

  12. Preparation and biological evaluation of 64Cu-CB-TE2A-sst2-ANT, a somatostatin antagonist for PET imaging of somatostatin receptor-positive tumors.

    PubMed

    Wadas, Thaddeus J; Eiblmaier, Martin; Zheleznyak, Alexander; Sherman, Christopher D; Ferdani, Riccardo; Liang, Kexian; Achilefu, Samuel; Anderson, Carolyn J

    2008-11-01

    Recently, the somatostatin receptor subtype 2 (SSTR2) selective antagonist sst2-ANT was determined to have a high affinity for SSTR2. Additionally, 111In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-sst2-ANT showed high uptake in an SSTR2-transfected, tumor-bearing mouse model and suggested that radiolabeled SSTR2 antagonists may be superior to agonists for imaging SSTR2-positive tumors. This report describes the synthesis and evaluation of 64Cu-CB-4,11-bis(carboxymethyl)-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-sst2-ANT (64Cu-CB-TE2A-sst2-ANT) as a PET radiopharmaceutical for the in vivo imaging of SSTR2-positive tumors. Receptor-binding studies were performed to determine the dissociation constant of the radiopharmaceutical 64Cu-CB-TE2A-sst2-ANT using AR42J rat pancreatic tumor cell membranes. The internalization of 64Cu-CB-TE2A-sst2-ANT was compared with that of the 64Cu-labeled agonist 64Cu-CB-TE2A-tyrosine3-octreotate (64Cu-CB-TE2A-Y3-TATE) in AR42J cells. Both radiopharmaceuticals were also compared in vivo through biodistribution studies using healthy rats bearing AR42J tumors, and small-animal PET/CT of 64Cu-CB-TE2A-sst2-ANT was performed. The dissociation constant value for the radiopharmaceutical was determined to be 26 +/- 2.4 nM, and the maximum number of binding sites was 23,000 fmol/mg. 64Cu-CB-TE2A-sst2-ANT showed significantly less internalization than did 64Cu-CB-TE2A-Y3-TATE at time points from 15 min to 4 h. Biodistribution studies revealed that the clearance of 64Cu-CB-TE2A-sst2-ANT from the blood was rapid, whereas the clearance of 64Cu-CB-TE2A-sst2-ANT from the liver and kidneys was more modest at all time points. Tumor-to-blood and tumor-to-muscle ratios were determined to be better for 64Cu-CB-TE2A-sst2-ANT than those for 64Cu-CB-TE2A-Y3-TATE at the later time points, although liver and kidney uptake was significantly higher. Small-animal imaging using 64Cu-CB-TE2A-sst2-ANT revealed excellent tumor-to-background contrast at 4 h

  13. Medicinal chemistry of adenosine, P2Y and P2X receptors.

    PubMed

    Jacobson, Kenneth A; Müller, Christa E

    2016-05-01

    Pharmacological tool compounds are now available to define action at the adenosine (ARs), P2Y and P2X receptors. We present a selection of the most commonly used agents to study purines in the nervous system. Some of these compounds, including A1 and A3 AR agonists, P2Y1R and P2Y12R antagonists, and P2X3, P2X4 and P2X7 antagonists, are potentially of clinical use in treatment of disorders of the nervous system, such as chronic pain, neurodegeneration and brain injury. Agonists of the A2AAR and P2Y2R are already used clinically, P2Y12R antagonists are widely used antithrombotics and an antagonist of the A2AAR is approved in Japan for treating Parkinson's disease. The selectivity defined for some of the previously introduced compounds has been revised with updated pharmacological characterization, for example, various AR agonists and antagonists were deemed A1AR or A3AR selective based on human data, but species differences indicated a reduction in selectivity ratios in other species. Also, many of the P2R ligands still lack bioavailability due to charged groups or hydrolytic (either enzymatic or chemical) instability. X-ray crystallographic structures of AR and P2YRs have shifted the mode of ligand discovery to structure-based approaches rather than previous empirical approaches. The X-ray structures can be utilized either for in silico screening of chemically diverse libraries for the discovery of novel ligands or for enhancement of the properties of known ligands by chemical modification. Although X-ray structures of the zebrafish P2X4R have been reported, there is scant structural information about ligand recognition in these trimeric ion channels. In summary, there are definitive, selective agonists and antagonists for all of the ARs and some of the P2YRs; while the pharmacochemistry of P2XRs is still in nascent stages. The therapeutic potential of selectively modulating these receptors is continuing to gain interest in such fields as cancer, inflammation, pain

  14. Selective dry etching of III-V nitrides in Cl{sub 2}/Ar, CH{sub 4}/H{sub 2}/Ar, ICi/Ar, and IBr/Ar

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vartuli, C.B.; Pearton, S.J.; MacKenzie, J.D.

    1996-10-01

    The selectivity for etching the binary (GaN, AlN, and InN) and ternary nitrides (InGaN and InAlN) relative to each other in Cl{sub 2}/Ar, CH{sub 4}/H{sub 2}/Ar, ICl/Ar, or IBr/Ar electron cyclotron resonance (ECR) plasmas, and Cl{sub 2}/Ar or CH{sub 4}/H{sub 2}/Ar reactive ion (RIE) plasmas was investigated. Cl-based etches appear to be the best choice for maximizing the selectivity of GaN over the other nitrides. GaN/AlN and GaN/InGaN etch rate ratios of {approximately} 10 were achieved at low RF power in Cl{sub 2}/Ar under ECR and RIE conditions, respectively. GaN/InN selectivity of 10 was found in ICl under ECR conditions.more » A relatively high selectivity (> 6) of InN/GaN was achieved in CH{sub 4}/H{sub 2}/Ar under ECR conditions at low RF powers (50 W). Since the high bond strengths of the nitrides require either high ion energies or densities to achieve practical etch rates it is difficult to achieve high selectivities.« less

  15. Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

    PubMed

    Müller, C E; Maurinsh, J; Sauer, R

    2000-01-01

    The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

  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. Control of yeast mating signal transduction by a mammalian. beta. sub 2 -adrenergic receptor and G sub s. alpha. subunit

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    King, K.; Caron, M.G.; Lefkowitz, R.J.

    1990-10-05

    To facilitate functional and mechanistic studies of receptor-G protein interactions by expression of the human {beta}{sub 2}-adrenergic receptor (h{beta}-AR) has been expressed in Saccharomyces cerevisiae. This was achieved by placing a modified h{beta}-AR gene under control of the galactose-inducible GAL1 promoter. After induction by galactose, functional h{beta}-AR was expressed at a concentration several hundred times as great as that found in any human tissue. As determined from competitive ligand binding experiments, h{beta}-AR expressed in yeast displayed characteristic affinities, specificity, and stereoselectivity. Partial activation of the yeast pheromone response pathway by {beta}-adrenergic receptor agonists was achieved in cells coexpressing h{beta}-AR andmore » a mammalian G protein (G{sub s}) {alpha} subunit - demonstrating that these components can couple to each other and to downstream effectors when expressed in yeast. This in vivo reconstitution system provides a new approach for examining ligand binding and G protein coupling to cell surface receptors.« less

  18. Comparative molecular field analysis of fenoterol derivatives: A platform towards highly selective and effective beta(2)-adrenergic receptor agonists.

    PubMed

    Jozwiak, Krzysztof; Woo, Anthony Yiu-Ho; Tanga, Mary J; Toll, Lawrence; Jimenez, Lucita; Kozocas, Joseph A; Plazinska, Anita; Xiao, Rui-Ping; Wainer, Irving W

    2010-01-15

    To use a previously developed CoMFA model to design a series of new structures of high selectivity and efficacy towards the beta(2)-adrenergic receptor. Out of 21 computationally designed structures 6 compounds were synthesized and characterized for beta(2)-AR binding affinities, subtype selectivities and functional activities. the best compound is (R,R)-4-methoxy-1-naphthylfelnoterol with K(i)beta(2)-AR=0.28microm, K(i)beta(1)-AR/K(i)beta(2)-AR=573, EC(50cAMP)=3.9nm, EC(50cardio)=16nm. The CoMFA model appears to be an effective predictor of the cardiomocyte contractility of the studied compounds which are targeted for use in congestive heart failure. Copyright 2009 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

    PubMed Central

    Yang, Wanzhi; Wang, Qi; Zhao, Han; Yang, Feng; Lv, Xiongwen; Li, Jun

    2014-01-01

    Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III. PMID:24682220

  1. 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.

  2. Adenosine A1 receptors link to smooth muscle contraction via CYP4a, protein kinase C-α, and ERK1/2.

    PubMed

    Kunduri, Swati S; Mustafa, S Jamal; Ponnoth, Dovenia S; Dick, Gregory M; Nayeem, Mohammed A

    2013-07-01

    Adenosine A1 receptor (A1AR) activation contracts smooth muscle, although signaling mechanisms are not thoroughly understood. Activation of A1AR leads to metabolism of arachidonic acid, including the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504a (CYP4a). The 20-HETE can activate protein kinase C-α (PKC-α), which crosstalks with extracellular signal-regulated kinase (ERK1/2) pathway. Both these pathways can regulate smooth muscle contraction, we tested the hypothesis that A1AR contracts smooth muscle through a pathway involving CYP4a, PKC-α, and ERK1/2. Experiments included isometric tension recordings of aortic contraction and Western blots of signaling molecules in wild type (WT) and A1AR knockout (A1KO) mice. Contraction to the A1-selective agonist 2-chloro-N cyclopentyladenosine (CCPA) was absent in A1KO mice aortae, indicating the contractile role of A1AR. Inhibition of CYP4a (HET0016) abolished 2-chloro-N cyclopentyladenosine-induced contraction in WT aortae, indicating a critical role for 20-HETE. Both WT and A1KO mice aortae contracted in response to exogenous 20-HETE. Inhibition of PKC-α (Gö6976) or ERK1/2 (PD98059) attenuated 20-HETE-induced contraction equally, suggesting that ERK1/2 is downstream of PKC-α. Contractions to exogenous 20-HETE were significantly less in A1KO mice; reduced protein levels of PKC-α, p-ERK1/2, and total ERK1/2 supported this observation. Our data indicate that A1AR mediates smooth muscle contraction via CYP4a and a PKC-α-ERK1/2 pathway.

  3. In Vitro Mutational Analysis of the β2 Adrenergic Receptor, an In Vivo Surrogate Odorant Receptor

    PubMed Central

    Pfister, Patrick; Tomoiaga, Delia; Rogers, Matthew E.; Feinstein, Paul

    2015-01-01

    Many G-protein coupled receptors (GPCRs), such as odorant receptors (ORs), cannot be characterized in heterologous cells because of their difficulty in trafficking to the plasma membrane. In contrast, a surrogate OR, the GPCR mouse β2-adrenergic-receptor (mβ2AR), robustly traffics to the plasma membrane. We set out to characterize mβ2AR mutants in vitro for their eventual use in olfactory axon guidance studies. We performed an extensive mutational analysis of mβ2AR using a Green Fluorescent Protein-tagged mβ2AR (mβ2AR::GFP) to easily assess the extent of its plasma membrane localization. In order to characterize mutants for their ability to successfully transduce ligand-initiated signal cascades, we determined the half maximal effective concentrations (EC50) and maximal response to isoprenaline, a known mβ2AR agonist. Our analysis reveals that removal of amino terminal (Nt) N-glycosylation sites and the carboxy terminal (Ct) palmitoylation site of mβ2AR do not affect its plasma membrane localization. By contrast, when both the Nt and Ct of mβ2AR are replaced with those of M71 OR, plasma membrane trafficking is impaired. We further analyze three mβ2AR mutants (RDY, E268A, and C327R) used in olfactory axon guidance studies and are able to decorrelate their plasma membrane trafficking with their capacity to respond to isoprenaline. A deletion of the Ct prevents proper trafficking and abolishes activity, but plasma membrane trafficking can be selectively rescued by a Tyrosine to Alanine mutation in the highly conserved GPCR motif NPxxY. This new loss-of-function mutant argues for a model in which residues located at the end of transmembrane domain 7 can act as a retention signal when unmasked. Additionally, to our surprise, amongst our set of mutations only Ct mutations appear to lower mβ2AR EC50s revealing their critical role in G-protein coupling. We propose that an interaction between the Nt and Ct is necessary for proper folding and/or transport of GPCRs

  4. α(2A)-adrenergic receptors filter parabrachial inputs to the bed nucleus of the stria terminalis.

    PubMed

    Flavin, Stephanie A; Matthews, Robert T; Wang, Qin; Muly, E Chris; Winder, Danny G

    2014-07-09

    α2-adrenergic receptors (AR) within the bed nucleus of the stria terminalis (BNST) reduce stress-reward interactions in rodent models. In addition to their roles as autoreceptors, BNST α(2A)-ARs suppress glutamatergic transmission. One prominent glutamatergic input to the BNST originates from the parabrachial nucleus (PBN) and consists of asymmetric axosomatic synapses containing calcitonin gene-related peptide (CGRP) and vGluT2. Here we provide immunoelectron microscopic data showing that many asymmetric axosomatic synapses in the BNST contain α(2A)-ARs. Further, we examined optically evoked glutamate release ex vivo in BNST from mice with virally delivered channelrhodopsin2 (ChR2) expression in PBN. In BNST from these animals, ChR2 partially colocalized with CGRP, and activation generated EPSCs in dorsal anterolateral BNST neurons that elicited two cell-type-specific outcomes: (1) feedforward inhibition or (2) an EPSP that elicited firing. We found that the α(2A)-AR agonist guanfacine selectively inhibited this PBN input to the BNST, preferentially reducing the excitatory response in ex vivo mouse brain slices. To begin to assess the overall impact of α(2A)-AR control of this PBN input on BNST excitatory transmission, we used a Thy1-COP4 mouse line with little postsynaptic ChR2 expression nor colocalization of ChR2 with CGRP in the BNST. In slices from these mice, we found that guanfacine enhanced, rather than suppressed, optogenetically initiated excitatory drive in BNST. Thus, our study reveals distinct actions of PBN afferents within the BNST and suggests that α(2A)-AR agonists may filter excitatory transmission in the BNST by inhibiting a component of the PBN input while enhancing the actions of other inputs. Copyright © 2014 the authors 0270-6474/14/349319-13$15.00/0.

  5. Study of the physical discharge properties of a Ar/O2 DC plasma jet

    NASA Astrophysics Data System (ADS)

    Barkhordari, A.; Ganjovi, A.; Mirzaei, I.; Falahat, A.

    2018-03-01

    In this paper, the physical properties of plasma discharge in a manufactured DC plasma jet operating with the Ar/O2 gaseous mixture are studied. Moreover, the optical emission spectroscopy technique is used to perform the experimental measurements. The obtained emission spectra are analyzed and, the plasma density, rotational, vibrational and electronic temperature are calculated. The NO emission lines from {NO }γ( A2 Σ^{+} \\to {X}2 Πr ) electronic transition are observed. It is seen that, at the higher argon contributions in Ar/O2 gaseous mixture, the emission intensities from argon ions will increase. Moreover, while the vibrational and excitation temperatures are increased at the higher input DC currents, they will decrease at the higher Ar percentages in the Ar/O2 gaseous mixture. Furthermore, at the higher DC currents and Ar contributions, both the plasma electron density and dissociation fraction of oxygen atoms are increased.

  6. 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.

  7. Tricyclic antidepressants exhibit variable pharmacological profiles at the α2A adrenergic receptor

    PubMed Central

    Cottingham, Christopher; Percival, Stefanie; Birky, Tana; Wang, Qin

    2014-01-01

    Antidepressant mechanisms of action remain shrouded in mystery, greatly hindering our ability to develop therapeutics which can fully treat patients suffering from depressive disorders. In an attempt to shed new light on this topic, we have undertaken a series of studies investigating actions of tricyclic antidepressant drugs (TCAs) at the α2A adrenergic receptor (AR), a centrally important receptor, dysregulation of which has been linked to depression. Our previous work established a particular TCA, desipramine, as an arrestin-biased α2AAR ligand driving receptor endocytosis and downregulation but not canonical heterotrimeric G protein-mediated signaling. The present work is aimed at broadening our understanding of how members of the TCA drug class act at the α2AAR, as we have selected the closely related but subtly different TCAs imipramine and amitriptyline for evaluation. Our data demonstrate that these drugs do also function as direct arrestin-biased α2AAR ligands. However, these data reveal differences in receptor affinity and in the extent/nature of arrestin recruitment to and endocytosis of α2AARs. Specifically, amitriptyline exhibits an approximately 14-fold stronger interaction with the receptor, is a weaker driver of arrestin recruitment, and preferentially recruits a different arrestin subtype. Extent of endocytosis is similar for all TCAs studied so far, and occurs in an arrestin-dependent manner, although imipramine uniquely retains a slight ability to drive α2AAR endocytosis in arrestin-null cells. These findings signify an important expansion of our mechanistic understanding of antidepressant pharmacology, and provide useful insights for future medicinal chemistry efforts. PMID:25128275

  8. NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer

    PubMed Central

    Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

    2017-01-01

    Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer. PMID:28212551

  9. NRIP/DCAF6 stabilizes the androgen receptor protein by displacing DDB2 from the CUL4A-DDB1 E3 ligase complex in prostate cancer.

    PubMed

    Chen, Hsin-Hsiung; Fan, Ping; Chang, Szu-Wei; Tsao, Yeou-Ping; Huang, Hsiang-Po; Chen, Show-Li

    2017-03-28

    Both nuclear receptor interaction protein (NRIP) and DNA damage binding protein 2 (DDB2) belong to the Cullin 4 (CUL4)-DDB1 binding protein family and are androgen receptor (AR)-interacting proteins. Here, we investigated the expression patterns of the NRIP, DDB2 and AR proteins in human prostate cancer tissues and found that the expression levels of NRIP and AR were higher, but the DDB2 level was lower, in prostate cancer tissues than in non-neoplastic controls, suggesting NRIP as a candidate tumor promoter and DDB2 as a tumor suppressor in prostate cancer. Furthermore, both NRIP and DDB2 shared the same AR binding domain; they were competitors for the AR, but not for DDB1 binding, in the AR-DDB2-DDB1-CUL4A complex. Conclusively, NRIP stabilizes the AR protein by displacing DDB2 from the AR-DDB2 complex. Consistent with our hypothesis, a specific expression pattern with high levels of NRIP and AR, together with a low level of DDB2, was found more frequently in the human prostate cancer tissues with a cribriform pattern than in non-cribriform tumors, suggesting that disruption of the balance between NRIP and DDB2 may change AR protein homeostasis and contribute to pathogenesis in certain aggressive types of prostate cancer.

  10. Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice.

    PubMed

    Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y; DeMayo, Francesco J; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W

    2005-07-05

    Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, AR(GAL4DBD), which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators.

  11. Roles of steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF) 2 in androgen receptor activity in mice

    PubMed Central

    Ye, Xiangcang; Han, Sang Jun; Tsai, Sophia Y.; DeMayo, Francesco J.; Xu, Jianming; Tsai, Ming-Jer; O'Malley, Bert W.

    2005-01-01

    Genetic disruption of the steroid receptor coactivator (SRC)-1 and transcriptional intermediary factor (TIF)2/SRC-2 in mouse resulted in distinctive mutant phenotypes. To quantify their roles in the function of androgen receptor (AR) transcriptional activity in vivo, we generated a unique transgenic AR-reporter mouse and analyzed the cell-specific contributions of SRC-1 and TIF2 to the activity of AR in mouse testis. Transgenic AR-luciferase and transgenic AR-lacZ mice harbor a recombinant mouse AR gene, ARGAL4DBD, which is functionally coupled with a upstream activation sequence-mediated reporter gene (AR activity indicator). After characterization of these mice in terms of AR function, we further derived bigenic mice by crossing AR activity indicator mice with the SRC-1-/- or TIF2+/- mutant mice. Analyses of the resultant bigenic mice by in vivo imaging and luciferase assays showed that testicular AR activity was decreased significantly in those with the TIF2+/- mutation but not in the SRC-1+/- background, suggesting that TIF2 serves as the preferential coactivator for AR in testis. Immunohistological analysis confirmed that AR and TIF2 coexist in mouse testicular Sertoli cell nuclei under normal conditions. Although SRC-1 concentrates in Sertoli cell nuclei in the absence of TIF2, nuclear SRC-1 is not able to rescue AR activity in the TIF2 mutant background. Interestingly, SRC-1 appears to negatively influence AR activity, thereby counterbalancing the TIF2-stimulated AR activity. Our results provide unique in vivo insights to the multidimensional cell-type-specific interactions between AR and coregulators. PMID:15983373

  12. 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.

  13. 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.

  14. β2-adrenergic receptor-mediated negative regulation of group 2 innate lymphoid cell responses.

    PubMed

    Moriyama, Saya; Brestoff, Jonathan R; Flamar, Anne-Laure; Moeller, Jesper B; Klose, Christoph S N; Rankin, Lucille C; Yudanin, Naomi A; Monticelli, Laurel A; Putzel, Gregory Garbès; Rodewald, Hans-Reimer; Artis, David

    2018-03-02

    The type 2 inflammatory response is induced by various environmental and infectious stimuli. Although recent studies identified group 2 innate lymphoid cells (ILC2s) as potent sources of type 2 cytokines, the molecular pathways controlling ILC2 responses are incompletely defined. Here we demonstrate that murine ILC2s express the β 2 -adrenergic receptor2 AR) and colocalize with adrenergic neurons in the intestine. β 2 AR deficiency resulted in exaggerated ILC2 responses and type 2 inflammation in intestinal and lung tissues. Conversely, β 2 AR agonist treatment was associated with impaired ILC2 responses and reduced inflammation in vivo. Mechanistically, we demonstrate that the β 2 AR pathway is a cell-intrinsic negative regulator of ILC2 responses through inhibition of cell proliferation and effector function. Collectively, these data provide the first evidence of a neuronal-derived regulatory circuit that limits ILC2-dependent type 2 inflammation. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  15. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

  16. A functional selectivity mechanism at the serotonin-2A GPCR involves ligand-dependent conformations of intracellular loop 2

    DOE PAGES

    Perez-Aguilar, Jose Manuel; Shan, Jufang; LeVine, Michael V.; ...

    2014-10-14

    With recent progress in determination of G protein-coupled receptor (GPCR) structure with crystallography, a variety of other experimental approaches (e.g., NMR spectroscopy, fluorescent-based assays, mass spectrometry techniques) are also being used to characterize state-specific and ligand-specific conformational states. MD simulations offer a powerful complementary approach to elucidate the dynamic features associated with ligand-specific GPCR conformations. To shed light on the conformational elements and dynamics of the important aspect of GPCR functional selectivity, we carried out unbiased microsecond-length MD simulations of the human serotonin 2A receptor (5-HT 2AR) in the absence of ligand and bound to four distinct serotonergic agonists. Themore » 5-HT 2AR is a suitable system to study the structural features involved in the ligand-dependent conformational heterogeneity of GPCRs because it is well-characterized experimentally and exhibits a strong agonist-specific phenotype in that some 5-HT 2AR agonists induce LSD-like hallucinations, while others lack this psychoactive property entirely. Here we report evidence for structural and dynamic differences in 5-HT 2AR interacting with such pharmacologically distinct ligands, hallucinogens, and nonhallucinogens obtained from all-atom MD simulations. Differential ligand binding contacts were identified for structurally similar hallucinogens and nonhallucinogens and found to correspond to different conformations in the intracellular loop 2 (ICL2). From the different ICL2 conformations, functional selective phenotypes are suggested through effects on dimerization and/or distinct direct interaction with effector proteins. Lastly, the findings are presented in the context of currently proposed hallucinogenesis mechanisms, and ICL2 is proposed as a fine-tuning selective switch that can differentiates modes of 5-HT 2AR activation.« less

  17. Dependence of purinergic P2X receptor activity on ectodomain structure.

    PubMed

    He, Mu-Lan; Zemkova, Hana; Stojilkovic, Stanko S

    2003-03-21

    Purinergic receptors (P2XRs) activate and desensitize in response to the binding of extracellular nucleotides in a receptor- and ligand-specific manner, but the structural bases of their ligand preferences and channel kinetics have been incompletely characterized. Here we tested the hypothesis that affinity of agonists for binding domain accounts for a ligand-specific desensitization pattern. We generated chimeras using receptors with variable sensitivity to ATP in order: P2X(4)R > P2X(2a)R = P2X(2b)R P2X(7)R. Chimeras having the ectodomain Ile(66)-Tyr(310) sequence of P2X(2)R and Val(61)-Phe(313) sequence of P2X(7)R in the backbone of P2X(4)R were expressed but were non-functioning channels. P2X(2a) + X(4)R and P2X(2b) + X(4)R chimeras having the Val(66)-Tyr(315) ectodomain sequence of P2X(4)R in the backbones of P2X(2a)R and P2X(2b)R were functional and exhibited increased sensitivity to ligands as compared with both parental receptors. These chimeras also desensitized faster than parental receptors and in a ligand-nonspecific manner. However, like parental P2X(2b)R and P2X(2a)R, chimeric P2X(2b) + X(4)R desensitized more rapidly than P2X(2a) + X(4)R, and the rate of desensitization of P2X(2a)+X(4)R increased by substituting its Arg(371)-Pro(376) intracellular C-terminal sequence with the Glu(376)-Gly(381) sequence of P2X(4)R. These results indicate the relevance of interaction between the ectodomain and flanking regions around the transmembrane domains on ligand potency and receptor activation. Furthermore, the ligand potency positively correlates with the rate of receptor desensitization but does not affect the C-terminal-specific pattern of desensitization.

  18. Two classes of cholesterol binding sites for the β2AR revealed by thermostability and NMR.

    PubMed

    Gater, Deborah L; Saurel, Olivier; Iordanov, Iordan; Liu, Wei; Cherezov, Vadim; Milon, Alain

    2014-11-18

    Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the ?2 adrenergic receptor2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

  19. 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.

  20. Structure-activity relationships of truncated C2- or C8-substituted adenosine derivatives as dual acting A₂A and A₃ adenosine receptor ligands.

    PubMed

    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

    2012-01-12

    Truncated N(6)-substituted-4'-oxo- and 4'-thioadenosine derivatives with C2 or C8 substitution were studied as dual acting A(2A) and A(3) 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 hA(2A)AR, but hydrophobic C8 substitution abolished binding at the hA(2A)AR. However, most of synthesized compounds displayed medium to high binding affinity at the hA(3)AR, regardless of C2 or C8 substitution, and low efficacy in a functional cAMP assay. Several compounds tended to be full hA(2A)AR agonists. C2 substitution probed geometrically through hA(2A)AR docking was important for binding in order of hexynyl > hexenyl > hexanyl. Compound 4g was the most potent ligand acting dually as hA(2A)AR agonist and hA(3)AR antagonist, which might be useful for treatment of asthma or other inflammatory diseases.

  1. Aggravated brain damage after hypoxic ischemia in immature adenosine A2A knockout mice.

    PubMed

    Adén, Ulrika; Halldner, Linda; Lagercrantz, Hugo; Dalmau, Ishar; Ledent, Catherine; Fredholm, Bertil B

    2003-03-01

    Cerebral hypoxic ischemia (HI) is an important cause of brain injury in the newborn infant. Adenosine is believed to protect against HI brain damage. However, the roles of the different adenosine receptors are unclear, particularly in young animals. We examined the role of adenosine A2A receptors (A2AR) using 7-day-old A2A knockout (A2AR(-/-)) mice in a model of HI. HI was induced in 7-day-old CD1 mice by exposure to 8% oxygen for 30 minutes after occlusion of the left common carotid artery. The resulting unilateral focal lesion was evaluated with the use of histopathological scoring and measurements of residual brain areas at 5 days, 3 weeks, and 3 months after HI. Behavioral evaluation of brain injury by locomotor activity, rotarod, and beam-walking test was made 3 weeks and 3 months after HI. Cortical cerebral blood flow, assessed by laser-Doppler flowmetry, and rectal temperature were measured during HI. Reduction in cortical cerebral blood flow during HI and rectal temperature did not differ between wild-type (A2AR(+/+)) and knockout mice. In the A2AR(-/-) animals, brain injury was aggravated compared with wild-type mice. The A2AR(-/-) mice subjected to HI displayed increased forward locomotion and impaired rotarod performance in adulthood compared with A2AR(+/+) mice subjected to HI, whereas beam-walking performance was similarly defective in both groups. These results suggest that, in contrast to the situation in adult animals, A2AR play an important protective role in neonatal HI brain injury.

  2. Platelet and intestinal 5-HT2A receptor mRNA in autistic spectrum disorders - results of a pilot study.

    PubMed

    Kazek, Beata; Huzarska, Małgorzata; Grzybowska-Chlebowczyk, Urszula; Kajor, Maciej; Ciupińska-Kajor, Monika; Woś, Halina; Marszał, Elzbieta

    2010-01-01

    The etiology and pathogenesis of autistic spectrum disorders (ASD) are still unknown. Platelet hyperserotonemia has been detected in 25-60% of autistic children. Higher incidence of gastrointestinal problems in people with autism is observed. The aim was compare the expression of platelet 5-HT(2A)r mRNA in autistic and non autistic groups. In a subgroup of patients with gastrointestinal problems an upper gastrointestinal tract endoscopy was performed and additionally the expression of 5-HT(2A) receptor mRNA in the duodenum was assessed. The examination was conducted in 79 children - 51 with ASD and 28 without autistic traits. Statistically significant differences between the study and control groups were proven in gastrointestinal problems. The analyses reveal a significantly higher level of 5-HT(2A)r mRNA in platelets of the study group patients, which could suggest serotonin system dysregulation.

  3. Inhibiting Insulin-Mediated β2-Adrenergic Receptor Activation Prevents Diabetes-Associated Cardiac Dysfunction.

    PubMed

    Wang, Qingtong; Liu, Yongming; Fu, Qin; Xu, Bing; Zhang, Yuan; Kim, Sungjin; Tan, Ruensern; Barbagallo, Federica; West, Toni; Anderson, Ethan; Wei, Wei; Abel, E Dale; Xiang, Yang K

    2017-01-03

    Type 2 diabetes mellitus (DM) and obesity independently increase the risk of heart failure by incompletely understood mechanisms. We propose that hyperinsulinemia might promote adverse consequences in the hearts of subjects with type-2 DM and obesity. High-fat diet feeding was used to induce obesity and DM in wild-type mice or mice lacking β 2 -adrenergic receptor2 AR) or β-arrestin2. Wild-type mice fed with high-fat diet were treated with a β-blocker carvedilol or a GRK2 (G-protein-coupled receptor kinase 2) inhibitor. We examined signaling and cardiac contractile function. High-fat diet feeding selectively increases the expression of phosphodiesterase 4D (PDE4D) in mouse hearts, in concert with reduced protein kinase A phosphorylation of phospholamban, which contributes to systolic and diastolic dysfunction. The expression of PDE4D is also elevated in human hearts with DM. The induction of PDE4D expression is mediated by an insulin receptor, insulin receptor substrate, and GRK2 and β-arrestin2-dependent transactivation of a β 2 AR-extracellular regulated protein kinase signaling cascade. Thus, pharmacological inhibition of β 2 AR or GRK2, or genetic deletion of β 2 AR or β-arrestin2, all significantly attenuate insulin-induced phosphorylation of extracellular regulated protein kinase and PDE4D induction to prevent DM-related contractile dysfunction. These studies elucidate a novel mechanism by which hyperinsulinemia contributes to heart failure by increasing PDE4D expression and identify β 2 AR or GRK2 as plausible therapeutic targets for preventing or treating heart failure in subjects with type 2 DM. © 2016 American Heart Association, Inc.

  4. Insilico study of the A(2A)R-D (2)R kinetics and interfacial contact surface for heteromerization.

    PubMed

    Prakash, Amresh; Luthra, Pratibha Mehta

    2012-10-01

    G-protein-coupled receptors (GPCRs) are cell surface receptors. The dynamic property of receptor-receptor interactions in GPCRs modulates the kinetics of G-protein signaling and stability. In the present work, the structural and dynamic study of A(2A)R-D(2)R interactions was carried to acquire the understanding of the A(2A)R-D(2)R receptor activation and deactivation process, facilitating the design of novel drugs and therapeutic target for Parkinson's disease. The structure-based features (Alpha, Beta, SurfAlpha, and SurfBeta; GapIndex, Leakiness and Gap Volume) and slow mode model (ENM) facilitated the prediction of kinetics (K (off), K (on), and K (d)) of A(2A)R-D(2)R interactions. The results demonstrated the correlation coefficient 0.294 for K (d) and K (on) and the correlation coefficient 0.635 for K (d) and K (off), and indicated stable interfacial contacts in the formation of heterodimer. The coulombic interaction involving the C-terminal tails of the A(2A)R and intracellular loops (ICLs) of D(2)R led to the formation of interfacial contacts between A(2A)R-D(2)R. The properties of structural dynamics, ENM and KFC server-based hot-spot analysis illustrated the stoichiometry of A(2A)R-D(2)R contact interfaces as dimer. The propensity of amino acid residues involved in A(2A)R-D(2)R interaction revealed the presence of positively (R, H and K) and negatively (E and D) charged structural motif of TMs and ICL3 of A(2A)R and D(2)R at interface of dimer contact. Essentially, in silico structural and dynamic study of A(2A)R-D(2)R interactions will provide the basic understanding of the A(2A)R-D(2)R interfacial contact surface for activation and deactivation processes, and could be used as constructive model to recognize the protein-protein interactions in receptor assimilations.

  5. 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

  6. Repeat polymorphisms in ESR2 and AR and colorectal cancer risk and prognosis: results from a German population-based case-control study.

    PubMed

    Rudolph, Anja; Shi, Hong; Försti, Asta; Hoffmeister, Michael; Sainz, Juan; Jansen, Lina; Hemminki, Kari; Brenner, Hermann; Chang-Claude, Jenny

    2014-11-07

    Evidence has accumulated which suggests that sex steroids influence colorectal cancer development and progression. We therefore assessed the association of repeat polymorphisms in the estrogen receptor β gene (ESR2) and the androgen receptor gene (AR) with colorectal cancer risk and prognosis. The ESR2 CA and AR CAG repeat polymorphisms were genotyped in 1798 cases (746 female, 1052 male) and 1810 controls (732 female, 1078 male), matched for sex, age and county of residence. Colorectal cancer risk associations overall and specific for gender were evaluated using multivariate logistic regression models adjusted for sex, county of residence and age. Associations with overall and disease-specific survival were evaluated using Cox proportional hazard models adjusted for established prognostic factors (diagnosis of other cancer after colorectal cancer diagnosis, detection by screening, treatment with adjuvant chemotherapy, tumour extent, nodal status, distant metastasis, body mass index, age at diagnosis and year of diagnosis) and stratified for grade of differentiation. Heterogeneity in gender specific associations was assessed by comparing models with and without a multiplicative interaction term by means of a likelihood ratio test. The average number of ESR2 CA repeats was associated with a small 5% increase in colorectal cancer risk (OR = 1.05, 95% CI 1.01-1.10) without significant heterogeneity according to gender or tumoural ESR2 expression. We found no indication for an association between the AR CAG repeat polymorphisms and risk of colorectal cancer. The ESR2 CA and AR CAG repeat polymorphisms were not associated with overall survival or disease specific survival after colorectal cancer diagnosis. Higher numbers of ESR2 CA repeats are potentially associated with a small increase in colorectal cancer risk. Our study does not support an association between colorectal cancer prognosis and the investigated repeat polymorphisms.

  7. Crosstalk between beta-2-adrenoceptor and muscarinic acetylcholine receptors in the airway.

    PubMed

    Pera, Tonio; Penn, Raymond B

    2014-06-01

    The M3 and M2 muscarinic acetylcholine receptors (mAChRs) and beta-2-adrenoceptors (β2ARs) are important regulators of airway cell function, and drugs targeting these receptors are among the first line drugs in the treatment of the obstructive lung diseases asthma and chronic obstructive lung disease (COPD). Cross-regulation or crosstalk between mAChRs and β2ARs in airway smooth muscle (ASM) helps determine the contractile state of the muscle, thus airway diameter and resistance to airflow. In this review we will detail mAChR and β2AR-signaling and crosstalk, focusing on events in the ASM cell but also addressing the function of these receptors in other cell types that impact airway physiology. We conclude by discussing how recent advances in GPCR pharmacology offer a unique opportunity to fine tune mAChR and β2AR signaling and their crosstalk, and thereby produce superior therapeutics for obstructive lung and other diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  8. β2-Adrenergic Receptor Agonists Inhibit the Proliferation of 1321N1 Astrocytoma CellsS⃞

    PubMed Central

    Toll, L.; Jimenez, L.; Waleh, N.; Jozwiak, K.; Woo, A.Y.-H.; Xiao, R.-P.; Bernier, M.

    2011-01-01

    Astrocytomas and glioblastomas have been particularly difficult to treat and refractory to chemotherapy. However, significant evidence has been presented that demonstrates a decrease in astrocytoma cell proliferation subsequent to an increase in cAMP levels. The 1321N1 astrocytoma cell line, as well as other astrocytomas and glioblastomas, expresses β2-adrenergic receptors2-ARs) that are coupled to Gs activation and consequent cAMP production. Experiments were conducted to determine whether the β2-AR agonist (R,R′)-fenoterol and other β2-AR agonists could attenuate mitogenesis and, if so, by what mechanism. Receptor binding studies were conducted to characterize β2-AR found in 1321N1 and U118 cell membranes. In addition, cells were incubated with (R,R′)-fenoterol and analogs to determine their ability to stimulate intracellular cAMP accumulation and inhibit [3H]thymidine incorporation into the cells. 1321N1 cells contain significant levels of β2-AR as determined by receptor binding. (R,R′)-fenoterol and other β2-AR agonists, as well as forskolin, stimulated cAMP accumulation in a dose-dependent manner. Accumulation of cAMP induced a decrease in [3H]thymidine incorporation. There was a correlation between concentration required to stimulate cAMP accumulation and inhibit [3H]thymidine incorporation. U118 cells have a reduced number of β2-ARs and a concomitant reduction in the ability of β2-AR agonists to inhibit cell proliferation. These studies demonstrate the efficacy of β2-AR agonists for inhibition of growth of the astrocytoma cell lines. Because a significant portion of brain tumors contain β2-ARs to a greater extent than whole brain, (R,R′)-fenoterol, or some analog, may be useful in the treatment of brain tumors after biopsy to determine β2-AR expression. PMID:21071556

  9. A new empirical potential energy function for Ar2

    NASA Astrophysics Data System (ADS)

    Myatt, Philip T.; Dham, Ashok K.; Chandrasekhar, Pragna; McCourt, Frederick R. W.; Le Roy, Robert J.

    2018-06-01

    A critical re-analysis of all available spectroscopic and virial coefficient data for Ar2 has been used to determine an improved empirical analytic potential energy function that has been 'tuned' to optimise its agreement with viscosity, diffusion and thermal diffusion data, and whose short-range behaviour is in reasonably good agreement with the most recent ab initio calculations for this system. The recommended Morse/long-range potential function is smooth and differentiable at all distances, and incorporates both the correct theoretically predicted long-range behaviour and the correct limiting short-range functional behaviour. The resulting value of the well depth is ? cm-1 and the associated equilibrium distance is re = 3.766 (±0.002) Å, while the 40Ar s-wave scattering length is -714 Å.

  10. 5-HT2a receptor in mPFC influences context-guided reconsolidation of object memory in perirhinal cortex.

    PubMed

    Morici, Juan Facundo; Miranda, Magdalena; Gallo, Francisco Tomás; Zanoni, Belén; Bekinschtein, Pedro; Weisstaub, Noelia V

    2018-05-02

    Context-dependent memories may guide adaptive behavior relaying in previous experience while updating stored information through reconsolidation. Retrieval can be triggered by partial and shared cues. When the cue is presented, the most relevant memory should be updated. In a contextual version of the object recognition task, we examined the effect of medial PFC (mPFC) serotonin 2a receptor (5-HT2aR) blockade during retrieval in reconsolidation of competing objects memories. We found that mPFC 5-HT2aR controls retrieval and reconsolidation of object memories in the perirhinal cortex (PRH), but not in the dorsal hippocampus in rats. Also, reconsolidation of objects memories in PRH required a functional interaction between the ventral hippocampus and the mPFC. Our results indicate that in the presence of conflicting information at retrieval, mPFC 5-HT2aR may facilitate top-down context-guided control over PRH to control the behavioral response and object memory reconsolidation. © 2018, Morici et al.

  11. 5-HT2a receptor in mPFC influences context-guided reconsolidation of object memory in perirhinal cortex

    PubMed Central

    Morici, Juan Facundo; Miranda, Magdalena; Gallo, Francisco Tomás; Zanoni, Belén; Bekinschtein, Pedro

    2018-01-01

    Context-dependent memories may guide adaptive behavior relaying in previous experience while updating stored information through reconsolidation. Retrieval can be triggered by partial and shared cues. When the cue is presented, the most relevant memory should be updated. In a contextual version of the object recognition task, we examined the effect of medial PFC (mPFC) serotonin 2a receptor (5-HT2aR) blockade during retrieval in reconsolidation of competing objects memories. We found that mPFC 5-HT2aR controls retrieval and reconsolidation of object memories in the perirhinal cortex (PRH), but not in the dorsal hippocampus in rats. Also, reconsolidation of objects memories in PRH required a functional interaction between the ventral hippocampus and the mPFC. Our results indicate that in the presence of conflicting information at retrieval, mPFC 5-HT2aR may facilitate top-down context-guided control over PRH to control the behavioral response and object memory reconsolidation. PMID:29717980

  12. Inerting of magnesium dust cloud with Ar, N2 and CO2.

    PubMed

    Li, G; Yuan, C M; Fu, Y; Zhong, Y P; Chen, B Z

    2009-10-15

    Experiments were conducted on the inerting of magnesium dust with N(2), CO(2), and Ar. Comparing the maximum explosion pressure, maximum rate of pressure rise, and limiting oxygen concentration with different inertants, it was determined that Ar is not the best inert gas under all conditions as commonly believed. N(2) was more effective than Ar as an inertant. CO(2) provided more inerting effect than either Ar and N(2) in low magnesium dust concentrations, although explosibility was increased at higher dust concentrations. Both N(2) and CO(2) as inerting agents showed higher LOC values than Ar. These results indicated that N(2) is a more economical inerting gas than Ar for the tested coarse magnesium dust.

  13. Novel mutations of the SRD5A2 and AR genes in Thai patients with 46, XY disorders of sex development.

    PubMed

    Ittiwut, Chupong; Pratuangdejkul, Jaturong; Supornsilchai, Vichit; Muensri, Sasipa; Hiranras, Yodporn; Sahakitrungruang, Taninee; Watcharasindhu, Suttipong; Suphapeetiporn, Kanya; Shotelersuk, Vorasuk

    2017-01-01

    Abnormalities of dihydrotestosterone conversion [5α-reductase deficiency: online Mendelian inheritance in man (OMIM) 607306] or actions of androgens [partial androgen insensitivity syndrome (PAIS): OMIM 312300] during the 8th-12th weeks of gestation cause varying degrees of undervirilized external genitalia in 46, XY disorders of sex development (DSD) with increased testosterone production. The objective of the study was to determine clinical and genetic characteristics of Thai patients with 46, XY DSD. A cross-sectional study was conducted in 46, XY DSD with increased testosterone production (n=43) evaluated by a human chorionic gonadotropin (hCG) stimulation test or clinical features consistent with 5α-reductase deficiency or PAIS. PCR sequencing of the entire coding regions of the SRD5A2 and AR genes was performed. Molecular modeling analysis of the androgen receptor-ligand-binding domain (AR-LBD) of a novel mutation was constructed. Mutations were found in seven patients (16.3%): five (11.6%) and two (4.7%) patients had mutations in SRD5A2 and AR, respectively. Two novel mutations, SRD5A2 c.383A>G (p.Y128C) and AR c.2176C>T (p.R726C), were identified. Dimensional structural analysis of the novel mutated AR (p.R726C) revealed that it affected the co-activator binding [binding function-3 (BF-3)], not the testosterone binding site. Short phallus length was associated with 5α-reductase deficiency. Around 16.3% of our patients with 46, XY DSD had 5α-reductase deficiency or PAIS. Two novel mutations of SRD5A2 and AR were identified. The novel mutated AR (p.R726C) might affect the co-activator binding (BF-3), not the testosterone binding site.

  14. 5-HT2A receptor activation is necessary for CO2-induced arousal

    PubMed Central

    Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

    2015-01-01

    Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

  15. Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage.

    PubMed

    Goth, Christoffer K; Tuhkanen, Hanna E; Khan, Hamayun; Lackman, Jarkko J; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H; Overall, Christopher M; Clausen, Henrik; Schjoldager, Katrine T; Petäjä-Repo, Ulla E

    2017-03-17

    The β 1 -adrenergic receptor (β 1 AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β 1 AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O -glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O -glycosylates β 1 AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O -glycosylation and proteolytic cleavage assays, a cell line deficient in O -glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β 1 AR. Furthermore, we demonstrate that impaired O -glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O -glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β 1 AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage*

    PubMed Central

    Goth, Christoffer K.; Tuhkanen, Hanna E.; Khan, Hamayun; Lackman, Jarkko J.; Wang, Shengjun; Narimatsu, Yoshiki; Hansen, Lasse H.; Overall, Christopher M.; Clausen, Henrik; Schjoldager, Katrine T.; Petäjä-Repo, Ulla E.

    2017-01-01

    The β1-adrenergic receptor (β1AR) is a G protein-coupled receptor (GPCR) and the predominant adrenergic receptor subtype in the heart, where it mediates cardiac contractility and the force of contraction. Although it is the most important target for β-adrenergic antagonists, such as β-blockers, relatively little is yet known about its regulation. We have shown previously that β1AR undergoes constitutive and regulated N-terminal cleavage participating in receptor down-regulation and, moreover, that the receptor is modified by O-glycosylation. Here we demonstrate that the polypeptide GalNAc-transferase 2 (GalNAc-T2) specifically O-glycosylates β1AR at five residues in the extracellular N terminus, including the Ser-49 residue at the location of the common S49G single-nucleotide polymorphism. Using in vitro O-glycosylation and proteolytic cleavage assays, a cell line deficient in O-glycosylation, GalNAc-T-edited cell line model systems, and a GalNAc-T2 knock-out rat model, we show that GalNAc-T2 co-regulates the metalloproteinase-mediated limited proteolysis of β1AR. Furthermore, we demonstrate that impaired O-glycosylation and enhanced proteolysis lead to attenuated receptor signaling, because the maximal response elicited by the βAR agonist isoproterenol and its potency in a cAMP accumulation assay were decreased in HEK293 cells lacking GalNAc-T2. Our findings reveal, for the first time, a GPCR as a target for co-regulatory functions of site-specific O-glycosylation mediated by a unique GalNAc-T isoform. The results provide a new level of β1AR regulation that may open up possibilities for new therapeutic strategies for cardiovascular diseases. PMID:28167537

  17. Visualization of arrestin recruitment by a G Protein-Coupled Receptor

    PubMed Central

    Reis, Rosana I.; Huang, Li-Yin; Tripathi-Shukla, Prachi; Qian, Jiang; Li, Sheng; Blanc, Adi; Oleskie, Austin N.; Dosey, Anne M.; Su, Min; Liang, Cui-Rong; Gu, Ling-Ling; Shan, Jin-Ming; Chen, Xin; Hanna, Rachel; Choi, Minjung; Yao, Xiao Jie; Klink, Bjoern U.; Kahsai, Alem W.; Sidhu, Sachdev S.; Koide, Shohei; Penczek, Pawel A.; Kossiakoff, Anthony A.; Jr, Virgil L. Woods; Kobilka, Brian K.; Skiniotis, Georgios; Lefkowitz, Robert J.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are critically regulated by β-arrestins (βarrs), which not only desensitize G protein signaling but also initiate a G protein independent wave of signaling1-5. A recent surge of structural data on a number of GPCRs, including the β2 adrenergic receptor2AR)-G protein complex, has provided novel insights into the structural basis of receptor activation6-11. Lacking however has been complementary information on recruitment of βarrs to activated GPCRs primarily due to challenges in obtaining stable receptor-βarr complexes for structural studies. Here, we devised a strategy for forming and purifying a functional β2AR-βarr1 complex that allowed us to visualize its architecture by single particle negative stain electron microscopy (EM) and to characterize the interactions between β2AR and βarr1 using hydrogen-deuterium exchange mass spectrometry (HDXMS) and chemical cross-linking. EM 2D averages and 3D reconstructions reveal bimodal binding of βarr1 to the β2AR, involving two separate sets of interactions, one with the phosphorylated carboxy-terminus of the receptor and the other with its seven-transmembrane core. Areas of reduced HDX together with identification of cross-linked residues suggest engagement of the finger loop of βarr1 with the seven-transmembrane core of the receptor. In contrast, focal areas of increased HDX indicate regions of increased dynamics in both N and C domains of βarr1 when coupled to the β2AR. A molecular model of the β2AR-βarr signaling complex was made by docking activated βarr1 and β2AR crystal structures into the EM map densities with constraints provided by HDXMS and cross-linking, allowing us to obtain valuable insights into the overall architecture of a receptor-arrestin complex. The dynamic and structural information presented herein provides a framework for better understanding the basis of GPCR regulation by arrestins. PMID:25043026

  18. PDK1/Akt/PDE4D axis identified as a target for asthma remedy synergistic with β2 AR agonists by a natural agent arctigenin.

    PubMed

    Fang, R; Cui, Q; Sun, J; Duan, X; Ma, X; Wang, W; Cheng, B; Liu, Y; Hou, Y; Bai, G

    2015-12-01

    Asthma is a heterogenetic disorder characterized by chronic inflammation with variable airflow obstruction and airway hyper-responsiveness. As the most potent and popular bronchodilators, β2 adrenergic receptor2 AR) agonists bind to the β2 ARs that are coupled via a stimulatory G protein to adenylyl cyclase, thereby improving cAMP accumulation and resulting in airway smooth muscle relaxation. We previously demonstrated arctigenin had a synergistic function with the β2 AR agonist, but the target for this remained elusive. Chemical proteomics capturing was used to enrich and uncover the target of arctigenin in human bronchial smooth muscle cells, and reverse docking and molecular dynamic stimulation were performed to evaluate the binding of arctigenin and its target. In vitro enzyme activities and protein levels were demonstrated with special kits and Western blotting. Finally, guinea pig tracheal muscle segregation and ex vivo function were analysed. Arctigenin bound to PDK1 with an ideal binding free energy -25.45 kcal/mol and inhibited PDK1 kinase activity without changing its protein level. Additionally, arctigenin reduced PKB/Akt-induced phosphorylation of PDE4D, which was first identified in this study. Attenuation of PDE4D resulted in cAMP accumulation in human bronchial smooth muscle. The inhibition of PDK1 showed a synergistic function with β2 AR agonists and relaxed the constriction of segregated guinea pig tracheal muscle. The PDK1/Akt/PDE4D axis serves as a novel asthma target, which may benefit airflow obstruction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  19. Molecular Recognition at Purine and Pyrimidine Nucleotide (P2) Receptors

    PubMed Central

    Jacobson, Kenneth A.; Constanzi, Stefano; Ohno, Michihiro; Joshi, Bhalchandra V.; Besada, Pedro; Xu, Bin; Tchilibon, Susanna

    2015-01-01

    In comparison to other classes of cell surface receptors, the medicinal chemistry at P2X (ligand-gated ion channels) and P2Y (G protein-coupled) nucleotide receptors has been relatively slow to develop. Recent effort to design selective agonists and antagonists based on a combination of library screening, empirical modification of known ligands, and rational design have led to the introduction of potent antagonists of the P2X1 (derivatives of pyridoxal phosphates and suramin), P2X3 (A-317491), P2X7 (derivatives of the isoquinoline KN-62), P2Y1 (nucleotide analogues MRS 2179 and MRS 2279), P2Y2 (thiouracil derivatives such as AR-C126313), and P2Y12 (nucleotide/nucleoside analogues AR-C69931X and AZD6140) receptors. A variety of native agonist ligands (ATP, ADP, UTP, UDP, and UDP-glucose) are currently the subject of structural modification efforts to improve selectivity. MRS2365 is a selective agonist for P2Y1 receptors. The dinucleotide INS 37217 potently activates the P2Y2 receptor. UTP-γ-S and UDP-β-S are selective agonists for P2Y2/P2Y4 and P2Y6 receptors, respectively. The current knowledge of the structures of P2X and P2Y receptors, is derived mainly from mutagenesis studies. Site-directed mutagenesis has shown that ligand recognition in the human P2Y1 receptor involves individual residues of both the TMs (3, 5, 6, and 7), as well as EL 2 and 3. The binding of the negatively-charged phosphate moiety is dependent on positively charged lysine and arginine residues near the exofacial side of TMs 3 and 7. PMID:15078212

  20. CgA1AR-1 acts as an alpha-1 adrenergic receptor in oyster Crassostrea gigas mediating both cellular and humoral immune response.

    PubMed

    Liu, Zhaoqun; Zhou, Zhi; Wang, Lingling; Qiu, Limei; Zhang, Huan; Wang, Hao; Song, Linsheng

    2016-11-01

    We have now cloned an alpha-1 adrenergic receptor (A1AR) from the cDNA library of oyster Crassostrea gigas, designating as CgA1AR-1. The full length of CgA1AR-1 was 1149 bp and it encodes a protein of 382 amino acids containing a 7 transmembrane domain, whose putative topology was similar to the A1ARs in higher organisms and shared similarity of 19% with mammalian A1ARs according to the phylogenic analysis. After cell transfection of CgA1AR-1 into HEK293T cells and the incubation with its specific agonist norepinephrine (NE), the concentration of second messenger Ca 2+ increased significantly (p < 0.05). But, this increasing of Ca 2+ could be inhibited by adding A1AR antagonist DOX. Tissue distribution assays using qRT-PCR suggested that CgA1AR-1 mRNA was ubiquitously expressed in all the major tissues of oyster. LPS stimulation could induce the up-regulation of CgA1AR-1 mRNA in haemocytes from 12 h to 24 h post stimulation. Moreover, the blocking of CgA1AR-1 by DOX before LPS stimulation affected the mRNA expression of oyster TNF (CGI_10005109 and CGI_10006440) in haemocytes, resulting in the rise of haemocyte phagocytic rate and apoptosis index. In addition to cellular immunity, CgA1AR-1 was also involved in humoral immunity of oyster. Inhibition of CgA1AR-1 with DOX could repress the up-regulation of LZY and SOD activities caused by LPS stimulation. These results suggested that CgA1AR-1 acted as an α-1 adrenergic receptor in cetacholaminergic neuroendocrine-immune network mediating both cellular and humoral immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Ligand Entry and Exit Pathways in the β2-adrenergic Receptor

    PubMed Central

    Wang, Ting; Duan, Yong

    2009-01-01

    The recently determined crystal structure of the human β2-adrenergic (β2AR) G-protein coupled receptor provides an excellent structural basis for exploring β2AR -ligand binding and dissociation process. Based on this crystal structure, we simulated ligand exit from the β2AR receptor by applying the random acceleration molecular dynamics (RAMD) simulation method. The simulation results showed that the extracellular opening on the receptor surface was the most frequently observed egress point (referred to as pathway A) and a few other pathways through inter-helical clefts were also observed with significantly lower frequencies. In the egress trajectories along pathway A, the D192-K305 salt bridge between the extracellular loop 2 (ECL2) and the apex of the transmembrane helix 7 (TM7) was exclusively broken. The spatial occupancy maps of the ligand computed from the 100 RAMD simulation trajectories indicated that the receptor-ligand interactions that restrained the ligand in the binding pocket were the major resistance encountered by the ligand during exit and no second barrier was notable. We next performed RAMD simulations by using a putative ligand-free conformation of the receptor as input structure. This conformation was obtained in a standard MD simulation in the absence of the ligand and it differed from the ligand-bound conformation in a hydrophobic patch bridging ECL2 and TM7 due to the rotation of F193 of ECL2. Results from the RAMD simulations with this putative ligand-free conformation suggest that the cleft formed by the hydrophobic bridge, TM2, TM3 and TM7 on the extracellular surface likely serves as a more specific ligand-entry site and the ECL2-TM7 hydrophobic junction can be partially interrupted upon the entry of ligand that pushes F193 to rotate, resulting in a conformation as observed in the ligand-bound crystal structure. These results may help design β2AR-targeting drugs with improved efficacy as well as understand the receptor subtype

  2. Nanoscale organization of {beta}{sub 2}-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vobornik, Dusan; Rouleau, Yanouchka; Haley, Jennifer

    2009-04-24

    Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize {beta}{sub 2}-adrenergic receptors ({beta}{sub 2}AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the {beta}{sub 2}AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline,more » the size of domains containing the {beta}{sub 2}AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of {beta}{sub 2}AR are observed in the membrane of the HEK293 cells that stably overexpress {beta}{sub 2}AR-GFP and {beta}{sub 2}AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use {beta}{sub 2}AR-Venus fusion proteins as models for {beta}{sub 2}AR function. These observations are critical for designing future cell models and assays based on {beta}{sub 2}AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.« less

  3. Preoptimised VB: a fast method for the ground and excited states of ionic clusters II. Delocalised preoptimisation for He 2+, Ar 2+, He 3+ and Ar 3+

    NASA Astrophysics Data System (ADS)

    Archirel, Pierre

    1997-09-01

    We generalise the preoptimisation of orbitals within VB (Part I of this series) through letting the orbitals delocalise on the neighbouring fragments. The method is more accurate than the local preoptimisation. The method is tested on the rare gas clusters He 2+, Ar 2+, He 3+ and Ar 3+. The results are in good agreement with previously published data on these systems. We complete these data with higher excited states. The binding energies of (ArCO) +, (ArN 2) + and N 4+ are revisited. The simulation of the SCF method is extended to Cu +H 2O.

  4. α2 Adrenergic receptor-mediated inhibition of thermogenesis.

    PubMed

    Madden, Christopher J; Tupone, Domenico; Cano, Georgina; Morrison, Shaun F

    2013-01-30

    α2 adrenergic receptor2-AR) agonists have been used as antihypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist clonidine (1.2 nmol) into the rostral raphe pallidus area (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist idazoxan (6 nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists dexmedetomidine (25 μg/kg, i.v.) and clonidine (100 μg/kg, i.v.) inhibited shivering EMGs, BAT SNA, and BAT thermogenesis, effects that were reversed by nanoinjection of idazoxan (6 nmol) into the rRPa. Dexmedetomidine (100 μg/kg, i.p.) prevented and reversed lipopolysaccharide-evoked (10 μg/kg, i.p.) thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of ventrolateral medulla neurons expressing the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially lethal elevations in body temperature during excessive fever.

  5. Alpha-2 adrenergic receptor-mediated inhibition of thermogenesis

    PubMed Central

    Madden, Christopher J.; Tupone, Domenico; Cano, Georgina; Morrison, Shaun F.

    2013-01-01

    Alpha2-adrenergic receptor2-AR) agonists have been use as anti-hypertensive agents, in the management of drug withdrawal, and as sedative analgesics. Since α2-AR agonists also influence the regulation of body temperature, we explored their potential as antipyretic agents. This study delineates the central neural substrate for the inhibition of rat brown adipose tissue (BAT) and shivering thermogenesis by α2-AR agonists. Nanoinjection of the α2-AR agonist, clonidine (1.2 nmol), into the rostral raphe pallidus (rRPa) inhibited BAT sympathetic nerve activity (SNA) and BAT thermogenesis. Subsequent nanoinjection of the α2-AR antagonist, idazoxan (6nmol) into the rRPa reversed the clonidine-evoked inhibition of BAT SNA and BAT thermogenesis. Systemic administration of the α2-AR agonists, dexmedetomidine (25ug/kg, iv) or clonidine (100ug/kg, iv) inhibited shivering EMGs, BAT SNA and BAT thermogenesis effects that were reversed by nanoinjection of idazoxan (6nmol) into the rRPa. Dexmedetomidine (100µg/kg, ip) prevented and reversed lipopolysaccharide (10µg/kg ip)-evoked thermogenesis in free-behaving rats. Cholera toxin subunit b retrograde tracing from rRPa and pseudorabies virus transynaptic retrograde tracing from BAT combined with immunohistochemistry for catecholaminergic biosynthetic enzymes revealed the ventrolateral medulla as the source of catecholaminergic input to the rRPa and demonstrated that these catecholaminergic neurons are synaptically connected to BAT. Photostimulation of VLM neurons expressing of the PRSx8-ChR2-mCherry lentiviral vector inhibited BAT SNA via activation of α2-ARs in the rRPa. These results indicate a potent inhibition of BAT and shivering thermogenesis by α2-AR activation in the rRPa, and suggest a therapeutic potential of α2-AR agonists for reducing potentially-lethal elevations in body temperature during excessive fever. PMID:23365239

  6. Arf6 negatively controls the rapid recycling of the β2 adrenergic receptor.

    PubMed

    Macia, Eric; Partisani, Mariagrazia; Paleotti, Olivia; Luton, Frederic; Franco, Michel

    2012-09-01

    β2-adrenergic receptor2AR), a member of the GPCR (G-protein coupled receptor) family, is internalized in a ligand- and β-arrestin-dependent manner into early endosomes, and subsequently recycled back to the plasma membrane. Here, we report that β-arrestin promotes the activation of the small G protein Arf6, which regulates the recycling and degradation of β2AR. We demonstrate in vitro that the C-terminal region of β-arrestin1 interacts directly and simultaneously with Arf6GDP and its specific exchange factor EFA6, to promote Arf6 activation. Similarly, the ligand-mediated activation of β2AR leads to the formation of Arf6GTP in vivo in a β-arrestin-dependent manner. Expression of either EFA6 or an activated Arf6 mutant caused accumulation of β2AR in the degradation pathway. This phenotype could be rescued by the expression of an activated mutant of Rab4, suggesting that Arf6 acts upstream of Rab4. We propose a model in which Arf6 plays an essential role in β2AR desensitization. The ligand-mediated stimulation of β2AR relocates β-arrestin to the plasma membrane, and triggers the activation of Arf6 by EFA6. The activation of Arf6 leads to accumulation of β2AR in the degradation pathway, and negatively controls Rab4-dependent fast recycling to prevent the re-sensitization of β2AR.

  7. Rational Design, Pharmacomodulation, and Synthesis of Dual 5-Hydroxytryptamine 7 (5-HT7)/5-Hydroxytryptamine 2A (5-HT2A) Receptor Antagonists and Evaluation by [(18)F]-PET Imaging in a Primate Brain.

    PubMed

    Deau, Emmanuel; Robin, Elodie; Voinea, Raluca; Percina, Nathalie; Satała, Grzegorz; Finaru, Adriana-Luminita; Chartier, Agnès; Tamagnan, Gilles; Alagille, David; Bojarski, Andrzej J; Morisset-Lopez, Séverine; Suzenet, Franck; Guillaumet, Gérald

    2015-10-22

    We report the synthesis of 46 tertiary amine-bearing N-alkylated benzo[d]imidazol-2(3H)-ones, imidazo[4,5-b]pyridin-2(3H)-ones, imidazo[4,5-c]pyridin-2(3H)-ones, benzo[d]oxazol-2(3H)-ones, oxazolo[4,5-b]pyridin-2(3H)-ones and N,N'-dialkylated benzo[d]imidazol-2(3H)-ones. These compounds were evaluated against 5-HT7R, 5-HT2AR, 5-HT1AR, and 5-HT6R as potent dual 5-HT7/5-HT2A serotonin receptors ligands. A thorough study of the structure-activity relationship of the aromatic rings and their substituents, the alkyl chain length and the tertiary amine was conducted. 1-(4-(4-(4-Fluorobenzoyl)piperidin-1-yl)butyl)-1H-benzo[d]imidazol-2(3H)-one (79) and 1-(6-(4-(4-fluorobenzoyl)piperidin-1-yl)hexyl)-1H-benzo[d]imidazol-2(3H)-one (81) were identified as full antagonist ligands on cyclic adenosine monophosphate (cAMP, KB = 4.9 and 5.9 nM, respectively) and inositol monophosphate (IP1, KB = 0.6 and 16 nM, respectively) signaling pathways of 5-HT7R and 5-HT2AR. Both antagonists crossed the blood-brain barrier as evaluated with [(18)F] radiolabeled compounds [(18)F]79 and [(18)F]81 in a primate's central nervous system using positron emission tomography. Both radioligands showed standard uptake values ranging from 0.8 to 1.1, a good plasmatic stability, and a distribution consistent with 5-HT7R and 5-HT2AR in the CNS.

  8. Androgen receptor polyglutamine repeat length affects receptor activity and C2C12 cell development.

    PubMed

    Sheppard, Ryan L; Spangenburg, Espen E; Chin, Eva R; Roth, Stephen M

    2011-10-20

    Testosterone (T) has an anabolic effect on skeletal muscle and is believed to exert its local effects via the androgen receptor (AR). The AR harbors a polymorphic stretch of glutamine repeats demonstrated to inversely affect receptor transcriptional activity in prostate and kidney cells. The effects of AR glutamine repeat length on skeletal muscle are unknown. In this study we examined the effect of AR CAG repeat length on AR function in C2C12 cells. AR expression vectors harboring 14, 24, and 33 CAG repeats were used to assess AR transcriptional activity. C2C12 cell proliferation, differentiation, gene expression, myotube formation, and myonuclear fusion index were assessed. Transcriptional activity increased with increasing repeat length and in response to testosterone (AR14 = 3.91 ± 0.26, AR24 = 25.21 ± 1.72, AR33 = 36.08 ± 3.22 relative light units; P < 0.001). Ligand activation was increased for AR33 (2.10 ± 0.04) compared with AR14 (1.54 ± 0.09) and AR24 (1.57 ± 0.05, P < 0.001). AR mRNA expression was elevated in each stably transfected line. AR33 cell proliferation (20,512.3 ± 1,024.0) was decreased vs. AR14 (27,604.17 ± 1,425.3; P < 0.001) after 72 h. Decreased CK activity in AR14 cells (54.9 ± 2.9 units/μg protein) in comparison to AR33 (70.8 ± 8.1) (P < 0.05) was noted. The myonuclear fusion index was lower for AR14 (15.21 ± 3.24%) and AR33 (9.97 ± 3.14%) in comparison to WT (35.07 ± 5.60%, P < 0.001). AR14 and AR33 cells also displayed atypical myotube morphology. RT-PCR revealed genotype differences in myostatin and myogenin expression. We conclude that AR polyglutamine repeat length is directly associated with transcriptional activity and alters the growth and development of C2C12 cells. This polymorphism may contribute to the heritability of muscle mass in humans.

  9. 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

  10. High-fat diet induces protein kinase A and G-protein receptor kinase phosphorylation of β2 -adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts.

    PubMed

    Fu, Qin; Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan; Xiang, Yang K

    2017-03-15

    Patients with diabetes show a blunted cardiac inotropic response to β-adrenergic stimulation despite normal cardiac contractile reserve. Acute insulin stimulation impairs β-adrenergically induced contractile function in isolated cardiomyocytes and Langendorff-perfused hearts. In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high-fat diet (HFD) feeding on the cardiac β 2 -adrenergic receptor signalling and the impacts on cardiac contractile function. We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β-adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β 2 -adrenergic receptor phosphorylation at protein kinase A and G-protein receptor kinase sites in the myocardium. The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high-fat diet (HFD) on the insulin-adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD-fed mice displayed a significant elevation of phosphorylation of the β 2 -adrenergic receptor2 AR) at both the protein kinase A site serine 261/262 and the G-protein-coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD-fed mice also displayed a reduced contractile response to

  11. Serotonin hyperinnervation and upregulated 5-HT2A receptor expression and motor-stimulating function in nigrostriatal dopamine-deficient Pitx3 mutant mice.

    PubMed

    Li, Li; Qiu, Guozhen; Ding, Shengyuan; Zhou, Fu-Ming

    2013-01-23

    The striatum receives serotonin (5-hydroxytryptamine, 5-HT) innervation and expresses 5-HT2A receptors (5-HT2ARs) and other 5-HT receptors, raising the possibility that the striatal 5-HT system may undergo adaptive changes after chronic severe dopamine (DA) loss and contribute to the function and dysfunction of the striatum. Here we show that in transcription factor Pitx3 gene mutant mice with a selective, severe DA loss in the dorsal striatum mimicking the DA denervation in late Parkinson's disease (PD), both the 5-HT innervation and the 5-HT2AR mRNA expression were increased in the dorsal striatum. Functionally, while having no detectable motor effect in wild type mice, the 5-HT2R agonist 2,5-dimethoxy-4-iodoamphetamine increased both the baseline and l-dopa-induced normal ambulatory and dyskinetic movements in Pitx3 mutant mice, whereas the selective 5-HT2AR blocker volinanserin had the opposite effects. These results demonstrate that Pitx3 mutant mice are a convenient and valid mouse model to study the compensatory 5-HT upregulation following the loss of the nigrostriatal DA projection and that the upregulated 5-HT2AR function in the DA deficient dorsal striatum may enhance both normal and dyskinetic movements. Copyright © 2012 Elsevier B.V. All rights reserved.

  12. Androgen and Progesterone Receptors Are Targets for Bisphenol A (BPA), 4-Methyl-2,4-bis-(P-Hydroxyphenyl)Pent-1-Ene--A Potent Metabolite of BPA, and 4-Tert-Octylphenol: A Computational Insight.

    PubMed

    Rehan, Mohd; Ahmad, Ejaz; Sheikh, Ishfaq A; Abuzenadah, Adel M; Damanhouri, Ghazi A; Bajouh, Osama S; AlBasri, Samera F; Assiri, Mansour M; Beg, Mohd A

    2015-01-01

    Exposure to toxic industrial chemicals that have capacity to disrupt the endocrine system, also known as endocrine disrupting chemicals (EDCs), has been increasingly associated with reproductive problems in human population. Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol) and 4-tert-octylphenol (OP; 4-(1,1,3,3-tetramethylbutyl)phenol) are among the most common environmental contaminants possessing endocrine disruption properties and are present in plastics, epoxy resins, detergents and other commercial products of common personal and industrial use. A metabolite of BPA, 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is about 1000 times more biologically active compared to BPA. Epidemiological, clinical, and experimental studies have shown association of BPA and OP with adverse effects on male and female reproductive system in human and animals. The endocrine disruption activity can occur through multiple pathways including binding to steroid receptors. Androgen receptor (AR) and progesterone receptor (PR) are critical for reproductive tract growth and function. Structural binding characterization of BPA, MBP, and OP with AR and PR using molecular docking simulation approaches revealed novel interactions of BPA with PR, and MBP and OP with AR and PR. For BPA, MBP, and OP, five AR interacting residues Leu-701, Leu-704, Asn-705, Met-742, and Phe-764 overlapped with those of native AR ligand testosterone, and four PR interacting residues Leu-715, Leu-718, Met-756, and Met-759 overlapped with those of PR co-complex ligand, norethindrone. For both the receptors the binding strength of MBP was maximum among the three compounds. Thus, these compounds have the potential to block or interfere in the binding of the endogenous native AR and PR ligands and, hence, resulting in dysfunction. The knowledge of the key interactions and the important amino-acid residues also allows better prediction of potential of xenobiotic molecules for disrupting AR- and PR

  13. Androgen and Progesterone Receptors Are Targets for Bisphenol A (BPA), 4-Methyl-2,4-bis-(P-Hydroxyphenyl)Pent-1-Ene—A Potent Metabolite of BPA, and 4-Tert-Octylphenol: A Computational Insight

    PubMed Central

    Rehan, Mohd; Ahmad, Ejaz; Sheikh, Ishfaq A.; Abuzenadah, Adel M.; Damanhouri, Ghazi A.; Bajouh, Osama S.; AlBasri, Samera F.; Assiri, Mansour M.; Beg, Mohd A.

    2015-01-01

    Exposure to toxic industrial chemicals that have capacity to disrupt the endocrine system, also known as endocrine disrupting chemicals (EDCs), has been increasingly associated with reproductive problems in human population. Bisphenol A (BPA; 4,4'-(propane-2,2-diyl)diphenol) and 4-tert-octylphenol (OP; 4-(1,1,3,3-tetramethylbutyl)phenol) are among the most common environmental contaminants possessing endocrine disruption properties and are present in plastics, epoxy resins, detergents and other commercial products of common personal and industrial use. A metabolite of BPA, 4-Methyl-2,4-bis(4-hydroxyphenyl)pent-1-ene (MBP) is about 1000 times more biologically active compared to BPA. Epidemiological, clinical, and experimental studies have shown association of BPA and OP with adverse effects on male and female reproductive system in human and animals. The endocrine disruption activity can occur through multiple pathways including binding to steroid receptors. Androgen receptor (AR) and progesterone receptor (PR) are critical for reproductive tract growth and function. Structural binding characterization of BPA, MBP, and OP with AR and PR using molecular docking simulation approaches revealed novel interactions of BPA with PR, and MBP and OP with AR and PR. For BPA, MBP, and OP, five AR interacting residues Leu-701, Leu-704, Asn-705, Met-742, and Phe-764 overlapped with those of native AR ligand testosterone, and four PR interacting residues Leu-715, Leu-718, Met-756, and Met-759 overlapped with those of PR co-complex ligand, norethindrone. For both the receptors the binding strength of MBP was maximum among the three compounds. Thus, these compounds have the potential to block or interfere in the binding of the endogenous native AR and PR ligands and, hence, resulting in dysfunction. The knowledge of the key interactions and the important amino-acid residues also allows better prediction of potential of xenobiotic molecules for disrupting AR- and PR

  14. Androgen Receptor (AR) in Cardiovascular Diseases

    PubMed Central

    Huang, Chiung-Kuei; Lee, Soo Ok; Chang, Eugene; Pang, Haiyan; Chang, Chawnshang

    2016-01-01

    Cardiovascular diseases (CVDs) are still the highest leading cause of death worldwide. Several risk factors have been linked to CVDs, including smoking, diabetes, hyperlipidemia, and gender among others. Sex hormones, especially the androgen and its receptor, androgen receptor (AR), have been linked to many diseases with a clear gender difference. Here, we summarize androgen/AR effects on CVDs, including hypertension, stroke, atherosclerosis, abdominal aortic aneurysm (AAA), myocardial hypertrophy, and heart failure, as well as metabolic syndrome/diabetes and their impacts on CVDs. Androgen/AR signaling exacerbates hypertension and anti-androgens may suppress hypertension. Androgen/AR signaling plays dual roles in strokes, depending on different kinds of factors, but generally males have a higher incidence of strokes than females. Androgen and AR differentially modulate atherosclerosis. Androgen deficiency causes elevated lipid accumulation to enhance atherosclerosis, but targeting AR in selective cells without altering serum androgen levels would suppress atherosclerosis progression. Androgen/AR signaling is crucial in AAA development and progression, and targeting androgen/AR profoundly restricts AAA progression. Men have increased cardiac hypertrophy as compared to age-matched women that may be due to androgens. Finally, androgen/AR plays important roles in contributing to obesity and insulin/leptin resistance to increase the metabolic syndrome. PMID:26769913

  15. 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.

  16. Palmitoylation regulates intracellular trafficking of β2 adrenergic receptor/arrestin/phosphodiesterase 4D complexes in cardiomyocytes.

    PubMed

    Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K

    2012-01-01

    β(2) adrenergic receptor (β(2)AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β(2)AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β(2)AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β(2)AR association with arrestin/PDE4D. Replacing cysteine 341 on β(2)AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β(2)AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β(2)AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β(2)AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β(2)AR.

  17. Palmitoylation Regulates Intracellular Trafficking of β2 Adrenergic Receptor/Arrestin/Phosphodiesterase 4D Complexes in Cardiomyocytes

    PubMed Central

    Liu, Ruijie; Wang, Dayong; Shi, Qian; Fu, Qin; Hizon, Steven; Xiang, Yang K.

    2012-01-01

    β2 adrenergic receptor2AR) is a prototypical G-protein coupled receptor that stimulates the classic cAMP-protein kinase A (PKA) signaling pathway. Recent studies indicate that the cAMP-PKA activities are spatiotemporally regulated in part due to dynamic association of β2AR with phosphodiesterase 4D (PDE4D), a group of cAMP degradation enzymes. Here, we demonstrate that in cardiomyocytes, palmitoylation of β2AR, the covalent acylation of cysteine residue 341, plays a critical role in shaping subcellular cAMP-PKA activities in cardiomyocytes via regulating β2AR association with arrestin/PDE4D. Replacing cysteine 341 on β2AR with alanine (C341A) leads to an impaired binding to β arrestin 2. Surprisingly, the C341A mutant is able to internalize via an arrestin-independent pathway at saturated concentration of agonist stimulation; the internalization becomes caveolae-dependent and requires dynamin GTPase. However, the impaired binding to β arrestin 2 also leads to an impaired recruitment of PDE4D to the C341A mutant. Thus, the mutant C341A β2AR is transported alone from the plasma membrane to the endosome without recruiting PDE4D. This alteration leads to an enhanced cytoplasmic cAMP signal for PKA activation under β2AR stimulation. Functionally, Mutation of the C341 residue or inhibition of palmitoylation modification of β2AR enhances the receptor-induced PKA activities in the cytoplasm and increases in myocyte contraction rate. Our data reveal a novel function of palmitoylation in shaping subcellular cAMP-PKA signaling in cardiomyocytes via modulating the recruitment of β arrestin 2-PDE4D complexes to the agonist-stimulated β2AR. PMID:22912718

  18. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    PubMed

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  19. Functional somatostatin receptors on a rat pancreatic acinar cell line

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Viguerie, N.; Tahiri-Jouti, N.; Esteve, J.P.

    1988-07-01

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of {sup 125}I-(Tyr{sup 11})Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 {plus minus} 20 fmol/10{sup 6} cells. Somatostatin receptor structure was analyzed by covalently cross-linking {sup 125}I-(Tyr{sup 11})somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3{prime},5{prime}-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibitionmore » of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein N{sub i} to inhibit adenylate cyclase.« less

  20. Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese A{sup y} mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nonogaki, Katsunori; Nozue, Kana; Oka, Yoshitomo

    2006-12-29

    Serotonin (5-hydroxytryptamine; 5-HT) 2A receptors contribute to the effects of 5-HT on platelet aggregation and vascular smooth muscle cell proliferation, and are reportedly involved in decreases in plasma levels of adiponectin, an adipokine, in diabetic subjects. Here, we report that systemic administration of sarpogrelate, a 5-HT2A receptor antagonist, suppressed appetite and increased hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, 5-HT2C, and 5-HT1B receptor gene expression. A{sup y} mice, which have ectopic expression of the agouti protein, significantly increased hypothalamic 5-HT2A receptor gene expression in association with obesity compared with wild-type mice matched for age. Systemic administration ofmore » sarpogrelate suppressed overfeeding, body weight gain, and hyperglycemia in obese A{sup y} mice, whereas it did not increase plasma adiponectin levels. These results suggest that obesity increases hypothalamic 5-HT2A receptor gene expression, and pharmacologic inactivation of 5-HT2A receptors inhibits overfeeding and obesity in A{sup y} mice, but did not increase plasma adiponectin levels.« less

  1. ADRA2A is involved in neuro-endocrine regulation of bone resorption.

    PubMed

    Mlakar, Vid; Jurkovic Mlakar, Simona; Zupan, Janja; Komadina, Radko; Prezelj, Janez; Marc, Janja

    2015-07-01

    Adrenergic stimulation is important for osteoclast differentiation and bone resorption. Previous research shows that this happens through β2-adrenergic receptor (AR), but there are conflicting evidence on presence and role of α2A-AR in bone. The aim of this study was to investigate the presence of α2A-AR and its involvement in neuro-endocrine signalling of bone remodelling in humans. Real-time polymerase chain reaction (PCR) and immunohistochemistry were used to investigate α2A-AR receptor presence and localization in bone cells. Functionality of rs553668 and rs1800544 single nucleotide polymorphism SNPs located in α2A-AR gene was analysed by qPCR expression on bone samples and luciferase reporter assay in human osteosarcoma HOS cells. Using real-time PCR, genetic association study between rs553668 A>G and rs1800544 C>G SNPs and major bone markers was performed on 661 Slovenian patients with osteoporosis. α2A-AR is expressed in osteoblasts and lining cells but not in osteocytes. SNP rs553668 has a significant influence on α2A-AR mRNA level in human bone samples through the stability of mRNA. α2A-AR gene locus associates with important bone remodelling markers (BMD, CTX, Cathepsin K and pOC). The results of this study are providing comprehensive new evidence that α2A-AR is involved in neuro-endocrine signalling of bone turnover and development of osteoporosis. As shown by our results the neurological signalling is mediated through osteoblasts and result in bone resorption. Genetic study showed association of SNPs in α2A-AR gene locus with bone remodelling markers, identifying the individuals with higher risk of development of osteoporosis. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. ADRA2A is involved in neuro-endocrine regulation of bone resorption

    PubMed Central

    Mlakar, Vid; Jurkovic Mlakar, Simona; Zupan, Janja; Komadina, Radko; Prezelj, Janez; Marc, Janja

    2015-01-01

    Adrenergic stimulation is important for osteoclast differentiation and bone resorption. Previous research shows that this happens through β2-adrenergic receptor (AR), but there are conflicting evidence on presence and role of α2A-AR in bone. The aim of this study was to investigate the presence of α2A-AR and its involvement in neuro-endocrine signalling of bone remodelling in humans. Real-time polymerase chain reaction (PCR) and immunohistochemistry were used to investigate α2A-AR receptor presence and localization in bone cells. Functionality of rs553668 and rs1800544 single nucleotide polymorphism SNPs located in α2A-AR gene was analysed by qPCR expression on bone samples and luciferase reporter assay in human osteosarcoma HOS cells. Using real-time PCR, genetic association study between rs553668 A>G and rs1800544 C>G SNPs and major bone markers was performed on 661 Slovenian patients with osteoporosis. α2A-AR is expressed in osteoblasts and lining cells but not in osteocytes. SNP rs553668 has a significant influence on α2A-AR mRNA level in human bone samples through the stability of mRNA. α2A-AR gene locus associates with important bone remodelling markers (BMD, CTX, Cathepsin K and pOC). The results of this study are providing comprehensive new evidence that α2A-AR is involved in neuro-endocrine signalling of bone turnover and development of osteoporosis. As shown by our results the neurological signalling is mediated through osteoblasts and result in bone resorption. Genetic study showed association of SNPs in α2A-AR gene locus with bone remodelling markers, identifying the individuals with higher risk of development of osteoporosis. PMID:25818344

  3. 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

  4. High‐fat diet induces protein kinase A and G‐protein receptor kinase phosphorylation of β2‐adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts

    PubMed Central

    Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan

    2017-01-01

    Key points Patients with diabetes show a blunted cardiac inotropic response to β‐adrenergic stimulation despite normal cardiac contractile reserve.Acute insulin stimulation impairs β‐adrenergically induced contractile function in isolated cardiomyocytes and Langendorff‐perfused hearts.In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high‐fat diet (HFD) feeding on the cardiac β2‐adrenergic receptor signalling and the impacts on cardiac contractile function.We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β‐adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2‐adrenergic receptor phosphorylation at protein kinase A and G‐protein receptor kinase sites in the myocardium.The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Abstract Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high‐fat diet (HFD) on the insulin–adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD‐fed mice displayed a significant elevation of phosphorylation of the β2‐adrenergic receptor2AR) at both the protein kinase A site serine 261/262 and the G‐protein‐coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD‐fed mice also displayed a

  5. HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis.

    PubMed

    Osiński, Maciej; Wirstlein, Przemysław; Wender-Ożegowska, Ewa; Mikołajczyk, Mateusz; Jagodziński, Paweł Piotr; Szczepańska, Małgorzata

    2018-01-01

    The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.

  6. 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

  7. Aurora Kinase A Promotes AR Degradation via the E3 Ligase CHIP.

    PubMed

    Sarkar, Sukumar; Brautigan, David L; Larner, James M

    2017-08-01

    Reducing the levels of the androgen receptor (AR) is one of the most viable approaches to combat castration-resistant prostate cancer. Previously, we observed that proteasomal-dependent degradation of AR in response to 2-methoxyestradiol (2-ME) depends primarily on the E3 ligase C-terminus of HSP70-interacting protein (STUB1/CHIP). Here, 2-ME stimulation activates CHIP by phosphorylation via Aurora kinase A (AURKA). Aurora A kinase inhibitors and RNAi knockdown of Aurora A transcript selectively blocked CHIP phosphorylation and AR degradation. Aurora A kinase is activated by 2-ME in the S-phase as well as during mitosis, and phosphorylates CHIP at S273. Prostate cancer cells expressing an S273A mutant of CHIP have attenuated AR degradation upon 2-ME treatment compared with cells expressing wild-type CHIP, supporting the idea that CHIP phosphorylation by Aurora A activates its E3 ligase activity for the AR. These results reveal a novel 2-ME→Aurora A→CHIP→AR pathway that promotes AR degradation via the proteasome that may offer novel therapeutic opportunities for prostate cancer. Mol Cancer Res; 15(8); 1063-72. ©2017 AACR . ©2017 American Association for Cancer Research.

  8. Prostanoid receptor EP2 as a therapeutic target.

    PubMed

    Ganesh, Thota

    2014-06-12

    Cycoloxygenase-2 (COX-2) induction is prevalent in a variety of (brain and peripheral) injury models where COX-2 levels correlate with disease progression. Thus, COX-2 has been widely explored for anti-inflammatory therapy with COX-2 inhibitors, which proved to be effective in reducing the pain and inflammation in patients with arthritis and menstrual cramps, but they have not provided any benefit to patients with chronic inflammatory neurodegenerative disease. Recently, two COX-2 drugs, rofecoxib and valdecoxib, were withdrawn from the United States market due to cardiovascular side effects. Thus, future anti-inflammatory therapy could be targeted through a specific prostanoid receptor downstream of COX-2. The PGE2 receptor EP2 is emerging as a pro-inflammatory target in a variety of CNS and peripheral diseases. Here we highlight the latest developments on the role of EP2 in diseases, mechanism of activation, and small molecule discovery targeted either to enhance or to block the function of this receptor.

  9. State selected ion--molecule reactions by a TESICO technique. V. N/sub 2//sup +/(v)+Ar. -->. N/sub 2/+Ar/sup +/

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kato, T.; Tanaka, K.; Koyano, I.

    1982-07-15

    Charge transfer reactions N/sub 2//sup +/(v)+Ar..-->..Ar/sup +/+N/sub 2/ (1) have been studied by selecting the vibrational states of N/sub 2//sup +/ using the threshold electron--secondary ion coincidence (TESICO) technique. Relative cross sections sigma(v) for the individual vibrational states v = 0--3 have been determined at three collision energies, 0.3, 1.5, and 11.8 eV. Results show that Reaction (1), which is endoergic for v = 0, is considerably enhanced by the vibrational excitation of N/sub 2//sup +/ at all collision energies. While excitation of one vibrational quantum enhances the cross section substantially, excitation of additional quanta further increases the cross sectionmore » up to v = 3. The ratios sigma(2)/sigma(1) and sigma(3)/sigma(2) are, however, much smaller than sigma(1)/sigma(0) and are significantly larger at the highest collision energy than at the other two collision energies. These results are discussed in conjunction with the calculated results based on the simple two-state theory of Rapp and Francis and the Franck--Condon factors.« less

  10. Bisphenol A affects androgen receptor function via multiple mechanisms.

    PubMed

    Teng, Christina; Goodwin, Bonnie; Shockley, Keith; Xia, Menghang; Huang, Ruili; Norris, John; Merrick, B Alex; Jetten, Anton M; Austin, Christopher P; Tice, Raymond R

    2013-05-25

    Bisphenol A (BPA), is a well-known endocrine disruptor compound (EDC) that affects the normal development and function of the female and male reproductive system, however the mechanisms of action remain unclear. To investigate the molecular mechanisms of how BPA may affect ten different nuclear receptors, stable cell lines containing individual nuclear receptor ligand binding domain (LBD)-linked to the β-Gal reporter were examined by a quantitative high throughput screening (qHTS) format in the Tox21 Screening Program of the NIH. The results showed that two receptors, estrogen receptor alpha (ERα) and androgen receptor (AR), are affected by BPA in opposite direction. To confirm the observed effects of BPA on ERα and AR, we performed transient transfection experiments with full-length receptors and their corresponding response elements linked to luciferase reporters. We also included in this study two BPA analogs, bisphenol AF (BPAF) and bisphenol S (BPS). As seen in African green monkey kidney CV1 cells, the present study confirmed that BPA and BPAF act as ERα agonists (half maximal effective concentration EC50 of 10-100 nM) and as AR antagonists (half maximal inhibitory concentration IC50 of 1-2 μM). Both BPA and BPAF antagonized AR function via competitive inhibition of the action of synthetic androgen R1881. BPS with lower estrogenic activity (EC50 of 2.2 μM), did not compete with R1881 for AR binding, when tested at 30 μM. Finally, the effects of BPA were also evaluated in a nuclear translocation assays using EGPF-tagged receptors. Similar to 17β-estradiol (E2) which was used as control, BPA was able to enhance ERα nuclear foci formation but at a 100-fold higher concentration. Although BPA was able to bind AR, the nuclear translocation was reduced. Furthermore, BPA was unable to induce functional foci in the nuclei and is consistent with the transient transfection study that BPA is unable to activate AR. Published by Elsevier Ireland Ltd.

  11. The Role of 5-HT2A, 5-HT2C and mGlu2 Receptors in the Behavioral Effects of Tryptamine Hallucinogens N,N-Dimethyltryptamine and N,N-Diisopropyltryptamine in Rats and Mice

    PubMed Central

    Carbonaro, Theresa M.; Eshleman, Amy J.; Forster, Michael J.; Cheng, Kejun; Rice, Kenner C.; Gatch, Michael B.

    2014-01-01

    Rationale: Serotonin 5-HT2A and 5-HT2C receptors are thought to be the primary pharmacological mechanisms for serotonin-mediated hallucinogenic drugs, but recently there has been interest in metabotropic glutamate (mGluR2) receptors as contributors to the mechanism of hallucinogens. Objective: The present study assesses the role of these 5-HT and glutamate receptors as molecular targets for two tryptamine hallucinogens, N,N-dimethyltryptamine (DMT) and N,N-diisopropyltryptamine (DiPT). Methods: Drug discrimination, head twitch and radioligand binding assays were used. A 5-HT2AR inverse agonist (MDL100907), 5-HT2CR antagonist (SB242084) and mGluR2/3 agonist (LY379268) were tested for their ability to attenuate the discriminative stimulus effects of DMT and DiPT; an mGluR2/3 antagonist (LY341495) was tested for potentiation. MDL100907 was used to attenuate head twitches induced by DMT and DiPT. Radioligand binding studies and inosital-1-phosphate (IP-1) accumulation were performed at the 5-HT2CR for DiPT. Results: MDL100907 fully blocked the discriminative stimulus effects of DMT, but only partially blocked DiPT. SB242084 partially attenuated the discriminative stimulus effects of DiPT, but produced minimal attenuation of DMT’s effects. LY379268 produced potent, but only partial blockade of the discriminative stimulus effects of DMT. LY341495 facilitated DMT- and DiPT-like effects. Both compounds elicited head twitches (DiPT>DMT) which were blocked by MDL1000907. DiPT was a low potency full agonist at 5-HT2CR in vitro. Conclusions: The 5-HT2AR likely plays a major role in mediating the effects of both compounds. 5-HT2C and mGluR2 receptors likely modulate the discriminative stimulus effects of both compounds to some degree. PMID:24985890

  12. The role of 5-HT2A, 5-HT 2C and mGlu2 receptors in the behavioral effects of tryptamine hallucinogens N,N-dimethyltryptamine and N,N-diisopropyltryptamine in rats and mice.

    PubMed

    Carbonaro, Theresa M; Eshleman, Amy J; Forster, Michael J; Cheng, Kejun; Rice, Kenner C; Gatch, Michael B

    2015-01-01

    Serotonin 5-HT2A and 5-HT2C receptors are thought to be the primary pharmacological mechanisms for serotonin-mediated hallucinogenic drugs, but recently there has been interest in metabotropic glutamate (mGluR2) receptors as contributors to the mechanism of hallucinogens. The present study assesses the role of these 5-HT and glutamate receptors as molecular targets for two tryptamine hallucinogens, N,N-dimethyltryptamine (DMT) and N,N-diisopropyltryptamine (DiPT). Drug discrimination, head twitch, and radioligand binding assays were used. A 5-HT2AR inverse agonist (MDL100907), 5-HT2CR antagonist (SB242084), and mGluR2/3 agonist (LY379268) were tested for their ability to attenuate the discriminative stimulus effects of DMT and DiPT; an mGluR2/3 antagonist (LY341495) was tested for potentiation. MDL100907 was used to attenuate head twitches induced by DMT and DiPT. Radioligand binding studies and inosital-1-phosphate (IP-1) accumulation were performed at the 5-HT2CR for DiPT. MDL100907 fully blocked the discriminative stimulus effects of DMT, but only partially blocked DiPT. SB242084 partially attenuated the discriminative stimulus effects of DiPT, but produced minimal attenuation of DMT's effects. LY379268 produced potent, but only partial blockade of the discriminative stimulus effects of DMT. LY341495 facilitated DMT- and DiPT-like effects. Both compounds elicited head twitches (DiPT>DMT) which were blocked by MDL1000907. DiPT was a low-potency full agonist at 5-HT2CR in vitro. The 5-HT2AR likely plays a major role in mediating the effects of both compounds. 5-HT2C and mGluR2 receptors likely modulate the discriminative stimulus effects of both compounds to some degree.

  13. Lung injury pathways: Adenosine receptor 2B signaling limits development of ischemic bronchiolitis obliterans organizing pneumonia.

    PubMed

    Densmore, John C; Schaid, Terry R; Jeziorczak, Paul M; Medhora, Meetha; Audi, Said; Nayak, Shraddha; Auchampach, John; Dwinell, Melinda R; Geurts, Aron M; Jacobs, Elizabeth R

    2017-02-01

    Purpose/Aim of the Study: Adenosine signaling was studied in bronchiolitis obliterans organizing pneumonia (BOOP) resulting from unilateral lung ischemia. Ischemia was achieved by either left main pulmonary artery or complete hilar ligation. Sprague-Dawley (SD) rats, Dahl salt sensitive (SS) rats and SS mutant rat strains containing a mutation in the A 2B adenosine receptor gene (Adora2b) were studied. Adenosine concentrations were measured in bronchoalveolar lavage (BAL) by HPLC. A 2A (A 2A AR) and A 2B adenosine receptor (A 2B AR) mRNA and protein were quantified. Twenty-four hours after unilateral PA ligation, BAL adenosine concentrations from ischemic lungs were increased relative to contralateral lungs in SD rats. A 2B AR mRNA and protein concentrations were increased after PA ligation while miR27a, a negatively regulating microRNA, was decreased in ischemic lungs. A 2A AR mRNA and protein concentrations remained unchanged following ischemia. A 2B AR protein was increased in PA ligated lungs of SS rats after 7 days, and 4 h after complete hilar ligation in SD rats. SS-Adora2b mutants showed a greater extent of BOOP relative to SS rats, and greater inflammatory changes. Increased A 2B AR and adenosine following unilateral lung ischemia as well as more BOOP in A 2B AR mutant rats implicate a protective role for A 2B AR signaling in countering ischemic lung injury.

  14. The deubiquitinases USP33 and USP20 coordinate beta2 adrenergic receptor recycling and resensitization.

    PubMed

    Berthouze, Magali; Venkataramanan, Vidya; Li, Yi; Shenoy, Sudha K

    2009-06-17

    Agonist-induced ubiquitination of the beta(2) adrenergic receptor (beta(2)AR) functions as an important post-translational modification to sort internalized receptors to the lysosomes for degradation. We now show that this ubiquitination is reversed by two deubiquitinating enzymes, ubiquitin-specific proteases (USPs) 20 and 33, thus, inhibiting lysosomal trafficking when concomitantly promoting receptor recycling from the late-endosomal compartments as well as resensitization of recycled receptors at the cell surface. Dissociation of constitutively bound endogenously expressed USPs 20 and 33 from the beta(2)AR immediately after agonist stimulation and reassociation on prolonged agonist treatment allows receptors to first become ubiquitinated and then deubiquitinated, thus, providing a 'trip switch' between degradative and recycling pathways at the late-endosomal compartments. Thus, USPs 20 and 33 serve as novel regulators that dictate both post-endocytic sorting as well as the intensity and extent of beta(2)AR signalling from the cell surface.

  15. Amelioration of sexual behavior and motor activity deficits in a castrated rodent model with a selective androgen receptor modulator SARM-2f

    PubMed Central

    Morimoto, Megumi; Amano, Yuichiro; Oka, Masahiro; Harada, Ayako; Fujita, Hisashi; Hikichi, Yukiko; Tozawa, Ryuichi; Yamaoka, Masuo

    2017-01-01

    Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical

  16. Opposing Effects of Cyclooxygenase-2 (COX-2) on Estrogen Receptor β (ERβ) Response to 5α-Reductase Inhibition in Prostate Epithelial Cells*

    PubMed Central

    Liu, Teresa T.; Grubisha, Melanie J.; Frahm, Krystle A.; Wendell, Stacy G.; Liu, Jiayan; Ricke, William A.; Auchus, Richard J.; DeFranco, Donald B.

    2016-01-01

    Current pharmacotherapies for symptomatic benign prostatic hyperplasia (BPH), an androgen receptor-driven, inflammatory disorder affecting elderly men, include 5α-reductase (5AR) inhibitors (i.e. dutasteride and finasteride) to block the conversion of testosterone to the more potent androgen receptor ligand dihydrotestosterone. Because dihydrotestosterone is the precursor for estrogen receptor β (ERβ) ligands, 5AR inhibitors could potentially limit ERβ activation, which maintains prostate tissue homeostasis. We have uncovered signaling pathways in BPH-derived prostate epithelial cells (BPH-1) that are impacted by 5AR inhibition. The induction of apoptosis and repression of the cell adhesion protein E-cadherin by the 5AR inhibitor dutasteride requires both ERβ and TGFβ. Dutasteride also induces cyclooxygenase type 2 (COX-2), which functions in a negative feedback loop in TGFβ and ERβ signaling pathways as evidenced by the potentiation of apoptosis induced by dutasteride or finasteride upon pharmacological inhibition or shRNA-mediated ablation of COX-2. Concurrently, COX-2 positively impacts ERβ action through its effect on the expression of a number of steroidogenic enzymes in the ERβ ligand metabolic pathway. Therefore, effective combination pharmacotherapies, which have included non-steroidal anti-inflammatory drugs, must take into account biochemical pathways affected by 5AR inhibition and opposing effects of COX-2 on the tissue-protective action of ERβ. PMID:27226548

  17. CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations.

    PubMed

    Khanna, Anchit; Rane, Jayant K; Kivinummi, Kati K; Urbanucci, Alfonso; Helenius, Merja A; Tolonen, Teemu T; Saramäki, Outi R; Latonen, Leena; Manni, Visa; Pimanda, John E; Maitland, Norman J; Westermarck, Jukka; Visakorpi, Tapio

    2015-08-14

    Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

  18. PDE4 and mAKAPβ are nodal organizers of β2-ARs nuclear PKA signaling in cardiac myocytes.

    PubMed

    Bedioune, Ibrahim; Lefebvre, Florence; Lechêne, Patrick; Varin, Audrey; Domergue, Valérie; Kapiloff, Michael S; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2018-05-03

    β1- and β2-adrenergic receptors (β-ARs) produce different acute contractile effects on the heart partly because they impact on different cytosolic pools of cAMP-dependent protein kinase (PKA). They also exert different effects on gene expression but the underlying mechanisms remain unknown. The aim of this study was to understand the mechanisms by which β1- and β2-ARs regulate nuclear PKA activity in cardiomyocytes. We used cytoplasmic and nuclear targeted biosensors to examine cAMP signals and PKA activity in adult rat ventricular myocytes upon selective β1- or β2-ARs stimulation. Both β1- and β2-AR stimulation increased cAMP and activated PKA in the cytoplasm. While the two receptors also increased cAMP in the nucleus, only β1-ARs increased nuclear PKA activity and up-regulated the PKA target gene and pro-apoptotic factor, inducible cAMP element repressor (ICER). Inhibition of PDE4, but not Gi, PDE3, GRK2 nor caveolae disruption disclosed nuclear PKA activation and ICER induction by β2-ARs. Both nuclear and cytoplasmic PKI prevented nuclear PKA activation and ICER induction by β1-ARs, indicating that PKA activation outside the nucleus is required for subsequent nuclear PKA activation and ICER mRNA expression. Cytoplasmic PKI also blocked ICER induction by β2-AR stimulation (with concomitant PDE4 inhibition). However, in this case nuclear PKI decreased ICER up-regulation by only 30%, indicating that other mechanisms are involved. Down-regulation of mAKAPβ partially inhibited nuclear PKA activation upon β1-AR stimulation, and drastically decreased nuclear PKA activation upon β2-AR stimulation in the presence of PDE4 inhibition. β1- and β2-ARs differentially regulate nuclear PKA activity and ICER expression in cardiomyocytes. PDE4 insulates a mAKAPβ-targeted PKA pool at the nuclear envelope that prevents nuclear PKA activation upon β2-AR stimulation.

  19. Expression of Hormone Receptors and HER-2 in Benign and Malignant Salivary Gland Tumors.

    PubMed

    Can, Nhu Thuy; Lingen, Mark W; Mashek, Heather; McElherne, James; Briese, Renee; Fitzpatrick, Carrie; van Zante, Annemieke; Cipriani, Nicole A

    2018-03-01

    With the advent of targeted therapies, expression of sex hormone receptors and HER-2 in salivary gland tumors (SGTs) is of clinical interest. Previous reports of estrogen (ER) and progesterone (PR) receptor expression have varied. Androgen receptor (AR) and HER-2 overexpression are frequently reported in salivary duct carcinoma (SDC), but have not been studied systematically in other SGTs. This study examines ER, PR, AR, and HER-2 expression in SGTs. Immunohistochemistry for ER, PR, AR, and HER-2 was performed on 254 SGTs (134 malignant). ER, PR, and AR expression was scored using Allred system. HER-2 expression was scored using Dako HercepTest guidelines. FISH for HER-2 amplification was performed on select cases with HER-2 overexpression (2-3+). No SGT demonstrated strong expression of ER or PR. Combined strong AR and HER-2 expression was seen in 22 carcinomas: 14/25 SDC, 3/16 poorly differentiated, two oncocytic, and one each carcinoma ex pleomorphic adenoma, squamous cell, and intraductal carcinoma. Eighteen additional high grade carcinomas had HER-2 overexpression with absent, weak, or moderate AR expression; eight high grade carcinomas had isolated strong AR expression with 0-1+ HER-2 staining. Of 15 tested cases, six demonstrated HER-2 amplification by FISH, all of which had 3+ immunoreactivity. Neither benign nor malignant SGTs had strong expression of ER or PR. None of the benign SGTs overexpressed AR or HER-2. Coexpression of AR and HER-2 should not define SDC, but immunostaining should be considered in high grade salivary carcinomas, as some show overexpression and may benefit from targeted therapy.

  20. 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.

  1. 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

  2. The role of DAB2IP in androgen receptor activation during prostate cancer progression.

    PubMed

    Wu, K; Liu, J; Tseng, S-F; Gore, C; Ning, Z; Sharifi, N; Fazli, L; Gleave, M; Kapur, P; Xiao, G; Sun, X; Oz, O K; Min, W; Alexandrakis, G; Yang, C-R; Hsieh, C-L; Wu, H-C; He, D; Xie, D; Hsieh, J-T

    2014-04-10

    Altered androgen-receptor (AR) expression and/or constitutively active AR are commonly associated with prostate cancer (PCa) progression. Targeting AR remains a focal point for designing new strategy of PCa therapy. Here, we have shown that DAB2IP, a novel tumor suppressor in PCa, can inhibit AR-mediated cell growth and gene activation in PCa cells via distinct mechanisms. DAB2IP inhibits the genomic pathway by preventing AR nuclear translocation or phosphorylation and suppresses the non-genomic pathway via its unique functional domain to inactivate c-Src. Also, DAB2IP is capable of suppressing AR activation in an androgen-independent manner. In addition, DAB2IP can inhibit several AR splice variants showing constitutive activity in PCa cells. In DAB2IP(-/-) mice, the prostate gland exhibits hyperplastic epithelia, in which AR becomes more active. Consistently, DAB2IP expression inversely correlates with AR activation status particularly in recurrent or metastatic PCa patients. Taken together, DAB2IP is a unique intrinsic AR modulator in normal cells, and likely can be further developed into a therapeutic agent for PCa.

  3. 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

  4. Cannabinoid receptor activation correlates with the proapoptotic action of the β2-adrenergic agonist (R,R')-4-methoxy-1-naphthylfenoterol in HepG2 hepatocarcinoma cells.

    PubMed

    Paul, Rajib K; Ramamoorthy, Anuradha; Scheers, Jade; Wersto, Robert P; Toll, Lawrence; Jimenez, Lucita; Bernier, Michel; Wainer, Irving W

    2012-10-01

    Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with β(2)-adrenergic receptor (β(2)-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the β(2)-AR agonists (R,R')-fenoterol (Fen) and (R,R')-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [(3)H]thymidine incorporation assays. Despite the expression of β(2)-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a β(2)-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of β(2)-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gα(i)/Gα(o)-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB(1)R and CB(2)R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The β(2)-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting β(2)-AR-CBR ligand.

  5. Can soil denitrification models be validated with the N2/Ar-method? Results from a comparison between DENUZ and the N2/Ar-method in Lower Saxony (Germany)

    NASA Astrophysics Data System (ADS)

    Eschenbach, Wolfram; Elbracht, Jörg; Höper, Heinrich; Kunkel, Ralf; Well, Reinhard; Wendland, Frank

    2014-05-01

    Diffuse NO3- emissions derived from agricultural N surpluses are the main cause of NO3- pollution of aquifers and open water bodies. Denitrification is the key process for the attenuation of these anthropogenic NO3- concentrations in soils and groundwater. Since the greenhouse gas N2O is an obligate intermediate of denitrification this process is also a major regulator of N2O emissions from soils and indirect N2O fluxes from aquifers and open water bodies which result from NO3--leaching. To predict NO3- leaching from the agricultural field and asses the maximal permissible agricultural N surplus to guarantee a mean long-term nitrate concentration in percolation water below 50 mg NO3-/l validated, soil denitrification models are needed. Validation of models predicting denitrification and NO3- leaching is difficult due to lack of suitable data sets and the complexity of denitrification. Moreover, existing groundwater well networks can currently not be used to check the modelled NO3- leaching because NO3- itself might be already partly or totally reduced in the groundwater below soils. In this study we assessed the possibility of validating the soil denitrification model DENUZ (Wendland et al., 2009) with calculated initial NO3- concentrations in the groundwater at the time of groundwater recharge (NO3-t0). NO3-t0 values can be derived from groundwater samples of normal groundwater monitoring wells using the N2/Ar-method (Weymann et al., 2008). Therefore we compare NO3- emission concentrations (pot-NO3-) obtained by groundwater modelled using DENUZ with NO3-t0 values, calculated from measured dissolved gas concentrations (N2, N2O, Ar) and measured NO3- in groundwater samples. We analysed groundwater samples from 484 groundwater monitoring wells throughout Lower-Saxony (Germany). Median NO3- and NO3-t0 concentrations were 0.3 and 30 mg NO3- l-1 respectively, showing that a considerable proportion of the anthropogenic N-surplus is denitrified within the saturated zone

  6. Conserved Binding Mode of Human [beta subscript 2] Adrenergic Receptor Inverse Agonists and Antagonist Revealed by X-ray Crystallography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wacker, Daniel; Fenalti, Gustavo; Brown, Monica A.

    2010-11-15

    G protein-coupled receptors (GPCRs) represent a large fraction of current pharmaceutical targets, and of the GPCRs, the {beta}{sub 2} adrenergic receptor ({beta}{sub 2}AR) is one of the most extensively studied. Previously, the X-ray crystal structure of {beta}{sub 2}AR has been determined in complex with two partial inverse agonists, but the global impact of additional ligands on the structure or local impacts on the binding site are not well-understood. To assess the extent of such ligand-induced conformational differences, we determined the crystal structures of a previously described engineered {beta}{sub 2}AR construct in complex with two inverse agonists: ICI 118,551 (2.8 {angstrom}),more » a recently described compound (2.8 {angstrom}) (Kolb et al, 2009), and the antagonist alprenolol (3.1 {angstrom}). The structures show the same overall fold observed for the previous {beta}{sub 2}AR structures and demonstrate that the ligand binding site can accommodate compounds of different chemical and pharmacological properties with only minor local structural rearrangements. All three compounds contain a hydroxy-amine motif that establishes a conserved hydrogen bond network with the receptor and chemically diverse aromatic moieties that form distinct interactions with {beta}{sub 2}AR. Furthermore, receptor ligand cross-docking experiments revealed that a single {beta}{sub 2}AR complex can be suitable for docking of a range of antagonists and inverse agonists but also indicate that additional ligand-receptor structures may be useful to further improve performance for in-silico docking or lead-optimization in drug design.« less

  7. 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.

  8. TBECH, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane, alters androgen receptor regulation in response to mutations associated with prostate cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kharlyngdoh, Joubert Banjop; Asnake, Solomon; Prad

    Point mutations in the AR ligand-binding domain (LBD) can result in altered AR structures leading to changes of ligand specificity and functions. AR mutations associated to prostate cancer (PCa) have been shown to result in receptor activation by non-androgenic substances and anti-androgenic drugs. Two AR mutations known to alter the function of anti-androgens are the AR{sub T877A} mutation, which is frequently detected mutation in PCa tumors and the AR{sub W741C} that is rare and has been derived in vitro following exposure of cells to the anti-androgen bicalutamide. AR activation by non-androgenic environmental substances has been suggested to affect PCa progression.more » In the present study we investigated the effect of AR mutations (AR{sub W741C} and AR{sub T877A}) on the transcriptional activation following exposure of cells to an androgenic brominated flame retardant, 1,2-dibromo-4-(1,2 dibromoethyl) cyclohexane (TBECH, also named DBE-DBCH). The AR mutations resulted in higher interaction energies and increased transcriptional activation in response to TBECH diastereomer exposures. The AR{sub T877A} mutation rendered AR highly responsive to low levels of DHT and TBECH and led to increased AR nuclear translocation. Gene expression analysis showed a stronger induction of AR target genes in LNCaP cells (AR{sub T877A}) compared to T-47D cells (AR{sub WT}) following TBECH exposure. Furthermore, AR knockdown experiments confirmed the AR dependency of these responses. The higher sensitivity of AR{sub T877A} and AR{sub W741C} to low levels of TBECH suggests that cells with these AR mutations are more susceptible to androgenic endocrine disrupters. - Highlights: • TBECH, is an endocrine disrupting compound that differ in activity depending on AR structure and sequence. • TBECH interaction with the human AR-LBD containing the mutations W741C and T877A is increased compared to the wild type receptor • The mutations, W741C and T877A, are more potent than the

  9. 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.

  10. 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.

  11. Interrelation of androgen receptor and miR-30a and miR-30a function in ER-, PR-, AR+ MDA-MB-453 breast cancer cells.

    PubMed

    Lyu, Shuhua; Liu, Han; Liu, Xia; Liu, Shan; Wang, Yahong; Yu, Qi; Niu, Yun

    2017-10-01

    The association between androgen-induced androgen receptor (AR) activating signal and microRNA (miR)-30a was investigated, as well as the function of miR-30a in estrogen receptor-negative (ER - ), progesterone receptor-negative (PR - ), and AR-positive (AR + ) MDA-MB-453 breast cancer cells. Androgen-induced AR activating signal upregulated the expression of AR, and downregulated the expression of miR-30a, b and c. Bioinformatics analysis indicated a putative miR-30a, b and c binding site in the 3'-untranslated region of AR mRNA. It was confirmed that the AR gene is a direct target of miR-30a, whereas AR does not target the miR-30a promoter, and AR activating signal may indirectly downregulate miR-30a through other cell signaling pathways. In this positive feedback mechanism AR is then upregulated through miR-30a. Overexpression of miR-30a inhibited cell proliferation, whereas inhibition of miR-30a expression by specific antisense oligonucleotides, increased cell growth. Previously, androgen-induced AR activating signal was demonstrated to inhibit cell proliferation in ER - , PR - and AR + MDA-MB-453 breast cancer cells, but AR activating signal downregulated the expression of miR-30a, relieving the inhibition of MDA-MB-453 cell growth. Therefore, in MDA-MB-453 breast cancer cells, miR-30a has two different functions regarding cell growth: Inhibition of cell proliferation through a positive feedback signaling pathway; and the relative promotion of cell proliferation through downregulation of miR-30a. Thus, the association between AR activating signal and microRNAs is complex, and microRNAs may possess different functions due to different signaling pathways. Although the results of the present study were obtained in one cell line, they contribute to subsequent studies on ER - , PR - and AR + breast cancer.

  12. Cannabinoid Receptor Activation Correlates with the Proapoptotic Action of the β2-Adrenergic Agonist (R,R′)-4-Methoxy-1-Naphthylfenoterol in HepG2 Hepatocarcinoma CellsS⃞

    PubMed Central

    Paul, Rajib K.; Ramamoorthy, Anuradha; Scheers, Jade; Wersto, Robert P.; Toll, Lawrence; Jimenez, Lucita; Wainer, Irving W.

    2012-01-01

    Inhibition of cell proliferation by fenoterol and fenoterol derivatives in 1321N1 astrocytoma cells is consistent with β2-adrenergic receptor2-AR) stimulation. However, the events that result in fenoterol-mediated control of cell proliferation in other cell types are not clear. Here, we compare the effect of the β2-AR agonists (R,R′)-fenoterol (Fen) and (R,R′)-4-methoxy-1-naphthylfenoterol (MNF) on signaling and cell proliferation in HepG2 hepatocarcinoma cells by using Western blotting and [3H]thymidine incorporation assays. Despite the expression of β2-AR, no cAMP accumulation was observed when cells were stimulated with isoproterenol or Fen, although the treatment elicited both mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt activation. Unexpectedly, isoproterenol and Fen promoted HepG2 cell growth, but MNF reduced proliferation together with increased apoptosis. The mitogenic responses of Fen were attenuated by 3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol (ICI 118,551), a β2-AR antagonist, whereas those of MNF were unaffected. Because of the coexpression of β2-AR and cannabinoid receptors (CBRs) and their impact on HepG2 cell proliferation, these Gαi/Gαo-linked receptors may be implicated in MNF signaling. Cell treatment with (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone (WIN 55,212-2), a synthetic agonist of CB1R and CB2R, led to growth inhibition, whereas inverse agonists of these receptors blocked MNF mitogenic responses without affecting Fen signaling. MNF responses were sensitive to pertussis toxin. The β2-AR-deficient U87MG cells were refractory to Fen, but responsive to the antiproliferative actions of MNF and WIN 55,212-2. The data indicate that the presence of the naphthyl moiety in MNF results in functional coupling to the CBR pathway, providing one of the first examples of a dually acting β2-AR-CBR ligand. PMID:22776956

  13. Androgen Receptor: A Complex Therapeutic Target for Breast Cancer

    PubMed Central

    Narayanan, Ramesh; Dalton, James T.

    2016-01-01

    Molecular and histopathological profiling have classified breast cancer into multiple sub-types empowering precision treatment. Although estrogen receptor (ER) and human epidermal growth factor receptor (HER2) are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR) is evolving as a molecular target for cancers that have developed resistance to conventional treatments. The high expression of AR in breast cancer and recent discovery and development of new nonsteroidal drugs targeting the AR provide a strong rationale for exploring it again as a therapeutic target in this disease. Ironically, both nonsteroidal agonists and antagonists for the AR are undergoing clinical trials, making AR a complicated target to understand in breast cancer. This review provides a detailed account of AR’s therapeutic role in breast cancer. PMID:27918430

  14. O2(a1Δ) quenching in O/O2/O3/CO2/He/Ar mixtures

    NASA Astrophysics Data System (ADS)

    Azyazov, V. N.; Mikheyev, P. A.; Postell, D.; Heaven, M. C.

    2010-02-01

    The development of discharge singlet oxygen generators (DSOG's) that can operate at high pressures is required for the power scaling of the discharge oxygen iodine laser. In order to achieve efficient high-pressure DSOG operation it is important to understand the mechanisms by which singlet oxygen (O2(a1Δ)) is quenched in these devices. It has been proposed that three-body deactivation processes of the type O2(a1Δ))+O+M-->2O2+M provide significant energy loss channels. To further explore these reactions the physical and reactive quenching of O2(a1Δ)) in O(3P)/O2/O3/CO2/He/Ar mixtures has been investigated. Oxygen atoms and singlet oxygen molecules were produced by the 248 nm laser photolysis of ozone. The kinetics of O2(a1Δ)) quenching were followed by observing the 1268 nm fluorescence of the O2 a1Δ-X3Ε transition. Fast quenching of O2(a1Δ)) in the presence of oxygen atoms and molecules was observed. The mechanism of the process has been examined using kinetic models, which indicate that quenching by vibrationally excited ozone is the dominant reaction.

  15. Increased Energy Demand during Adrenergic Receptor Stimulation Contributes to Ca(2+) Wave Generation.

    PubMed

    Bovo, Elisa; Mazurek, Stefan R; de Tombe, Pieter P; Zima, Aleksey V

    2015-10-20

    While β-adrenergic receptor (β-AR) stimulation ensures adequate cardiac output during stress, it can also trigger life-threatening cardiac arrhythmias. We have previously shown that proarrhythmic Ca(2+) waves during β-AR stimulation temporally coincide with augmentation of reactive oxygen species (ROS) production. In this study, we tested the hypothesis that increased energy demand during β-AR stimulation plays an important role in mitochondrial ROS production and Ca(2+)-wave generation in rabbit ventricular myocytes. We found that β-AR stimulation with isoproterenol (0.1 μM) decreased the mitochondrial redox potential and the ratio of reduced to oxidated glutathione. As a result, β-AR stimulation increased mitochondrial ROS production. These metabolic changes induced by isoproterenol were associated with increased sarcoplasmic reticulum (SR) Ca(2+) leak and frequent diastolic Ca(2+) waves. Inhibition of cell contraction with the myosin ATPase inhibitor blebbistatin attenuated oxidative stress as well as spontaneous SR Ca(2+) release events during β-AR stimulation. Furthermore, we found that oxidative stress induced by β-AR stimulation caused the formation of disulfide bonds between two ryanodine receptor (RyR) subunits, referred to as intersubunit cross-linking. Preventing RyR cross-linking with N-ethylmaleimide decreased the propensity of Ca(2+) waves induced by β-AR stimulation. These data suggest that increased energy demand during sustained β-AR stimulation weakens mitochondrial antioxidant defense, causing ROS release into the cytosol. By inducing RyR intersubunit cross-linking, ROS can increase SR Ca(2+) leak to the critical level that can trigger proarrhythmic Ca(2+) waves. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Reprogramming of G protein-coupled receptor recycling and signaling by a kinase switch

    PubMed Central

    Vistein, Rachel; Puthenveedu, Manojkumar A.

    2013-01-01

    The postendocytic recycling of signaling receptors is subject to multiple requirements. Why this is so, considering that many other proteins can recycle without apparent requirements, is a fundamental question. Here we show that cells can leverage these requirements to switch the recycling of the beta-2 adrenergic receptor (B2AR), a prototypic signaling receptor, between sequence-dependent and bulk recycling pathways, based on extracellular signals. This switch is determined by protein kinase A-mediated phosphorylation of B2AR on the cytoplasmic tail. The phosphorylation state of B2AR dictates its partitioning into spatially and functionally distinct endosomal microdomains mediating bulk and sequence-dependent recycling, and also regulates the rate of B2AR recycling and resensitization. Our results demonstrate that G protein-coupled receptor recycling is not always restricted to the sequence-dependent pathway, but may be reprogrammed as needed by physiological signals. Such flexible reprogramming might provide a versatile method for rapidly modulating cellular responses to extracellular signaling. PMID:24003153

  17. Role of androgen receptor and associated lysine-demethylase coregulators, LSD1 and JMJD2A, in localized and advanced human bladder cancer.

    PubMed

    Kauffman, Eric C; Robinson, Brian D; Downes, Martin J; Powell, Leagh G; Lee, Ming Ming; Scherr, Douglas S; Gudas, Lorraine J; Mongan, Nigel P

    2011-12-01

    Bladder cancer is approximately three times more common in men as compared to women. We and others have previously investigated the contribution of androgens and the androgen receptor (AR) to bladder cancer. JMJD2A and LSD1 are recently discovered AR coregulator proteins that mediate AR-dependent transcription via recently described histone lysine-demethylation (KDM) mechanisms. We used immunohistochemistry to examine JMJD2A, LSD1, and AR expression in 72 radical cystectomy specimens, resulting in evaluation of 129 tissue samples (59 urothelial carcinoma, 70 benign). We tested levels of these proteins for statistical association with clinicopathologic variables and patient survival. Expression of these markers was also assessed in human bladder cancer cell lines. The effects of pharmacological inhibition of LSD1 on the proliferation of these bladder cancer cells was determined. JMJD2A and AR levels were significantly lower in malignant versus benign urothelium, while increased LSD1 levels were observed in malignant urothelium relative to benign. A significant reduction in all three proteins occurred with cancer stage progression, including muscle invasion (JMJD2A/LSD1/AR), extravesical extension (JMJD2A/LSD1), and lymph node metastasis (JMJD2A/AR). Lower JMJD2A intensity correlated with additional poor prognostic features, including lymphovascular invasion, concomitant carcinoma in situ and tobacco usage, and predicted significantly worse overall survival. Pharmacological inhibition of LSD1 suppressed bladder cancer cell proliferation and androgen-induced transcription. Our results support a novel role for the AR-KDM complex in bladder cancer initiation and progression, identify JMJD2A as a promising prognostic biomarker, and demonstrate targeting of the KDM activity as an effective potential approach for bladder cancer growth inhibition. Copyright © 2011 Wiley Periodicals, Inc.

  18. Overexpression of androgen receptor and forkhead-box A1 protein in apocrine breast carcinoma.

    PubMed

    Sasahara, Manami; Matsui, Akira; Ichimura, Yoshiko; Hirakata, Yuuko; Murata, Yuuya; Marui, Eiji

    2014-03-01

    Apocrine breast carcinoma often lacks estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor type-2 (HER2) expression. Accordingly, development of a new treatment strategy is important for this type of cancer. The growth stimulus through the androgen receptor (AR) can be a candidate for targeted treatment. Therefore, we examined the factors related to AR transcription. We immunohistochemically evaluated 54 apocrine cancer lesions for ER, PgR, AR, HER2, Ki-67, forkhead-box protein A1 (FOXA1), and prostate-specific antigen (PSA) expression. ER, PgR, and HER2 were expressed at a low level, thus 44 out of 54 (81.4%) cases were of triple-negative breast cancer. AR, PSA and FOXA1 were expressed in 100% (54/54), 48% (26/54) and 93% (50/54) of cases, respectively. Most of apocrine breast carcinomas were immunohistochemically-positive for AR and FOXA1. Anti-androgenic therapies can potentially serve as a cancer-targeting therapy for apocrine breast carcinoma.

  19. Androgen Receptor-Mediated Growth Suppression of HPr-1AR and PC3-Lenti-AR Prostate Epithelial Cells

    PubMed Central

    Bolton, Eric C.

    2015-01-01

    The androgen receptor (AR) mediates the developmental, physiologic, and pathologic effects of androgens including 5α-dihydrotestosterone (DHT). However, the mechanisms whereby AR regulates growth suppression and differentiation of luminal epithelial cells in the prostate gland and proliferation of malignant versions of these cells are not well understood, though they are central to prostate development, homeostasis, and neoplasia. Here, we identify androgen-responsive genes that restrain cell cycle progression and proliferation of human prostate epithelial cell lines (HPr-1AR and PC3-Lenti-AR), and we investigate the mechanisms through which AR regulates their expression. DHT inhibited proliferation of HPr-1AR and PC3-Lenti-AR, and cell cycle analysis revealed a prolonged G1 interval. In the cell cycle, the G1/S-phase transition is initiated by the activity of cyclin D and cyclin-dependent kinase (CDK) complexes, which relieve growth suppression. In HPr-1AR, cyclin D1/2 and CDK4/6 mRNAs were androgen-repressed, whereas CDK inhibitor, CDKN1A, mRNA was androgen-induced. The regulation of these transcripts was AR-dependent, and involved multiple mechanisms. Similar AR-mediated down-regulation of CDK4/6 mRNAs and up-regulation of CDKN1A mRNA occurred in PC3-Lenti-AR. Further, CDK4/6 overexpression suppressed DHT-inhibited cell cycle progression and proliferation of HPr-1AR and PC3-Lenti-AR, whereas CDKN1A overexpression induced cell cycle arrest. We therefore propose that AR-mediated growth suppression of HPr-1AR involves cyclin D1 mRNA decay, transcriptional repression of cyclin D2 and CDK4/6, and transcriptional activation of CDKN1A, which serve to decrease CDK4/6 activity. AR-mediated inhibition of PC3-Lenti-AR proliferation occurs through a similar mechanism, albeit without down-regulation of cyclin D. Our findings provide insight into AR-mediated regulation of prostate epithelial cell proliferation. PMID:26372468

  20. Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype

    PubMed Central

    Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

    2016-01-01

    Study Objectives: We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Methods: Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Results: Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (−114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (−531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (−608/−618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (−194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. Conclusions: IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. Commentary: A

  1. [125I]2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), a high-affinity radioligand selective for I1 imidazoline receptors.

    PubMed

    Greney, Hugues; Urosevic, Dragan; Schann, Stephan; Dupuy, Laurence; Bruban, Véronique; Ehrhardt, Jean-Daniel; Bousquet, Pascal; Dontenwill, Monique

    2002-07-01

    The I1 subtype of imidazoline receptors (I1R) is a plasma membrane protein that is involved in diverse physiological functions. Available radioligands used so far to characterize the I(1)R were able to bind with similar affinities to alpha2-adrenergic receptors (alpha2-ARs) and to I1R. This feature was a major drawback for an adequate characterization of this receptor subtype. New imidazoline analogs were therefore synthesized and the present study describes one of these compounds, 2-(2-chloro-4-iodo-phenylamino)-5-methyl-pyrroline (LNP 911), which was of high affinity and selectivity for the I1R. LNP 911 was radioiodinated and its binding properties characterized in different membrane preparations. Saturation experiments with [125I]LNP 911 revealed a single high affinity binding site in PC-12 cell membranes (K(D) = 1.4 nM; B(max) = 398 fmol/mg protein) with low nonspecific binding. [125I]LNP 911 specific binding was inhibited by various imidazolines and analogs but was insensitive to guanosine-5'-O-(3-thio)triphosphate. The rank order of potency of some competing ligands [LNP 911, PIC, rilmenidine, 4-chloro-2-(imidazolin-2-ylamino)-isoindoline (BDF 6143), lofexidine, and clonidine] was consistent with the definition of [125I]LNP 911 binding sites as I1R. However, other high-affinity I1R ligands (moxonidine, efaroxan, and benazoline) exhibited low affinities for these binding sites in standard binding assays. In contrast, when [125I]LNP 911 was preincubated at 4 degrees C, competition curves of moxonidine became biphasic. In this case, moxonidine exhibited similar high affinities on [125I]LNP 911 binding sites as on I1R defined with [125I]PIC. Moxonidine proved also able to accelerate the dissociation of [125I]LNP 911 from its binding sites. These results suggest the existence of an allosteric modulation at the level of the I1R, which seems to be corroborated by the dose-dependent enhancement by LNP 911 of the agonist effects on the adenylate cyclase pathway

  2. Whole Genome DNA Methylation Analysis of Obstructive Sleep Apnea: IL1R2, NPR2, AR, SP140 Methylation and Clinical Phenotype.

    PubMed

    Chen, Yung-Che; Chen, Ting-Wen; Su, Mao-Chang; Chen, Chung-Jen; Chen, Kuang-Den; Liou, Chia-Wei; Tang, Petrus; Wang, Ting-Ya; Chang, Jen-Chieh; Wang, Chin-Chou; Lin, Hsin-Ching; Chin, Chien-Hung; Huang, Kuo-Tung; Lin, Meng-Chih; Hsiao, Chang-Chun

    2016-04-01

    We hypothesized that DNA methylation patterns may contribute to disease severity or the development of hypertension and excessive daytime sleepiness (EDS) in patients with obstructive sleep apnea (OSA). Illumina's (San Diego, CA, USA) DNA methylation 27-K assay was used to identify differentially methylated loci (DML). DNA methylation levels were validated by pyrosequencing. A discovery cohort of 15 patients with OSA and 6 healthy subjects, and a validation cohort of 72 patients with sleep disordered breathing (SDB). Microarray analysis identified 636 DMLs in patients with OSA versus healthy subjects, and 327 DMLs in patients with OSA and hypertension versus those without hypertension. In the validation cohort, no significant difference in DNA methylation levels of six selected genes was found between the primary snoring subjects and OSA patients (primary outcome). However, a secondary outcome analysis showed that interleukin-1 receptor 2 (IL1R2) promoter methylation (-114 cytosine followed by guanine dinucleotide sequence [CpG] site) was decreased and IL1R2 protein levels were increased in the patients with SDB with an oxygen desaturation index > 30. Androgen receptor (AR) promoter methylation (-531 CpG site) and AR protein levels were both increased in the patients with SDB with an oxygen desaturation index > 30. Natriuretic peptide receptor 2 (NPR2) promoter methylation (-608/-618 CpG sites) were decreased, whereas levels of both NPR2 and serum C type natriuretic peptide protein were increased in the SDB patients with EDS. Speckled protein 140 (SP140) promoter methylation (-194 CpG site) was increased, and SP140 protein levels were decreased in the patients with SDB and EDS. IL1R2 hypomethylation and AR hypermethylation may constitute an important determinant of disease severity, whereas NPR2 hypomethylation and SP140 hypermethylation may provide a biomarker for vulnerability to EDS in OSA. A commentary on this article appears in this issue on page 723. © 2016

  3. Understanding the conformation transition in the activation pathway of β2 adrenergic receptor via a targeted molecular dynamics simulation.

    PubMed

    Xiao, Xiuchan; Zeng, Xiaojun; Yuan, Yuan; Gao, Nan; Guo, Yanzhi; Pu, Xuemei; Li, Menglong

    2015-01-28

    G protein coupled receptors (GPCRs) play a crucial role in regulating signal recognition and transduction through their activation. The conformation transition in the activation pathway is of particular importance for their function. However, it has been poorly elucidated due to experimental difficulties in determining the conformations and the time limitation of conventional molecular dynamics (CMD) simulation. Thus, in this work, we employed a targeted molecular dynamic (TMD) simulation to study the activation process from an inactive structure to a fully active one for β2 adrenergic receptor2AR). As a reference, 110 ns CMD simulations on wild β2AR and its D130N mutant were also carried out. TMD results show that there is at least an intermediate conformation cluster in the activation process, evidenced by the principal component analysis and the structural and dynamic differences of some important motifs. It is noteworthy that the activation of the ligand binding site lags the G-protein binding site, displaying uncoupled correlation. Comparisons between the CMD and TMD results show that the D130N mutation significantly speeds up ICL2 and key ionic lock to enter into the intermediate state, which to some extent facilitates the activation involved in the NPxxY, DRY region and the separation between TM3 and TM6. However, the contribution from the D130N mutation to the activation of the ligand binding site could not be observed within the scale of 110 ns time. These observations could provide novel insights into previous studies for better understanding of the activation mechanism for β2AR.

  4. Function of beta 2-adrenergic receptors in chronic localized myalgia.

    PubMed

    Maekawa, Kenji; Kuboki, Takuo; Inoue, Eitoku; Inoue-Minakuchi, Mami; Suzuki, Koji; Yatani, Hirofumi; Clark, Glenn T

    2003-01-01

    To investigate alteration of beta 2-adrenergic receptor (beta 2 AR) function in chronic localized myalgia subjects by evaluating levels of the beta 2 AR second messenger, cyclic adenosine monophosphate (cAMP), in mononuclear cells after beta AR-agonist stimulation. Eleven chronic localized myalgia subjects and 21 matched healthy controls participated in this study. Peripheral blood (30 cc) was drawn from the subjects' anterocubital vein. Mononuclear cells were isolated from the total blood by using the Ficoll-Hypaque gradient technique. Basal and stimulated intracellular cAMP levels were determined by enzyme immunoassay using a commercially available kit. Aliquots of 5 x 10(6) cells were incubated with or without stimulation of the beta AR-agonist isoproterenol for 5 minutes. Five different concentrations of isoproterenol (10(-3) M to 10(-7) M) were utilized. cAMP levels in both groups were tested statistically by a 2-way repeated-measures ANOVA with 2 predictors, group difference and isoproterenol concentration difference. As with isoproterenol stimulation, the cAMP responses to forskolin, which activates adenylyl cyclase directly and produces cAMP, bypassing the cell surface receptors were also measured. The basal cAMP levels in both groups (myalgia: 0.33 +/- 0.02 pmol/5 x 10(6) cells; control: 0.43 +/- 0.10 pmol/5 x 10(6) cells) were almost identical, and isoproterenol-produced cAMP levels increased dose-dependently in both groups. No significant differences in the mean cAMP levels were observed between the groups (P = .909). Significant increases were observed according to the isoproterenol concentration increase (P < .0001). The cAMP responses to forskolin stimulation also showed no significant group difference (P = .971). These results suggest that beta 2 AR function is not different between localized myalgia subjects and healthy individuals.

  5. Complement C3a binding to its receptor as a negative modulator of Th2 response in liver injury in trichloroethylene-sensitized mice.

    PubMed

    Wang, Feng; Zha, Wan-sheng; Zhang, Jia-xiang; Li, Shu-long; Wang, Hui; Ye, Liang-ping; Shen, Tong; Wu, Chang-hao; Zhu, Qi-xing

    2014-08-17

    Trichloroethylene (TCE) is a major occupational health hazard and causes occupational medicamentosa-like dermatitis (OMLDT) and liver damage. Recent evidence suggests immune response as a distinct mode of action for TCE-induced liver damage. This study aimed to explore the role of the key complement activation product C3a and its receptor C3aR in TCE-induced immune liver injury. A mouse model of skin sensitization was induced by TCE in the presence and absence of the C3aR antagonist SB 290157. Liver function was evaluated by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in conjunction with histopathological characterizations. C3a and C3aR were detected by immunohistochemistry and C5b-9 was assessed by immunofluorescence. IFN-γ and IL4 expressions were determined by flow cytometry and ELISA. The total sensitization rate was 44.1%. TCE sensitization caused liver cell necrosis and inflammatory infiltration, elevated serum ALT and AST, expression of C3a and C3aR, and deposition of C5b-9 in the liver. IFN-γ and IL-4 expressions were up-regulated in spleen mononuclear cells and their serum levels were also increased. Pretreatment with SB 290157 resulted in more inflammatory infiltration in the liver, higher levels of AST, reduced C3aR expression on Kupffer cells, and decreased IL-4 levels while IFN-γ remained unchanged. These data demonstrate that blocking of C3a binding to C3aR reduces IL4, shifts IFN-γ and IL-4 balance, and aggravates TCE-sensitization induced liver damage. These findings reveal a novel mechanism whereby modulation of Th2 response by C3a binding to C3a receptor contributes to immune-mediated liver damage by TCE exposure. Copyright © 2014. Published by Elsevier Ireland Ltd.

  6. Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

    PubMed

    Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

    2012-05-11

    Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)ARA(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

  7. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONIST AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  8. A NOVEL CELL LINE THAT STABLY EXPRESSES AN ANDROGEN RESPONSIVE LUCIFERASE REPORTER FOR THE DETECTION OF ANDROGEN RECEPTOR (AR) AGONISTS AND ANTAGONISTS

    EPA Science Inventory

    The use of in vitro assays to screen chemicals for estrogen receptor (ER) and AR mediated actions is being evaluated by the USEPA for use in a Tier I screening battery to detect endocrine active chemicals. We have developed a stable cell line, MDA-MB-453-KB2, for screening of and...

  9. MEN15596, a novel nonpeptide tachykinin NK2 receptor antagonist.

    PubMed

    Cialdai, Cecilia; Tramontana, Manuela; Patacchini, Riccardo; Lecci, Alessandro; Catalani, Claudio; Catalioto, Rose-Marie; Meini, Stefania; Valenti, Claudio; Altamura, Maria; Giuliani, Sandro; Maggi, Carlo Alberto

    2006-11-07

    The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2a dose-related and persistent manner colon contractions induced by the selective tachykinin NK2 receptor agonist, [betaAla8]neurokinin A(4-10) (3 nmol/kg i.v.), either after intravenous (ED50 0.18 micromol/kg), intraduodenal (ED50 3.16 micromol/kg) or oral administration (10-30 micromol/kg) without affecting, at 3 micromol/kg, i.v., the colonic contractions produced by the NK1 receptor selective agonist [Sar9]substance P sulfone (3 nmol/kg i.v.). In addition MEN15596 was effective in inhibiting bronchoconstriction produced by i.v. administration of [betaAla8]neurokinin A(4-10). Overall the results indicate that MEN15596 is a potent and selective

  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. Allosterism in human complement component 5a ((h)C5a): a damper of C5a receptor (C5aR) signaling.

    PubMed

    Rana, Soumendra; Sahoo, Amita Rani; Majhi, Bharat Kumar

    2016-06-01

    The phenomena of allosterism continues to advance the field of drug discovery, by illuminating gainful insights for many key processes, related to the structure-function relationships in proteins and enzymes, including the transmembrane G-protein coupled receptors (GPCRs), both in normal as well as in the disease states. However, allosterism is completely unexplored in the native protein ligands, especially when a small covalent change significantly modulates the pharmacology of the protein ligands toward the signaling axes of the GPCRs. One such example is the human C5a ((h)C5a), the potent cationic anaphylatoxin that engages C5aR and C5L2 to elicit numerous immunological and non-immunological responses in humans. From the recently available structure-function data, it is clear that unlike the mouse C5a ((m)C5a), the (h)C5a displays conformational heterogeneity. However, the molecular basis of such conformational heterogeneity, otherwise allosterism in (h)C5a and its precise contribution toward the overall C5aR signaling is not known. This study attempts to decipher the functional role of allosterism in (h)C5a, by exploring the inherent conformational dynamics in (m)C5a, (h)C5a and in its point mutants, including the proteolytic mutant des-Arg(74)-(h)C5a. Prima facie, the comparative molecular dynamics study, over total 500 ns, identifies Arg(74)-Tyr(23) and Arg(37)-Phe(51) "cation-π" pairs as the molecular "allosteric switches" on (h)C5a that potentially functions as a damper of C5aR signaling.

  12. Human Freud-2/CC2D1B: a novel repressor of postsynaptic serotonin-1A receptor expression.

    PubMed

    Hadjighassem, Mahmoud R; Austin, Mark C; Szewczyk, Bernadeta; Daigle, Mireille; Stockmeier, Craig A; Albert, Paul R

    2009-08-01

    Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor. Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot. Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5' or 3' human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N-SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N-SH cells. Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.

  13. Alpha2-Adrenergic Receptors and Breast Tumor Stroma: A Novel Pathway Driving Breast Cancer Growth and Metastasis

    DTIC Science & Technology

    2014-10-01

    TAF ) were isolated to determine if α2AR activation directly modulates collagen microstructure. 15. SUBJECT TERMS Alpha2-adrenergic receptors, breast...group. 15 3) Significant results/key outcomes: Here we demonstrate the isolation of tumor associated fibroblasts ( TAF ) from 4T1 tumors. The goal...of these experiments was to determine the optimal conditions and appropriate markers for isolation of TAF and to optimize yield and purity of the

  14. Short-term effects of β2-AR blocker ICI 118,551 on sarcoplasmic reticulum SERCA2a and cardiac function of rats with heart failure.

    PubMed

    Gong, Haibin; Li, Yanfei; Wang, Lei; Lv, Qian; Wang, Xiuli

    2016-09-01

    The study was conducted to examine the effects of ICI 118,551 on the systolic function of cardiac muscle cells of rats in heart failure and determine the molecular mechanism of selective β2-adrenergic receptor2-AR) antagonist on these cells. The chronic heart failure model for rats was prepared through abdominal aortic constriction and separate cardiac muscle cells using the collagenase digestion method. The rats were then divided into Sham, HF and HF+ICI 50 nM goups and cultivated for 48 h. β2-AR, Gi/Gs and sarcoplasmic reticulum Ca 2+ -ATPase (SERCA2a) protein expression levels in the cardiac muscle cells were evaluated by western blotting and changes in the systolic function of cardiac muscle cells based on the boundary detection system of contraction dynamics for individual cells was measured. The results showed that compared with the Sham group, the survival rate, percentage of basic contraction and maximum contraction amplitude percentage of cardiac muscle cells with heart failure decreased, Gi protein expression increased while Gs and SERCA2a protein expression decreased. Compared with the HF group, the maximum contraction amplitude percentage of cardiac muscle cells in group HF+ICI 50 nM decreased, the Gi protein expression level increased while the SERCA2a protein expression level decreased. Following the stimulation of Ca 2+ and ISO, the maximum contraction amplitude percentage of cardiac muscle cells in the HF+ICI 50 nM group was lower than that in group HF. This indicated that ICI 118,551 has negative inotropic effects on cardiac muscle cells with heart failure, which may be related to Gi protein. Systolic function of cardiac muscle cells with heart failure can therefore be reduced by increasing Gi protein expression and lowering SERCA2a protein expression.

  15. Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development.

    PubMed

    Diaz-Tellez, Abigail; Zampedri, Cecilia; Ramos-Balderas, Jose L; García-Hernández, Fernando; Maldonado, Ernesto

    2016-04-01

    Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord. This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development. © 2016 Wiley Periodicals, Inc.

  16. Prognostic significance of β2-adrenergic receptor expression in malignant melanoma.

    PubMed

    Shimizu, Akira; Kaira, Kyoichi; Mori, Keita; Kato, Madoka; Shimizu, Kimihiro; Yasuda, Masahito; Takahashi, Ayumi; Oyama, Tetsunari; Asao, Takayuki; Ishikawa, Osamu

    2016-05-01

    Recent studies cite β2-adrenergic receptor2AR) antagonists as novel therapeutic agents for melanoma, as they may reduce the disease progression. The β2AR has shown to be expressed in malignant melanoma. However, it remains unclear whether the β2AR expression has a clinical and pathological significance in patients with cutaneous malignant melanoma. We herein conducted a clinicopathological study to investigate the protein expression of β2AR in malignant melanoma of the skin and its prognostic significance. One hundred thirty-three patients with surgically resected cutaneous malignant melanoma were evaluated. Tumor sections were stained by immunohistochemistry for β2AR, Ki-67, the microvessel density (MVD) determined by CD34, and p53. β2AR was highly expressed in 44.4 % (59 out of 133) of the patients. The expression of β2AR was significantly associated with the tumor thickness, ulceration, T factor, N factor, disease stage, tumor size, cell proliferation (Ki-67), and MVD (CD34). Using Spearman's rank test, the β2AR expression was correlated with Ki-67 (r = 0.278; 95 % CI, 0.108 to 0.432; P = 0.001), CD34 (r = 0.445; 95 %CI, 0.293 to 0.575; P < 0.001), and the tumor size (r = 0.226; 95 % CI, 0.053 to 0.386; P = 0.008). Using a univariate analysis, the tumor thickness, ulceration, disease stage, β2AR, Ki-67, and CD34 had a significant relationship with the overall and progression-free survivals. A multivariable analysis confirmed that β2AR was an independent prognostic factor for predicting a poor overall survival (HR 1.730; 95 % CI 1.221-2.515) and progression-free survival (HR 1.576; 95 % CI 1.176-2.143) of malignant melanoma of the skin. β2AR can serve as a promising prognostic factor for predicting a worse outcome after surgical treatment and may play an important role in the development and aggressiveness of malignant melanoma.

  17. The Complement C3a-C3aR Axis Promotes Development of Thoracic Aortic Dissection via Regulation of MMP2 Expression.

    PubMed

    Ren, Weihong; Liu, Yan; Wang, Xuerui; Piao, Chunmei; Ma, Youcai; Qiu, Shulan; Jia, Lixin; Chen, Boya; Wang, Yuan; Jiang, Wenjian; Zheng, Shuai; Liu, Chang; Dai, Nan; Lan, Feng; Zhang, Hongjia; Song, Wen-Chao; Du, Jie

    2018-03-01

    Thoracic aortic dissection (TAD), once ruptured, is devastating to patients, and no effective pharmaceutical therapy is available. Anaphylatoxins released by complement activation are involved in a variety of diseases. However, the role of the complement system in TAD is unknown. We found that plasma levels of C3a, C4a, and C5a were significantly increased in patients with TAD. Elevated circulating C3a levels were also detected in the developmental process of mouse TAD, which was induced by β-aminopropionitrile monofumarate (BAPN) treatment, with enhanced expression of C1q and properdin in mouse dissected aortas. These findings indicated activation of classical and alternative complement pathways. Further, expression of C3aR was obviously increased in smooth muscle cells of human and mouse dissected aortas, and knockout of C3aR notably inhibited BAPN-induced formation and rupture of TAD in mice. C3aR antagonist administered pre- and post-BAPN treatment attenuated the development of TAD. We found that C3aR knockout decreased matrix metalloproteinase 2 (MMP2) expression in BAPN-treated mice. Additionally, recombinant C3a stimulation enhanced MMP2 expression and activation in smooth muscle cells that were subjected to mechanical stretch. Finally, we generated MMP2-knockdown mice by in vivo MMP2 short hairpin RNA delivery using recombinant adeno-associated virus and found that MMP2 deficiency significantly reduced the formation of TAD. Therefore, our study suggests that the C3a - C3aR axis contributes to the development of TAD via regulation of MMP2 expression. Targeting the C3a-C3aR axis may represent a strategy for inhibiting the formation of TAD. Copyright © 2018 by The American Association of Immunologists, Inc.

  18. Agonist-induced conformational changes in the G-protein-coupling domain of the β2 adrenergic receptor

    PubMed Central

    Ghanouni, Pejman; Steenhuis, Jacqueline J.; Farrens, David L.; Kobilka, Brian K.

    2001-01-01

    The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the β2 adrenergic receptor2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-β2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the β2AR, which may reflect the different energetics of activation by diffusible ligands. PMID:11353823

  19. Neuropharmacology of Purinergic Receptors in Human Submucous Plexus: Involvement of P2X1, P2X2, P2X3 Channels, P2Y and A3 Metabotropic Receptors in Neurotransmission

    PubMed Central

    Liñán-Rico, A.; Wunderlich, JE.; Enneking, JT.; Tso, DR.; Grants, I.; Williams, KC.; Otey, A.; Michel, K.; Schemann, M.; Needleman, B.; Harzman, A.; Christofi, FL.

    2015-01-01

    Rationale The role of purinergic signaling in the human ENS is not well understood. We sought to further characterize the neuropharmacology of purinergic receptors in human ENS and test the hypothesis that endogenous purines are critical regulators of neurotransmission. Experimental Approach LSCM-Fluo-4-(Ca2+)-imaging of postsynaptic Ca2+ transients (PSCaTs) was used as a reporter of neural activity. Synaptic transmission was evoked by fiber tract electrical stimulation in human SMP surgical preparations. Pharmacological analysis of purinergic signaling was done in 1,556 neurons from 234 separate ganglia 107 patients; immunochemical labeling for P2XRs of neurons in ganglia from 19 patients. Real-time MSORT (Di-8-ANEPPS) imaging was used to test effects of adenosine on fast excitatory synaptic potentials (fEPSPs). Results Synaptic transmission is sensitive to pharmacological manipulations that alter accumulation of extracellular purines. Apyrase blocks PSCaTs in a majority of neurons. An ecto-NTPDase-inhibitor 6-N,N-diethyl-D-β,γ-dibromomethyleneATP or adenosine deaminase augments PSCaTs. Blockade of reuptake/deamination of eADO inhibits PSCaTs. Adenosine inhibits fEPSPs and PSCaTs (IC50=25μM), sensitive to MRS1220-antagonism (A3AR). A P2Y agonist ADPβS inhibits PSCaTs (IC50=111nM) in neurons without stimulatory ADPβS responses (EC50=960nM). ATP or a P2X1,2,2/3 (α,β-MeATP) agonist evokes fast, slow, biphasic Ca2+ transients or Ca2+ oscillations (EC50=400μM). PSCaTs are sensitive to P2X1 antagonist NF279. Low (20nM) or high (5μM) concentrations of P2X antagonist TNP-ATP block PSCaTs in different neurons; proportions of neurons with P2XR-ir follow the order P2X2>P2X1≫P2X3; P2X1+ P2X2 and P2X3+P2X2 are co-localized. RT-PCR identified mRNA-transcripts for P2X1-7,P2Y1,2,12-14R. Responsive neurons were also identified by HuC/D-ir. Conclusions Purines are critical regulators of neurotransmission in the human enteric nervous system. Purinergic signaling involves

  20. [AR-V7 Androgen Receptor Variant as a Predictor of Response to Androgen-receptor Targeting Agents Used to Treat Castration-refractory Metastatic Prostate Cancer].

    PubMed

    Büchler, Tomáš; Bobek, Vladimír; Kološtová, Katarína

    2017-01-01

    Several systemic treatment options are currently available for patients with metastatic castration-refractory prostate cancer (mCRPC), including the androgen-receptor targeting agents (ARTA) enzalutamide and abiraterone, the taxanes docetaxel and cabazitaxel, and the radioisotope drug 223-radium dichloride. In some patients with mCRCP, alternative splicing of androgen receptor (AR) mRNA occurs, resulting in the formation of a truncated AR lacking the androgen-binding domain. These receptors activate downstream signalling pathways even without the ligand. Recent studies show that the presence of the AR-V7 (ARV - AR variants) splicing variant is associated with resistance to ARTA. Bec>ause the presence of AR-V7 does not affect the efficacy of other systemic therapies used in mCRCPs, particularly taxanes, AR-V7 is a candidate predictive biomarker for the individualisation of mCRCP treatment. Two types of assays based on mRNA or abnormal protein detection are used to detect AR-V7 in circulating tumour cells. To describe the current status of AR-V7 testing in mCRPC and possible applications of this method for predicting outcomes of ARTA therapy. The percentage of CTC AR-V7+ in ARTA-naive men is relatively low at baseline, but in patients pretreated with ARTA, the prevalence of AR-V7 increases to 19-34%. Given the relatively high expected prevalence, AR-V7 testing may be economically feasible in this population. The proportion of AR-V7+ patients responding to ARTA retreatment appears to be very low, at only 4.8%. AR-V7 testing could thus be useful if an ARTA switch is considered in a patient progressing onto an ARTA drug. Both protein-based tests and mRNA-based tests are currently undergoing clinical validation in prospective studies, with results expected within a year.Key words: prostate cancer - abiraterone - enzalutamide - alternative splicing - drug resistanceSubmitted: 30. 8. 2017Accepted: 5. 11. 2017 doc. MUDr. Tomáš Büchler, Ph.D. received honorary lectures

  1. 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

  2. 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.

  3. Lubiprostone activates CFTR, but not ClC-2, via the prostaglandin receptor (EP(4)).

    PubMed

    Norimatsu, Yohei; Moran, Aurelia R; MacDonald, Kelvin D

    2012-09-28

    The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP(4) receptor has been described [2-5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP(4) receptor or β adrenergic receptorAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP(4) receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH = 6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP(4) did not respond to the presence of 0.1, 1, or 10 μM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1 μM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTR(inh)172. Co-expression of CFTR and EP(4) resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTR(inh)172. The EC(50) for lubiprostone mediated CFTR activation was ~10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP(4) receptor in oocytes. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Lubiprostone Activates CFTR, but not ClC-2, via the Prostaglandin Receptor (EP4)

    PubMed Central

    Norimatsu, Yohei; Moran, Aurelia R.; MacDonald, Kelvin D.

    2012-01-01

    The goal of this study was to determine the mechanism of lubiprostone activation of epithelial chloride transport. Lubiprostone is a bicyclic fatty acid approved for the treatment of constipation [1]. There is uncertainty, however, as to how lubiprostone increases epithelial chloride transport. Direct stimulation of ClC-2 and CFTR chloride channels as well as stimulation of these channels via the EP4 receptor has been described [2; 3; 4; 5]. To better define this mechanism, two-electrode voltage clamp was used to assay Xenopus oocytes expressing ClC-2, with or without co-expression of the EP4 receptor or β adrenergic receptorAR), for changes in conductance elicited by lubiprostone. Oocytes co-expressing CFTR and either βAR or the EP4 receptor were also studied. In oocytes co-expressing ClC-2 and βAR conductance was stimulated by hyperpolarization and acidic pH (pH=6), but there was no response to the β adrenergic agonist, isoproterenol. Oocytes expressing ClC-2 only or co-expressing ClC-2 and EP4 did not respond to the presence of 0.1, 1, or 10 µM lubiprostone in the superperfusate. Oocytes co-expressing CFTR and βAR did not respond to hyperpolarization, acidic pH, or 1µM lubiprostone. However, conductance was elevated by isoproterenol and inhibited by CFTRinh172. Co-expression of CFTR and EP4 resulted in lubiprostone-stimulated conductance, which was also sensitive to CFTRinh172. The EC50 for lubiprostone mediated CFTR activation was ~ 10 nM. These results demonstrate no direct action of lubiprostone on either ClC-2 or CFTR channels expressed in oocytes. However, the results confirm that CFTR can be activated by lubiprostone via the EP4 receptor in oocytes. PMID:22960173

  5. Decavanadate, a P2X receptor antagonist, and its use to study ligand interactions with P2X7 receptors.

    PubMed

    Michel, Anton D; Xing, Mengle; Thompson, Kyla M; Jones, Clare A; Humphrey, Patrick P A

    2006-03-18

    In this study we have studied decavanadate effects at P2X receptors. Decavanadate competitively blocked 2'- and 3'-O-(4benzoylbenzoyl) ATP (BzATP) stimulated ethidium accumulation in HEK293 cells expressing human recombinant P2X7 receptors (pK(B) 7.5). The effects of decavanadate were rapid (minutes) in both onset and offset and contrasted with the much slower kinetics of pyridoxal 5-phosphate (P5P), Coomassie brilliant blue (CBB) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62). Decavanadate competitively blocked the slowly reversible, or irreversible, blockade of the P2X7 receptor produced by P5P and oxidised ATP suggesting competition for a common binding site. However, the interaction between decavanadate and KN62 was non-competitive. Decavanadate also blocked P2X2 and P2X4 receptors but with slightly lower potency. These data demonstrate that decavanadate is the first reversible and competitive antagonist of the P2X7 receptor and is a useful tool for studying the mechanism of interaction of ligands with the P2X7 receptor.

  6. Adrenergic Receptors in Individual Ventricular Myocytes: The Beta-1 and Alpha-1B Are in All Cells, the Alpha-1A Is in a Subpopulation, and the Beta-2 and Beta-3 Are Mostly Absent.

    PubMed

    Myagmar, Bat-Erdene; Flynn, James M; Cowley, Patrick M; Swigart, Philip M; Montgomery, Megan D; Thai, Kevin; Nair, Divya; Gupta, Rumita; Deng, David X; Hosoda, Chihiro; Melov, Simon; Baker, Anthony J; Simpson, Paul C

    2017-03-31

    It is unknown whether every ventricular myocyte expresses all 5 of the cardiac adrenergic receptors (ARs), β1, β2, β3, α1A, and α1B. The β1 and β2 are thought to be the dominant myocyte ARs. Quantify the 5 cardiac ARs in individual ventricular myocytes. We studied ventricular myocytes from wild-type mice, mice with α1A and α1B knockin reporters, and β1 and β2 knockout mice. Using individual isolated cells, we measured knockin reporters, mRNAs, signaling (phosphorylation of extracellular signal-regulated kinase and phospholamban), and contraction. We found that the β1 and α1B were present in all myocytes. The α1A was present in 60%, with high levels in 20%. The β2 and β3 were detected in only ≈5% of myocytes, mostly in different cells. In intact heart, 30% of total β-ARs were β2 and 20% were β3, both mainly in nonmyocytes. The dominant ventricular myocyte ARs present in all cells are the β1 and α1B. The β2 and β3 are mostly absent in myocytes but are abundant in nonmyocytes. The α1A is in just over half of cells, but only 20% have high levels. Four distinct myocyte AR phenotypes are defined: 30% of cells with β1 and α1B only; 60% that also have the α1A; and 5% each that also have the β2 or β3. The results raise cautions in experimental design, such as receptor overexpression in myocytes that do not express the AR normally. The data suggest new paradigms in cardiac adrenergic signaling mechanisms. © 2017 American Heart Association, Inc.

  7. Serotonin and brain function: a tale of two receptors.

    PubMed

    Carhart-Harris, R L; Nutt, D J

    2017-09-01

    Previous attempts to identify a unified theory of brain serotonin function have largely failed to achieve consensus. In this present synthesis, we integrate previous perspectives with new and older data to create a novel bipartite model centred on the view that serotonin neurotransmission enhances two distinct adaptive responses to adversity, mediated in large part by its two most prevalent and researched brain receptors: the 5-HT1A and 5-HT2A receptors. We propose that passive coping (i.e. tolerating a source of stress) is mediated by postsynaptic 5-HT1AR signalling and characterised by stress moderation. Conversely, we argue that active coping (i.e. actively addressing a source of stress) is mediated by 5-HT2AR signalling and characterised by enhanced plasticity (defined as capacity for change). We propose that 5-HT1AR-mediated stress moderation may be the brain's default response to adversity but that an improved ability to change one's situation and/or relationship to it via 5-HT2AR-mediated plasticity may also be important - and increasingly so as the level of adversity reaches a critical point. We propose that the 5-HT1AR pathway is enhanced by conventional 5-HT reuptake blocking antidepressants such as the selective serotonin reuptake inhibitors (SSRIs), whereas the 5-HT2AR pathway is enhanced by 5-HT2AR-agonist psychedelics. This bipartite model purports to explain how different drugs (SSRIs and psychedelics) that modulate the serotonergic system in different ways, can achieve complementary adaptive and potentially therapeutic outcomes.

  8. Serotonin and brain function: a tale of two receptors

    PubMed Central

    Carhart-Harris, RL; Nutt, DJ

    2017-01-01

    Previous attempts to identify a unified theory of brain serotonin function have largely failed to achieve consensus. In this present synthesis, we integrate previous perspectives with new and older data to create a novel bipartite model centred on the view that serotonin neurotransmission enhances two distinct adaptive responses to adversity, mediated in large part by its two most prevalent and researched brain receptors: the 5-HT1A and 5-HT2A receptors. We propose that passive coping (i.e. tolerating a source of stress) is mediated by postsynaptic 5-HT1AR signalling and characterised by stress moderation. Conversely, we argue that active coping (i.e. actively addressing a source of stress) is mediated by 5-HT2AR signalling and characterised by enhanced plasticity (defined as capacity for change). We propose that 5-HT1AR-mediated stress moderation may be the brain’s default response to adversity but that an improved ability to change one’s situation and/or relationship to it via 5-HT2AR-mediated plasticity may also be important – and increasingly so as the level of adversity reaches a critical point. We propose that the 5-HT1AR pathway is enhanced by conventional 5-HT reuptake blocking antidepressants such as the selective serotonin reuptake inhibitors (SSRIs), whereas the 5-HT2AR pathway is enhanced by 5-HT2AR-agonist psychedelics. This bipartite model purports to explain how different drugs (SSRIs and psychedelics) that modulate the serotonergic system in different ways, can achieve complementary adaptive and potentially therapeutic outcomes. PMID:28858536

  9. Preclinical evaluation of 68Ga-DOTA-minigastrin for the detection of cholecystokinin-2/gastrin receptor-positive tumors.

    PubMed

    Brom, Maarten; Joosten, Lieke; Laverman, Peter; Oyen, Wim J G; Béhé, Martin; Gotthardt, Martin; Boerman, Otto C

    2011-04-01

    In comparison to somatostatin receptor scintigraphy, gastrin receptor scintigraphy using 111In-DTPA-minigastrin (MG0) showed added value in diagnosing neuroendocrine tumors. We investigated whether the 68Ga-labeled gastrin analogue DOTA-MG0 is suited for positron emission tomography (PET), which could improve image quality. Targeting of cholecystokinin-2 (CCK2)/gastrin receptor-positive tumor cells with DOTA-MG0 labeled with either 111In or 68Ga in vitro was investigated using the AR42J rat tumor cell line. Biodistribution was examined in BALB/c nude mice with a subcutaneous AR42J tumor. In vivo PET imaging was performed using a preclinical PET-computed tomographic scanner. DOTA-MG0 showed high receptor affinity in vitro. Biodistribution studies revealed high tumor uptake of 68Ga-DOTA-MG0: 4.4 ± 1.3 %ID/g at 1 hour postinjection. Coadministration of an excess unlabeled peptide blocked the tumor uptake (0.7 ± 0.1 %ID/g), indicating CCK2/gastrin receptor-mediated uptake (p  =  .0005). The biodistribution of 68Ga-DOTA-MG0 was similar to that of 111In-DOTA-MG0. Subcutaneous and intraperitoneal tumors were clearly visualized by small-animal PET imaging with 5 MBq 68Ga-DOTA-MG0. 111In- and 68Ga-labeled DOTA-MG0 specifically accumulate in CCK2/gastrin receptor-positive AR42J tumors with similar biodistribution apart from the kidneys. AR42J tumors were clearly visualized by microPET. Therefore, 68Ga-DOTA-MG0 is a promising tracer for PET imaging of CCK2/gastrin receptor-positive tumors in humans.

  10. Substituent effect in spin-crossover behavior of iron(II)-Ar-pybox complexes (Ar-pybox = 4-aryl-2,6-bis(oxazolin-2-yl)pyridine)

    NASA Astrophysics Data System (ADS)

    Kimura, Akifumi; Ishida, Takayuki

    2018-01-01

    Spin-crossover (SCO) is a reversible transition between low-spin (LS) and high-spin (HS) states by external stimuli like heat. The SCO behavior of [Fe(Ar-pybox)2](ClO4)2 was investigated, where Ar-pybox stands for 4-aryl-2,6-bis(oxazolin-2-yl)pyridine with Ar = 4-pyridyl (4Py), 3-thienyl (3Th), and phenyl (Ph). They were characterized by means of single-crystal X-ray diffraction study, being consistent with the results of the magnetic measurements. The SCO temperatures (T1/2) in the polycrystalline state were determined to be 360 and 230 K for Ar = 4Py and Ph, respectively. The 3Th derivative possessed a HS state in all the temperature range. The solution susceptometry was also performed to purge intermolecular interaction and rigid crystal lattice effects, affording T1/2 = 310, 240, and 240 K for Ar = 4Py, 3Th, and Ph, respectively, in acetone. The substituent effect analysis using the Hammett substituent constant (σp) clarified that electron-withdrawing groups raise T1/2. A plausible model describing the substituent effect on T1/2 is proposed based on d-π interaction. The present result is regarded as a successful example of crystal field engineering.

  11. Platelets Express Activated P2Y12 Receptor in Patients With Diabetes Mellitus.

    PubMed

    Hu, Liang; Chang, Lin; Zhang, Yan; Zhai, Lili; Zhang, Shenghui; Qi, Zhiyong; Yan, Hongmei; Yan, Yan; Luo, Xinping; Zhang, Si; Wang, Yiping; Kunapuli, Satya P; Ye, Hongying; Ding, Zhongren

    2017-08-29

    Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood. We measured P2Y 12 expression on platelets from patients with type 2 diabetes mellitus and on platelets from rats with diabetes mellitus. We also assayed platelet P2Y 12 activation by measuring cAMP and VASP phosphorylation. The antiplatelet and antithrombotic effects of AR-C78511 and cangrelor were compared in rats. Finally, we explored the role of the nuclear factor-κB pathway in regulating P2Y 12 receptor expression in megakaryocytes. Platelet P2Y 12 levels are 4-fold higher in patients with type 2 diabetes mellitus compared with healthy subjects. P2Y 12 expression correlates with ADP-induced platelet aggregation (r=0.89, P <0.01). P2Y 12 in platelets from patients with diabetes mellitus is constitutively activated. Although both AR-C78511, a potent P2Y 12 inverse agonist, and cangrelor have similar antiplatelet efficacy on platelets from healthy subjects, AR-C78511 exhibits more powerful antiplatelet effects on diabetic platelets than cangrelor (aggregation ratio 36±3% versus 49±5%, respectively, P <0.05). Using a FeCl 3 -injury mesenteric arteriole thrombosis model in rats and an arteriovenous shunt thrombosis model in rats, we found that the inverse agonist AR-C78511 has greater antithrombotic effects on GK rats with diabetes mellitus than cangrelor (thrombus weight 4.9±0.3 mg versus 8.3±0.4 mg, respectively, P <0.01). We also found that a pathway involving high glucose-reactive oxygen species-nuclear factor-κB increases platelet P2Y 12 receptor expression in diabetes mellitus. Platelet P2Y 12 receptor expression is significantly increased and the receptor is constitutively activated in patients with type 2 diabetes mellitus, which contributes to

  12. A case-based evaluation of SRD5A1, SRD5A2, AR, and ADRA1A as candidate genes for severity of BPH.

    PubMed

    Klotsman, M; Weinberg, C R; Davis, K; Binnie, C G; Hartmann, K E

    2004-01-01

    In men with a clinical diagnosis of benign prostatic hyperplasia (BPH), polytomous logistic regression analysis was conducted to evaluate associations between two silent polymorphisms in SRD5A1 (codon positions 30 and 116), two polymorphisms in SRD5A2 (Val89Leu substitution and C to T transition in intron 1), a trinucleotide (CAG)n repeat in androgen receptor (AR), and an Arg492Cys substitution in ADRA1A and clinical parameters that characterize severity of BPH. Candidate gene selection was based on two mechanistic pathways targeted by pharmacotherapy for BPH: (1) androgen metabolic loci contributing to prostate growth (static obstruction); and (2) factors affecting smooth muscle tone (dynamic obstruction). Polymorphisms in SRD5A2 were not associated with severity of BPH; however, SRD5A1 polymorphisms were associated with severity of BPH. The process(es) in which these silent single-nucleotide polymorphisms (SNPs) influence BPH phenotypes is unknown and additional studies will be needed to assess whether these SNPs have direct functional consequences. The characterization of additional molecular factors that contribute to static and dynamic obstruction may help predict response to pharmacotherapy and serve to identify novel drug targets for the clinical management of BPH.

  13. The transcriptional programme of the androgen receptor (AR) in prostate cancer.

    PubMed

    Lamb, Alastair D; Massie, Charlie E; Neal, David E

    2014-03-01

    The androgen receptor (AR) is essential for normal prostate and prostate cancer cell growth. AR transcriptional activity is almost always maintained even in hormone relapsed prostate cancer (HRPC) in the absence of normal levels of circulating testosterone. Current molecular techniques, such as chromatin-immunoprecipitation sequencing (ChIP-seq), have permitted identification of direct AR-binding sites in cell lines and human tissue with a distinct coordinate network evident in HRPC. The effectiveness of novel agents, such as abiraterone acetate (suppresses adrenal androgens) or enzalutamide (MDV3100, potent AR antagonist), in treating advanced prostate cancer underlines the on-going critical role of the AR throughout all stages of the disease. Persistent AR activity in advanced disease regulates cell cycle activity, steroid biosynthesis and anabolic metabolism in conjunction with regulatory co-factors, such as the E2F family, c-Myc and signal transducer and activator of transcription (STAT) transcription factors. Further treatment approaches must target these other factors. © 2013 The Authors. BJU International © 2013 BJU International.

  14. 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.

  15. Differences in potency and efficacy of a series of phenylisopropylamine/phenylethylamine pairs at 5-HT(2A) and 5-HT(2C) receptors.

    PubMed

    Acuña-Castillo, Claudio; Villalobos, Claudio; Moya, Pablo R; Sáez, Patricio; Cassels, Bruce K; Huidobro-Toro, J Pablo

    2002-06-01

    The pharmacological profile of a series of (+/-)-2,5-dimethoxy-4-(X)-phenylisopropylamines (X=I, Br, NO(2), CH(3), or H) and corresponding phenylethylamines, was determined in Xenopus laevis oocytes injected with cRNA coding for rat 5-HT(2A) or 5-HT(2C) receptors. The efficacy and relative potency of these drugs were determined and compared to classical 5-HT(2) receptor agonists and antagonists. The rank order of agonist potency at the 5-HT(2A) receptor was: alpha-methyl-5-HT=5-HT>m-CPP>MK-212; at the 5-HT(2C) receptor the order was: 5-HT>alpha-methyl-5-HT>MK-212>m-CPP. All these compounds were full agonists at the 5-HT(2C) receptor, but alpha-methyl-5-HT and m-CPP showed lower efficacy at the 5-HT(2A) receptor. 4-(4-Fluorobenzoyl)-1-(4-phenylbutyl)piperidine (4F 4PP) was 200 times more potent as a 5-HT(2A) antagonist than at 5-HT(2C) receptors. Conversely, RS 102221 was 100 times more potent as a 5-HT(2C) antagonist, confirming their relative receptor selectivities. The phenylisopropylamines were partial agonists at the 5-HT(2A) receptor, with I(max) relative to 5-HT in the 22+/-7 to 58+/-15% range; the corresponding phenylethylamines had lower or undetectable efficacies. All these drugs had higher efficacies at 5-HT(2C) receptors; DOI was a full 5-HT(2C) agonist. 2C-I and the other phenylethylamines examined showed relative efficacies at the 5-HT(2C) receptor ranging from 44+/-10% to 76+/-16%. 2C-N was a 5-HT(2) receptor antagonist; the mechanism was competitive at the 5-HT(2A), but non-competitive at the 5-HT(2C) receptor. The antagonism was time-dependent at the 5-HT(2C) receptor but independent of pre-incubation time at the 5-HT(2A) receptor subtype. The alpha-methyl group determines the efficacy of these phenylalkylamines at the 5-HT(2A) and 5-HT(2C) receptors.

  16. Cell-Free Expression, Purification, and Characterization of the Functional β2-Adrenergic Receptor for Multianalyte Detection of β-Agonists.

    PubMed

    Wang, Jian; Liu, Yuan; Zhang, Junhua; Han, Zhengzheng; Wang, Wei; Liu, Yang; Wei, Dong; Huang, Wei

    2017-11-01

    Large-scale expression of β 2 -adrenergic receptor2 -AR) in functional form is necessary for establishment of receptor assays for detecting illegally abused β-adrenergic agonists (β-agonists). Cell-based heterologous expression systems have manycritical difficulties in synthesizing this membrane protein, such as low protein yields and aberrant folding. To overcome these challenges, the main objective of the present work was to synthesize large amounts of functional β 2 -AR in a cell-free system based on Escherichia coli extracts. A codon-optimized porcine β 2 -AR gene (codon adaptation index: 0.96) suitable for high expression in E. coli was synthesized and transcribed to the cell-free system, which contributed to increase the expression up to 1.1 mg/ml. After purification using Ni-affinity chromatography, the bioactivity of the purified receptor was measured by novel enzyme-linked receptor assays. It was determined that the relative affinities of the purified β 2 -AR for β-agonists in descending order were as follows: clenbuterol > salbutamol > ractopamine. Moreover, their IC 50 values were 45.99, 60.38, and 78.02 µg/liter, respectively. Although activity of the cell-free system was slightly lower than activity of systems based on insect and mammalian cells, this system should allow production of β 2 -AR in bulk amounts sufficient for the development of multianalyte screening methods for detecting β-agonist residues.

  17. Fourier-transform MW spectroscopy of the SH({sup 2}{Pi}{sub i})-Ar and SD-Ar radical complexes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sumiyoshi, Yoshihiro; Endo, Yasuki; Ohshima, Yasuhiro

    1996-12-31

    The authors have studied the SH({sup 2}{Pi}{sub i})-Ar and SD-Ar radical complexes with FTMW spectroscopy. The complexes were produced in a supersonic free jet by a pulsed discharge of H{sub 2}S or D{sub 2}S, which was diluted to 0.35% in Ar with a stagnation pressure of 2 atm. R-branch transitions in the lower spin-orbit component ({Omega}=3/2) for the linear {sup 2}{Pi}{sub i} radicals were observed for J{double_prime} = 3/2 to J{double_prime} = 15/2 in the 8-26 GHz region. The transitions were split into two parity components owing to the parity doubling. Each parity component was split further due to themore » magnetic hyperfine interaction associated with the H/D nucleus. Rotational constants for SH-Ar and SD-Ar were determined to be 1569.656(2) and 1567.707(2)MHz respectively. The value for SH-Ar agrees well with that of a previous LIF study. From the SH/SD data, it was confirmed that the argon atom is located at the hydrogen side of the SH radical. With an assumption that the S-H bond length is equal to that in the monomer, the H-Ar distance is calculated to be 2.900 {Angstrom}, which is about 0.1 {Angstrom} longer than that in OH-Ar. The effective D{sub J} constants of SH-Ar and SD-Ar were found to have negative values of -58.4(7) and -50.7(6), kHz respectively.« less

  18. ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer.

    PubMed

    Mahajan, Kiran; Malla, Pavani; Lawrence, Harshani R; Chen, Zhihua; Kumar-Sinha, Chandan; Malik, Rohit; Shukla, Sudhanshu; Kim, Jongphil; Coppola, Domenico; Lawrence, Nicholas J; Mahajan, Nupam P

    2017-06-12

    The androgen receptor (AR) is critical for the progression of prostate cancer to a castration-resistant (CRPC) state. AR antagonists are ineffective due to their inability to repress the expression of AR or its splice variant, AR-V7. Here, we report that the tyrosine kinase ACK1 (TNK2) phosphorylates histone H4 at tyrosine 88 upstream of the AR transcription start site. The WDR5/MLL2 complex reads the H4-Y88-phosphorylation marks and deposits the transcriptionally activating H3K4-trimethyl marks promoting AR transcription. Reversal of the pY88-H4 epigenetic marks by the ACK1 inhibitor (R)-9bMS-sensitized naive and enzalutamide-resistant prostate cancer cells and reduced AR and AR-V7 levels to mitigate CRPC tumor growth. Thus, a feedforward ACK1/pY88-H4/WDR5/MLL2/AR epigenetic circuit drives CRPC and is necessary for maintenance of the malignant state. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Differential Effects of Inescapable Stress on Locus Coeruleus GRK3, Alpha2-Adrenoceptor and CRF1 Receptor Levels in Learned Helpless and Non-Helpless Rats: A potential link to stress resilience

    PubMed Central

    Taneja, Manish; Salim, Samina; Saha, Kaustuv; Happe, H. Kevin; Qutna, Nidal; Petty, Frederick; Bylund, David B.; Eikenburg, Douglas C.

    2011-01-01

    Exposure of rats to unpredictable, inescapable stress results in two distinct behaviors during subsequent escape testing. One behavior, suggestive of lack of stress resilience, is prolonged escape latency compared to non-stressed rats and is labeled learned helplessness (LH). The other behavior suggestive of stress resilience is normal escape latency and is labeled non-helpless (NH). This study examines the effects of unpredictable, inescapable tail-shock stress (TSS) on alpha2-adrenoceptor (α2-AR) and corticotropin-releasing factor 1 receptor (CRF1) regulation as well as protein levels of G protein-coupled receptor kinase 3 (GRK3), GRK2, tyrosine hydroxylase (TH) plus carbonylated protein levels in locus coeruleus (LC), amygdala (AMG), cortex (COR) and striatum (STR). In NH rats, α2-AR and CRF1 receptors were significantly down-regulated in LC after TSS. No changes in these receptor levels were observed in the LC of LH rats. GRK3, which phosphorylates receptors and thereby contributes to α2-AR and CRF1 receptor down-regulation, was reduced in the LC of LH but not NH rats. GRK2 levels were unchanged. In AMG, GRK3 but not GRK2 levels were reduced in LH but not NH rats, and receptor regulation was impaired in LH rats. In STR, no changes in GRK3 or GRK2 levels were observed. Finally, protein carbonylation, an index of oxidative stress, was increased in the LC and AMG of LH but not NH rats. We suggest that reduced stress resilience after TSS may be related to oxidative stress, depletion of GRK3 and impaired regulation of α2-AR and CRF1 receptor in LC. PMID:21333691

  20. Deletion of CB2 Cannabinoid Receptor Induces Schizophrenia-Related Behaviors in Mice

    PubMed Central

    Ortega-Alvaro, Antonio; Aracil-Fernández, Auxiliadora; García-Gutiérrez, María S; Navarrete, Francisco; Manzanares, Jorge

    2011-01-01

    The possible role of the CB2 receptor (CB2r) in psychiatric disorders has been considered. Several animal models use knockout (KO) mice that display schizophrenia-like behaviors and this study evaluated the role of CB2r in the regulation of such behaviors. Mice lacking the CB2r (CB2KO) were challenged in open field, light–dark box, elevated plus-maze, tail suspension, step down inhibitory avoidance, and pre-pulse inhibition tests (PPI). Furthermore, the effects of treatment with cocaine and risperidone were evaluated using the OF and the PPI test. Gene expression of dopamine D2 (D2r), adrenergic-α2C (α2Cr), serotonergic 5-HT2A and 5-HT2C receptors (5-HT2Ar and 5-HT2Cr) were studied by RT-PCR in brain regions related to schizophrenia. Deletion of CB2r decreased motor activity in the OF test, but enhanced response to acute cocaine and produced mood-related alterations, PPI deficit, and cognitive impairment. Chronic treatment with risperidone tended to impair PPI in WT mice, whereas it ‘normalized' the PPI deficit in CB2KO mice. CB2KO mice presented increased D2r and α2Cr gene expressions in the prefrontal cortex (PFC) and locus coeruleus (LC), decreased 5-HT2Cr gene expression in the dorsal raphe (DR), and 5-HT2Ar gene expression in the PFC. Chronic risperidone treatment in WT mice left α2Cr gene expression unchanged, decreased D2r gene expression (15 μg/kg), and decreased 5-HT2Cr and 5-HT2Ar in PFC and DR. In CB2KO, the gene expression of D2r in the PFC, of α2Cr in the LC, and of 5-HT2Cr and 5-HT2Ar in PFC was reduced; 5-HT2Cr and 5-HT2Ar gene expressions in DR were increased after treatment with risperidone. These results suggest that deletion of CB2r has a relation with schizophrenia-like behaviors. Pharmacological manipulation of CB2r may merit further study as a potential therapeutic target for the treatment of schizophrenia-related disorders. PMID:21430651

  1. Adenosine 2A Receptor Activation Attenuates Ischemia Reperfusion Injury During Extracorporeal Cardiopulmonary Resuscitation.

    PubMed

    Mehaffey, James H; Money, Dustin; Charles, Eric J; Schubert, Sarah; Piñeros, Angela Fernandez; Wu, Di; Bontha, Sai Vineela; Hawkins, Robert; Teman, Nicholas R; Laubach, Victor E; Mas, Valeria R; Tribble, Curtis G; Maluf, Daniel G; Sharma, Ashish K; Yang, Zequan; Kron, Irving L; Roeser, Mark E

    2018-01-25

    We tested the hypothesis that systemic administration of an A2AR agonist will reduce multiorgan IRI in a porcine model of ECPR. Advances in ECPR have decreased mortality after cardiac arrest; however, subsequent IRI contributes to late multisystem organ failure. Attenuation of IRI has been reported with the use of an A2AR agonist. Adult swine underwent 20 minutes of circulatory arrest, induced by ventricular fibrillation, followed by 6 hours of reperfusion with ECPR. Animals were randomized to vehicle control, low-dose A2AR agonist, or high-dose A2AR agonist. A perfusion specialist using a goal-directed resuscitation protocol managed all the animals during the reperfusion period. Hourly blood, urine, and tissue samples were collected. Biochemical and microarray analyses were performed to identify differential inflammatory markers and gene expression between groups. Both the treatment groups demonstrated significantly higher percent reduction from peak lactate after reperfusion compared with vehicle controls. Control animals required significantly more fluid, epinephrine, and higher final pump flow while having lower urine output than both the treatment groups. The treatment groups had lower urine NGAL, an early marker of kidney injury (P = 0.01), lower plasma aspartate aminotransferase, and reduced rate of troponin rise (P = 0.01). Pro-inflammatory cytokines were lower while anti-inflammatory cytokines were significantly higher in the treatment groups. Using a novel and clinically relevant porcine model of circulatory arrest and ECPR, we demonstrated that a selective A2AR agonist significantly attenuated systemic IRI and warrants clinical investigation.

  2. 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

  3. 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

  4. Role of the serotonin 5-HT(2A) receptor in learning.

    PubMed

    Harvey, John A

    2003-01-01

    This study reviews the role of the serotonin 5-HT2A receptor in learning as measured by the acquisition of the rabbit's classically conditioning nictitating membrane response, a component of the eyeblink response. Agonists at the 5-HT2A receptor including LSD (d-lysergic acid diethylamide) enhanced associative learning at doses that produce cognitive effects in humans. Some antagonists such as BOL (d-bromolysergic acid diethylamide), LY53,857, and ketanserin acted as neutral antagonists in that they had no effect on learning, whereas others (MDL11,939, ritanserin, and mianserin) acted as inverse agonists in that they retarded learning through an action at the 5-HT2A receptor. These results were placed in the context of what is known concerning the anatomical distribution and electrophysiological effects of 5-HT2A receptor activation in frontal cortex and hippocampus, as well as the role of cortical 5-HT2A receptors in schizophrenia. It was concluded that the 5-HT2A receptor demonstrates constitutive activity, and that variations in this activity can produce profound alterations in cognitive states.

  5. Selective regulation of nuclear orphan receptors 4A by adenosine receptor subtypes in human mast cells

    PubMed Central

    Zhang, Li; Paine, Catherine

    2010-01-01

    Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A2A (A2AAR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A2B and A3) and that A2AAR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A2AAR-silencing amplified NR4A induction by NECA. Interestingly, a similar A2AAR modulatory effect was observed on ERK1/2 phosphorylation because A2AAR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A2AAR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A2AAR activation, not only in the context of adenosine but of other mast cell activating stimuli as well. PMID:21234122

  6. Effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand-directed chiral recognition.

    PubMed

    Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

    2011-01-01

    The β(2) adrenergic receptor (β(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a β(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of β(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts. Copyright © 2011 Wiley-Liss, Inc.

  7. 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.

  8. Role of medial prefrontal cortex serotonin 2A receptors in the control of retrieval of recognition memory in rats.

    PubMed

    Bekinschtein, Pedro; Renner, Maria Constanza; Gonzalez, Maria Carolina; Weisstaub, Noelia

    2013-10-02

    Often, retrieval cues are not uniquely related to one specific memory, which could lead to memory interference. Controlling interference is particularly important during episodic memory retrieval or when remembering specific events in a spatiotemporal context. Despite a clear involvement of prefrontal cortex (PFC) in episodic memory in human studies, information regarding the mechanisms and neurotransmitter systems in PFC involved in memory is scarce. Although the serotoninergic system has been linked to PFC functionality and modulation, its role in memory processing is poorly understood. We hypothesized that the serotoninergic system in PFC, in particular the 5-HT2A receptor (5-HT2AR) could have a role in the control of memory retrieval. In this work we used different versions of the object recognition task in rats to study the role of the serotoninergic modulation in the medial PFC (mPFC) in memory retrieval. We found that blockade of 5-HT2AR in mPFC affects retrieval of an object in context memory in a spontaneous novelty preference task, while sparing single-item recognition memory. We also determined that 5-HT2ARs in mPFC are required for hippocampal-mPFC interaction during retrieval of this type of memory, suggesting that the mPFC controls the expression of memory traces stored in the hippocampus biasing retrieval to the most relevant one.

  9. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

    PubMed

    Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

    2013-05-17

    A prospective, large library virtual screen against an activated β2-adrenergic receptor2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

  10. p-( sup 125 I)iodoclonidine is a partial agonist at the alpha 2-adrenergic receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gerhardt, M.A.; Wade, S.M.; Neubig, R.R.

    1990-08-01

    The binding properties of p-(125I)iodoclonidine (( 125I)PIC) to human platelet membranes and the functional characteristics of PIC are reported. (125I)PIC bound rapidly and reversibly to platelet membranes, with a first-order association rate constant (kon) at room temperature of 8.0 +/- 2.7 x 10(6) M-1 sec-1 and a dissociation rate constant (koff) of 2.0 +/- 0.8 x 10(-3) sec-1. Scatchard plots of specific (125I)PIC binding (0.1-5 nM) were linear, with a Kd of 1.2 +/- 0.1 nM. (125I)PIC bound to the same number of high affinity sites as the alpha 2-adrenergic receptor (alpha 2-AR) full agonist (3H) bromoxidine (UK14,304), which representedmore » approximately 40% of the sites bound by the antagonist (3H)yohimbine. Guanosine 5'-(beta, gamma-imido)triphosphate greatly reduced the amount of (125I)PIC bound (greater than 80%), without changing the Kd of the residual binding. In competition experiments, the alpha 2-AR-selective ligands yohimbine, bromoxidine, oxymetazoline, clonidine, p-aminoclonidine, (-)-epinephrine, and idazoxan all had Ki values in the low nanomolar range, whereas prazosin, propranolol, and serotonin yielded Ki values in the micromolar range. Epinephrine competition for (125I)PIC binding was stereoselective. Competition for (3H)bromoxidine binding by PIC gave a Ki of 1.0 nM (nH = 1.0), whereas competition for (3H)yohimbine could be resolved into high and low affinity components, with Ki values of 3.7 and 84 nM, respectively. PIC had minimal agonist activity in inhibiting adenylate cyclase in platelet membranes, but it potentiated platelet aggregation induced by ADP with an EC50 of 1.5 microM. PIC also inhibited epinephrine-induced aggregation, with an IC50 of 5.1 microM. Thus, PIC behaves as a partial agonist in a human platelet aggregation assay. (125I)PIC binds to the alpha 2B-AR in NG-10815 cell membranes with a Kd of 0.5 +/- 0.1 nM.« less

  11. AT1 and AT2 Receptors in the Prelimbic Cortex Modulate the Cardiovascular Response Evoked by Acute Exposure to Restraint Stress in Rats.

    PubMed

    Brasil, Taíz F S; Fassini, Aline; Corrêa, Fernando M

    2018-01-01

    The prelimbic cortex (PL) is an important structure in the neural pathway integrating stress responses. Brain angiotensin is involved in cardiovascular control and modulation of stress responses. Blockade of angiotensin receptors has been reported to reduce stress responses. Acute restraint stress (ARS) is a stress model, which evokes sustained blood pressure increase, tachycardia, and reduction in tail temperature. We therefore hypothesized that PL locally generated angiotensin and angiotensin receptors modulate stress autonomic responses. To test this hypothesis, we microinjected an angiotensin-converting enzyme (ACE) inhibitor or angiotensin antagonists into the PL, prior to ARS. Male Wistar rats were used; guide cannulas were bilaterally implanted in the PL for microinjection of vehicle or drugs. A polyethylene catheter was introduced into the femoral artery to record cardiovascular parameters. Tail temperature was measured using a thermal camera. ARS was started 10 min after PL treatment with drugs. Pretreatment with ACE inhibitor lisinopril (0.5 nmol/100 nL) reduced the pressor response, but did not affect ARS-evoked tachycardia. At a dose of 1 nmol/100 nL, it reduced both ARS pressor and tachycardic responses. Pretreatment with candesartan, AT1 receptor antagonist reduced ARS-evoked pressor response, but not tachycardia. Pretreatment with PD123177, AT2 receptor antagonist, reduced tachycardia, but did not affect ARS pressor response. No treatment affected ARS fall in tail temperature. Results suggest involvement of PL angiotensin in the mediation of ARS cardiovascular responses, with participation of both AT1 and AT2 receptors. In conclusion, results indicate that PL AT1-receptors modulate the ARS-evoked pressor response, while AT2-receptors modulate the tachycardic component of the autonomic response.

  12. Agonist properties of N,N-dimethyltryptamine at serotonin 5-HT2A and 5-HT2C receptors.

    PubMed

    Smith, R L; Canton, H; Barrett, R J; Sanders-Bush, E

    1998-11-01

    Extensive behavioral and biochemical evidence suggests an agonist role at the 5-HT2A receptor, and perhaps the 5-HT2C receptor, in the mechanism of action of hallucinogenic drugs. However the published in vitro pharmacological properties of N,N-dimethyltryptamine (DMT), an hallucinogenic tryptamine analog, are not consistent with this hypothesis. We, therefore, undertook an extensive investigation into the properties of DMT at 5-HT2A and 5-HT2C receptors. In fibroblasts transfected with the 5-HT2A receptor or the 5-HT2C receptor, DMT activated the major intracellular signaling pathway (phosphoinositide hydrolysis) to an extent comparable to that produced by serotonin. Because drug efficacy changes with receptor density and cellular microenvironment, we also examined the properties of DMT in native preparations using a behavioral and biochemical approach. Rats were trained to discriminate an antagonist ketanserin from an agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) in a two-lever choice paradigm. Pharmacological studies showed that responding on the DOI and ketanserin lever reflected agonist and antagonist activity at 5-HT2A receptors, and hence, was a suitable model for evaluating the in vivo functional properties of DMT. Like other 5-HT2A receptor agonists, DMT substituted fully for DOI. Intact choroid plexus was used to evaluate the agonist properties at endogenous 5-HT2C receptors; DMT was a partial agonist at 5-HT2C receptors in this native preparation. Thus, we conclude that DMT behaves as an agonist at both 5-HT2A and 5-HT2A receptors. One difference was evident in that the 5-HT2C, but not the 5-HT2A, receptor showed a profound desensitization to DMT over time. This difference is interesting in light of the recent report that the hallucinogenic activity of DMT does not tolerate in humans and suggests the 5-HT2C receptor plays a less prominent role in the action of DMT.

  13. A genetic polymorphism of the alpha2-adrenergic receptor increases autonomic responses to stress.

    PubMed

    Finley, J Clayton; O'Leary, Michael; Wester, Derin; MacKenzie, Steven; Shepard, Neil; Farrow, Stephen; Lockette, Warren

    2004-06-01

    We hypothesized that individual differences in autonomic responses to psychological, physiological, or environmental stresses are inherited, and exaggerated autonomic responsiveness may represent an intermediate phenotype that can contribute to the development of essential hypertension in humans over time. alpha(2)-Adrenergic receptors (alpha(2)-ARs), encoded by a gene on chromosome 10, are found in the central nervous system and also mediate release of norepinephrine from the presynaptic nerve terminals of the peripheral sympathetic nervous system and the exocytosis of epinephrine from the adrenal medulla. We postulated that, because this receptor mediates central and peripheral autonomic responsiveness to stress, genetic mutations in the gene encoding this receptor may explain contrasting activity of the autonomic nervous system among individuals. The restriction enzyme Dra I identifies a polymorphic site in the 3'-transcribed, but not translated, portion of the gene encoding the chromosome 10 alpha(2)-AR. Southern blotting of genomic DNA with a cDNA probe after restriction enzyme digestion results in fragments that are either 6.7 kb or 6.3 kb in size. Transfection studies of these two genotypes resulted in contrasting expression of a reporter gene, and it is suggested from these findings that this is a functional polymorphism. In a study of 194 healthy subjects, we measured autonomic responses to provocative motion, a fall in blood pressure induced by decreasing venous return and cardiac output, or exercise. Specifically, we measured reactions to 1) Coriolis stress, a strong stimulus that induces motion sickness in man; 2) heart rate responses to the fall in blood pressure induced by the application of graded lower body negative pressure; and 3) exercise-induced sweat secretion. In all of these paradigms of stress, subjective and objective evidence of increased autonomic responsiveness was found in those individuals harboring the 6.3-kb allele. Specifically

  14. Structure of a nanobody-stabilized active state of the β(2) adrenoceptor.

    PubMed

    Rasmussen, Søren G F; Choi, Hee-Jung; Fung, Juan Jose; Pardon, Els; Casarosa, Paola; Chae, Pil Seok; Devree, Brian T; Rosenbaum, Daniel M; Thian, Foon Sun; Kobilka, Tong Sun; Schnapp, Andreas; Konetzki, Ingo; Sunahara, Roger K; Gellman, Samuel H; Pautsch, Alexander; Steyaert, Jan; Weis, William I; Kobilka, Brian K

    2011-01-13

    G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β(2) adrenergic receptor (β(2)AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β(2)AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.

  15. The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp; Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp; Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp

    2013-11-15

    Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHRmore » or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.« less

  16. Presynaptic serotonin 2A receptors modulate thalamocortical plasticity and associative learning

    PubMed Central

    Barre, Alexander; Berthoux, Coralie; De Bundel, Dimitri; Valjent, Emmanuel; Bockaert, Joël; Marin, Philippe; Bécamel, Carine

    2016-01-01

    Higher-level cognitive processes strongly depend on a complex interplay between mediodorsal thalamus nuclei and the prefrontal cortex (PFC). Alteration of thalamofrontal connectivity has been involved in cognitive deficits of schizophrenia. Prefrontal serotonin (5-HT)2A receptors play an essential role in cortical network activity, but the mechanism underlying their modulation of glutamatergic transmission and plasticity at thalamocortical synapses remains largely unexplored. Here, we show that 5-HT2A receptor activation enhances NMDA transmission and gates the induction of temporal-dependent plasticity mediated by NMDA receptors at thalamocortical synapses in acute PFC slices. Expressing 5-HT2A receptors in the mediodorsal thalamus (presynaptic site) of 5-HT2A receptor-deficient mice, but not in the PFC (postsynaptic site), using a viral gene-delivery approach, rescued the otherwise absent potentiation of NMDA transmission, induction of temporal plasticity, and deficit in associative memory. These results provide, to our knowledge, the first physiological evidence of a role of presynaptic 5-HT2A receptors located at thalamocortical synapses in the control of thalamofrontal connectivity and the associated cognitive functions. PMID:26903620

  17. AR Signaling in Breast Cancer.

    PubMed

    Rahim, Bilal; O'Regan, Ruth

    2017-02-24

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies.

  18. AR Signaling in Breast Cancer

    PubMed Central

    Rahim, Bilal; O’Regan, Ruth

    2017-01-01

    Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies. PMID:28245550

  19. The RNA binding protein Ars2 supports hematopoiesis at multiple levels.

    PubMed

    Elahi, Seerat; Egan, Shawn M; Holling, G Aaron; Kandefer, Rachel L; Nemeth, Michael J; Olejniczak, Scott H

    2018-05-15

    Recent biochemical characterization of Arsenic resistance protein 2 (Ars2) has established it as central to determining the fate of nascent RNA polymerase II (RNAPII) transcripts. Through interactions with the nuclear 5'-7-methylguanosine (7mG) cap binding complex (CBC), Ars2 promotes co-transcriptional processing coupled with nuclear export or degradation of several classes of RNAPII transcripts, allowing for gene expression programs that facilitate rapid and sustained proliferation of immortalized cells in culture. However, rapidly dividing cells in culture do not represent the physiological condition of the vast majority of cells in an adult mammal. To examine functions of Ars2 in a physiological setting we generated inducible Ars2 knockout mice and found that deletion of Ars2 from adult mice resulted in defective hematopoiesis in bone marrow and thymus. Importantly, only some of this defect could be explained by the requirement of Ars2 for rapid proliferation, which we found to be cell-type specific in vivo. Rather Ars2 was required for survival of developing thymocytes and for limiting differentiation of bone marrow resident long-term hematopoietic stem cells (LT-HSCs). As a result, Ars2 knockout led to rapid thymic involution and loss of the ability of mice to regenerate peripheral blood following myeloablation. These in vivo data demonstrate that Ars2 expression is important at several steps of hematopoiesis, likely because Ars2 acts on gene expression programs underlying essential cell fate decisions such as the decision to die, to proliferate, or to differentiate. Copyright © 2018. Published by Elsevier Inc.

  20. Converging evidence for the association of functional genetic variation in the serotonin receptor 2a gene with prefrontal function and olanzapine treatment.

    PubMed

    Blasi, Giuseppe; De Virgilio, Caterina; Papazacharias, Apostolos; Taurisano, Paolo; Gelao, Barbara; Fazio, Leonardo; Ursini, Gianluca; Sinibaldi, Lorenzo; Andriola, Ileana; Masellis, Rita; Romano, Raffaella; Rampino, Antonio; Di Giorgio, Annabella; Lo Bianco, Luciana; Caforio, Grazia; Piva, Francesco; Popolizio, Teresa; Bellantuono, Cesario; Todarello, Orlando; Kleinman, Joel E; Gadaleta, Gemma; Weinberger, Daniel R; Bertolino, Alessandro

    2013-09-01

    Serotonin (5-hydroxytryptamine) receptor 2a (5-HT2AR) signaling is important for modulation of corticostriatal pathways and prefrontal activity during cognition. Furthermore, newer antipsychotic drugs target 5-HT2AR. A single-nucleotide polymorphism in the 5-HT2AR gene (HTR2A rs6314, C>T; OMIM 182135) has been weakly associated with differential 5-HT2AR signaling and with physiologic as well as behavioral effects. To use a hierarchical approach to determine the functional effects of this single-nucleotide polymorphism on 5-HT2AR messenger RNA and protein expression, on prefrontal phenotypes linked with genetic risk for schizophrenia, and on treatment with olanzapine. In silico predictions, in vitro, and case-control investigations. Academic and clinical facilities. The postmortem study included 112 brains from healthy individuals; the in vivo investigation included a total sample of 371 healthy individuals and patients with schizophrenia. EXPOSURES Patients received olanzapine monotherapy for 8 weeks. In silico predictions, messenger RNA, and protein expression in postmortem human prefrontal cortex and HeLa cells, functional magnetic resonance imaging prefrontal activity and behavior during working memory and attention in healthy individuals, and response to an 8-week trial of olanzapine treatment in patients with schizophrenia. Bioinformatic analysis predicted that rs6314 alters patterns of splicing, with possible effects on HTR2A expression. Moreover, the T allele was associated with reduced prefrontal messenger RNA expression in postmortem prefrontal cortex, with reduced protein expression in vitro, inefficient prefrontal blood oxygen level-dependent functional magnetic resonance imaging response during working memory and attentional control processing, and impaired working memory and attention behavior, as well as with attenuated improvement in negative symptoms after olanzapine treatment. Our results suggest that HTR2A rs6314 affects 5-HT2AR expression and

  1. α2A- and α2C-Adrenoceptors as Potential Targets for Dopamine and Dopamine Receptor Ligands.

    PubMed

    Sánchez-Soto, Marta; Casadó-Anguera, Verònica; Yano, Hideaki; Bender, Brian Joseph; Cai, Ning-Sheng; Moreno, Estefanía; Canela, Enric I; Cortés, Antoni; Meiler, Jens; Casadó, Vicent; Ferré, Sergi

    2018-03-18

    The poor norepinephrine innervation and high density of Gi/o-coupled α 2A - and α 2C -adrenoceptors in the striatum and the dense striatal dopamine innervation have prompted the possibility that dopamine could be an effective adrenoceptor ligand. Nevertheless, the reported adrenoceptor agonistic properties of dopamine are still inconclusive. In this study, we analyzed the binding of norepinephrine, dopamine, and several compounds reported as selective dopamine D 2 -like receptor ligands, such as the D 3 receptor agonist 7-OH-PIPAT and the D 4 receptor agonist RO-105824, to α 2 -adrenoceptors in cortical and striatal tissue, which express α 2A -adrenoceptors and both α 2A - and α 2C -adrenoceptors, respectively. The affinity of dopamine for α 2 -adrenoceptors was found to be similar to that for D 1 -like and D 2 -like receptors. Moreover, the exogenous dopamine receptor ligands also showed high affinity for α 2A - and α 2C -adrenoceptors. Their ability to activate Gi/o proteins through α 2A - and α 2C -adrenoceptors was also analyzed in transfected cells with bioluminescent resonance energy transfer techniques. The relative ligand potencies and efficacies were dependent on the Gi/o protein subtype. Furthermore, dopamine binding to α 2 -adrenoceptors was functional, inducing changes in dynamic mass redistribution, adenylyl cyclase activity, and ERK1/2 phosphorylation. Binding events were further studied with computer modeling of ligand docking. Docking of dopamine at α 2A - and α 2C -adrenoceptors was nearly identical to its binding to the crystallized D 3 receptor. Therefore, we provide conclusive evidence that α 2A - and α 2C -adrenoceptors are functional receptors for norepinephrine, dopamine, and other previously assumed selective D 2 -like receptor ligands, which calls for revisiting previous studies with those ligands.

  2. The 2.6 Angstrom Crystal Structure of a Human A[subscript 2A] Adenosine Receptor Bound to an Antagonist

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jaakola, Veli-Pekka; Griffith, Mark T.; Hanson, Michael A.

    2009-01-15

    The adenosine class of heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) mediates the important role of extracellular adenosine in many physiological processes and is antagonized by caffeine. We have determined the crystal structure of the human A{sub 2A} adenosine receptor, in complex with a high-affinity subtype-selective antagonist, ZM241385, to 2.6 angstrom resolution. Four disulfide bridges in the extracellular domain, combined with a subtle repacking of the transmembrane helices relative to the adrenergic and rhodopsin receptor structures, define a pocket distinct from that of other structurally determined GPCRs. The arrangement allows for the binding of the antagonist in an extendedmore » conformation, perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core, in ligand recognition by this class of GPCRs and suggests a role for ZM241385 in restricting the movement of a tryptophan residue important in the activation mechanism of the class A receptors.« less

  3. Hacking and securing the AR.Drone 2.0 quadcopter: investigations for improving the security of a toy

    NASA Astrophysics Data System (ADS)

    Pleban, Johann-Sebastian; Band, Ricardo; Creutzburg, Reiner

    2014-02-01

    In this article we describe the security problems of the Parrot AR.Drone 2.0 quadcopter. Due to the fact that it is promoted as a toy with low acquisition costs, it may end up being used by many individuals which makes it a target for harmful attacks. In addition, the videostream of the drone could be of interest for a potential attacker due to its ability of revealing confidential information. Therefore, we will perform a security threat analysis on this particular drone. We will set the focus mainly on obvious security vulnerabilities like the unencrypted Wi-Fi connection or the user management of the GNU/Linux operating system which runs on the drone. We will show how the drone can be hacked in order to hijack the AR.Drone 2.0. Our aim is to sensitize the end-user of AR.Drones by describing the security vulnerabilities and to show how the AR.Drone 2.0 could be secured from unauthorized access. We will provide instructions to secure the drones Wi-Fi connection and its operation with the official Smartphone App and third party PC software.

  4. Development of a Sigma-2 Receptor affinity filter through a Monte Carlo based QSAR analysis.

    PubMed

    Rescifina, Antonio; Floresta, Giuseppe; Marrazzo, Agostino; Parenti, Carmela; Prezzavento, Orazio; Nastasi, Giovanni; Dichiara, Maria; Amata, Emanuele

    2017-08-30

    For the first time in sigma-22 ) receptor field, a quantitative structure-activity relationship (QSAR) model has been built using pK i values of the whole set of known selective σ 2 receptor ligands (548 compounds), taken from the Sigma-2 Receptor Selective Ligands Database (S2RSLDB) (http://www.researchdsf.unict.it/S2RSLDB/), through the Monte Carlo technique and employing the software CORAL. The model has been developed by using a large and structurally diverse set of compounds, allowing for a prediction of different populations of chemical compounds endpoint (σ 2 receptor pK i ). The statistical quality reached, suggested that model for pK i determination is robust and possesses a satisfactory predictive potential. The statistical quality is high for both visible and invisible sets. The screening of the FDA approved drugs, external to our dataset, suggested that sixteen compounds might be repositioned as σ 2 receptor ligands (predicted pK i ≥8). A literature check showed that six of these compounds have already been tested for affinity at σ 2 receptor and, of these, two (Flunarizine and Terbinafine) have shown an experimental σ 2 receptor pK i >7. This suggests that this QSAR model may be used as focusing screening filter in order to prospectively find or repurpose new drugs with high affinity for the σ 2 receptor, and overall allowing for an enhanced hit rate respect to a random screening. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. 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.

  6. S2RSLDB: a comprehensive manually curated, internet-accessible database of the sigma-2 receptor selective ligands.

    PubMed

    Nastasi, Giovanni; Miceli, Carla; Pittalà, Valeria; Modica, Maria N; Prezzavento, Orazio; Romeo, Giuseppe; Rescifina, Antonio; Marrazzo, Agostino; Amata, Emanuele

    2017-01-01

    Sigma (σ) receptors are accepted as a particular receptor class consisting of two subtypes: sigma-1 (σ 1 ) and sigma-22 ). The two receptor subtypes have specific drug actions, pharmacological profiles and molecular characteristics. The σ 2 receptor is overexpressed in several tumor cell lines, and its ligands are currently under investigation for their role in tumor diagnosis and treatment. The σ 2 receptor structure has not been disclosed, and researchers rely on σ 2 receptor radioligand binding assay to understand the receptor's pharmacological behavior and design new lead compounds. Here we present the sigma-2 Receptor Selective Ligands Database (S2RSLDB) a manually curated database of the σ 2 receptor selective ligands containing more than 650 compounds. The database is built with chemical structure information, radioligand binding affinity data, computed physicochemical properties, and experimental radioligand binding procedures. The S2RSLDB is freely available online without account login and having a powerful search engine the user may build complex queries, sort tabulated results, generate color coded 2D and 3D graphs and download the data for additional screening. The collection here reported is extremely useful for the development of new ligands endowed of σ 2 receptor affinity, selectivity, and appropriate physicochemical properties. The database will be updated yearly and in the near future, an online submission form will be available to help with keeping the database widely spread in the research community and continually updated. The database is available at http://www.researchdsf.unict.it/S2RSLDB.

  7. Silencing of P2Y2 receptor delays Ap4A-corneal re-epithelialization process

    PubMed Central

    Crooke, Almudena; Mediero, Aránzazu; Guzmán-Aránguez, Ana

    2009-01-01

    Purpose There are no selective antagonists for the metabotropic nucleotide P2Y2 receptor subtype. This implies that it is not possible to demonstrate the importance of such a receptor in the relevant process of corneal wound healing. Therefore, we have cloned and designed a small interference RNA (siRNA) against the rabbit P2Y2 receptor (P2Y2-R) mRNA, which clearly demonstrates the importance of this receptor in the process of wound healing triggered by nucleotides and dinucleotides both in vitro and in vivo. Methods Rabbit P2Y2-R cDNA was cloned using a combination of degenerate reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). To test the efficacy of synthesized siRNAs targeting P2Y2-R, immunocytochemistry, immunohistochemistry, and quantitative RT-PCR (qRT–PCR) assays were performed. Migration assays were performed both in vitro and in vivo by wounding the epithelium with a pipette tip and n-heptanol, respectively. These wounds were performed 72 h after siRNA transfection either in the presence or the absence of the P2Y2 agonist, 100 μM Ap4A (diadenosine tetraphosphate). Results The cloned receptor presents 93% homology compared to the human gene. Two siRNAs were designed and synthesized against a rabbit P2Y2-R sequence. After transfection (in vitro assays) or topical instillation (in vivo assays), we demonstrated P2Y2-R siRNA efficient transfection/delivery and its efficient gene silencing. Clear reduction of P2Y2-R expression was observed at both the mRNA and protein levels in corneas treated with siRNA. In vitro and in vivo migration analysis showed that the silencing process has concomitantly reduced the ability of corneal cells to close the wounds in the presence of the Ap4A. In addition, both synthesized siRNAs exert a delay effect on the Ap4A-induced migration rate in vitro. These results suggest the absence of non-specific (off-target) effects by our siRNA. Conclusions The application of P2Y2-R si

  8. Silencing of P2Y2 receptor delays Ap4A-corneal re-epithelialization process.

    PubMed

    Crooke, Almudena; Mediero, Aránzazu; Guzmán-Aránguez, Ana; Pintor, Jesús

    2009-06-11

    There are no selective antagonists for the metabotropic nucleotide P2Y(2) receptor subtype. This implies that it is not possible to demonstrate the importance of such a receptor in the relevant process of corneal wound healing. Therefore, we have cloned and designed a small interference RNA (siRNA) against the rabbit P2Y(2) receptor (P2Y(2)-R) mRNA, which clearly demonstrates the importance of this receptor in the process of wound healing triggered by nucleotides and dinucleotides both in vitro and in vivo. Rabbit P2Y(2)-R cDNA was cloned using a combination of degenerate reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). To test the efficacy of synthesized siRNAs targeting P2Y(2)-R, immunocytochemistry, immunohistochemistry, and quantitative RT-PCR (qRT-PCR) assays were performed. Migration assays were performed both in vitro and in vivo by wounding the epithelium with a pipette tip and n-heptanol, respectively. These wounds were performed 72 h after siRNA transfection either in the presence or the absence of the P2Y(2) agonist, 100 muM Ap(4)A (diadenosine tetraphosphate). The cloned receptor presents 93% homology compared to the human gene. Two siRNAs were designed and synthesized against a rabbit P2Y(2)-R sequence. After transfection (in vitro assays) or topical instillation (in vivo assays), we demonstrated P2Y(2)-R siRNA efficient transfection/delivery and its efficient gene silencing. Clear reduction of P2Y(2)-R expression was observed at both the mRNA and protein levels in corneas treated with siRNA. In vitro and in vivo migration analysis showed that the silencing process has concomitantly reduced the ability of corneal cells to close the wounds in the presence of the Ap(4)A. In addition, both synthesized siRNAs exert a delay effect on the Ap(4)A-induced migration rate in vitro. These results suggest the absence of non-specific (off-target) effects by our siRNA. The application of P2Y(2)-R siRNA has

  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. Ion Energy and Ion Flux Distributions of CF4/Ar/O2 Inductively Coupled Plasmas in a GEC Cell

    NASA Technical Reports Server (NTRS)

    Rao, M. V. V. S.; Cruden, Brett; Sharma, Surendra; Meyyappan, Meyya

    2001-01-01

    Knowledge of ion kinetics in plasma processing gas mixtures, such as CF4:Ar:O2, is important for understanding plasma assisted etching and deposition of materials. Ion energies and ion fluxes were measured in this mixture for 80:10:10, 60:20:20, and 40:30:30 mixture ratios in the pressure range of 10-50 mTorr, and at 200 and 300 W of RF power. Ions from plasma, sampled through a 10 micron orifice in the center of the lower plane electrode, were energy and mass analyzed by a combination of electrostatic energy and quadrupole mass filters. CFx(+) (x = 1 - 3), F2(+), F(+), C(+) from CF4, Ar(+) from Ar, and O2(+) and O(+) from O2, and by-product ions SiFx(+)(x = 1 - 3) from etching of quartz coupling window, COFx(+)(x = 1 - 3), CO(+), CO2(+), and OF(+) were detected. In all conditions ion flux decreases with increase of pressure but increase with increase of RF power. Ar(+) signal decreases with increase of pressure while CF3(+), which is the dominant ion at all conditions, increases with increase in pressure. The loss mechanism for Ar(+) and increase of CF3(+) is due to large cross section for Ar(+) + CF4 yields Ar + CF3(+) + F. Ion energies, which range from 15-25 eV depending on plasma operating conditions, are nearly Gaussian. By-product ion signals are higher at lower pressures indicating stronger plasma interaction with quartz window.

  11. β-Adrenergic Receptor-Mediated Cardiac Contractility is Inhibited via Vasopressin Type 1A-Receptor-Dependent Signaling

    PubMed Central

    Tilley, Douglas G.; Zhu, Weizhong; Myers, Valerie D.; Barr, Larry A.; Gao, Erhe; Li, Xue; Song, Jianliang; Carter, Rhonda L.; Makarewich, Catherine A.; Yu, Daohai; Troupes, Constantine D.; Grisanti, Laurel A.; Coleman, Ryan C.; Koch, Walter J.; Houser, Steven R.; Cheung, Joseph Y.; Feldman, Arthur M.

    2014-01-01

    Background Enhanced arginine vasopressin (AVP) levels are associated with increased mortality during end-stage human heart failure (HF), and cardiac AVP type 1A receptor (V1AR) expression becomes increased. Additionally, mice with cardiac-restricted V1AR overexpression develop cardiomyopathy and decreased β-adrenergic receptorAR) responsiveness. This led us to hypothesize that V1AR signaling regulated βAR responsiveness and in doing so contributes to HF development. Methods and Results Transaortic constriction resulted in decreased cardiac function and βAR density and increased cardiac V1AR expression, effects reversed by a V1AR-selective antagonist. Molecularly, V1AR stimulation led to decreased βAR ligand affinity, as well as βAR-induced Ca2+ mobilization and cAMP generation in isolated adult cardiomyocytes, effects recapitulated via ex vivo Langendorff analysis. V1AR-mediated regulation of βAR responsiveness was demonstrated to occur in a previously unrecognized Gq protein-independent/GRK-dependent manner. Conclusions This newly discovered relationship between cardiac V1AR and βAR may be informative for the treatment of patients with acute decompensated HF and elevated AVP. PMID:25205804

  12. Actions of alpha2 adrenoceptor ligands at alpha2A and 5-HT1A receptors: the antagonist, atipamezole, and the agonist, dexmedetomidine, are highly selective for alpha2A adrenoceptors.

    PubMed

    Newman-Tancredi, A; Nicolas, J P; Audinot, V; Gavaudan, S; Verrièle, L; Touzard, M; Chaput, C; Richard, N; Millan, M J

    1998-08-01

    This study examined the activity of chemically diverse alpha2 adrenoceptor ligands at recombinant human (h) and native rat (r) alpha2A adrenoceptors compared with 5-HT1A receptors. First, in competition binding experiments at h alpha2A and h5-HT1A receptors expressed in CHO cells, several compounds, including the antagonists 1-(2-pyrimidinyl)piperazine (1-PP), (+/-)-idazoxan, benalfocin (SKF 86466), yohimbine and RX 821,002, displayed preference for h alpha2A versus h5-HT1A receptors of only 1.4-, 3.6-, 4-, 10- and 11-fold, respectively (based on differences in pKi values). Clonidine, brimonidine (UK 14304), the benzopyrrolidine fluparoxan and the guanidines guanfacine and guanabenz exhibited intermediate selectivity (22- to 31-fold) for h alpha2A receptors. Only the antagonist atipamezole and the agonist dexmedetomidine (DMT) displayed high preference for alpha2 adrenoceptors (1290- and 91-fold, respectively). Second, the compounds were tested for their ability to induce h5-HT1A receptor-mediated G-protein activation, as indicated by the stimulation of [35S]GTPgammaS binding. All except atipamezole and RX 821,002 exhibited agonist activity, with potencies which correlated with their affinity for h5-HT1A receptors. Relative efficacies (Emax values) were 25-35% for guanabenz, guanfacine, WB 4101 and benalfocin, 50-65% for 1-PP, (+/-)-idazoxan and clonidine, and over 70% for fluparoxan, oxymetazoline and yohimbine (relative to 5-HT = 100%). Yohimbine-induced [35S]GTPgammaS binding was inhibited by the selective 5-HT1A receptor antagonist WAY 100,635. In contrast, RX 821,002 was the only ligand which exhibited antagonist activity at h5-HT1A receptors, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Atipamezole, which exhibited negligeable affinity for 5-HT1A receptors, was inactive. Third, the affinities for r alpha2A differed considerably from the affinities for h alpha2A receptors whereas the affinities for r5-HT1A differed much less from the affinities for h5-HT

  13. β2-Adrenergic Receptor Knockout Mice Exhibit A Diabetic Retinopathy Phenotype

    PubMed Central

    Jiang, Youde; Zhang, Qiuhua; Liu, Li; Tang, Jie; Kern, Timothy S.; Steinle, Jena J.

    2013-01-01

    There is considerable evidence from our lab and others for a functional link between β-adrenergic receptor and insulin receptor signaling pathways in retina. Furthermore, we hypothesize that this link may contribute to lesions similar to diabetic retinopathy in that the loss of adrenergic input observed in diabetic retinopathy may disrupt normal anti-apoptotic insulin signaling, leading to retinal cell death. Our studies included assessment of neural retina function (ERG), vascular degeneration, and Müller glial cells (which express only β1 and β2-adrenergic receptor subtypes). In the current study, we produced β2-adrenergic receptor knockout mice to examine this deletion on retinal neurons and vasculature, and to identify specific pathways through which β2-adrenergic receptor modulates insulin signaling. As predicted from our hypothesis, β2-adrenergic receptor knockout mice display certain features similar to diabetic retinopathy. In addition, loss of β2-adrenergic input resulted in an increase in TNFα, a key inhibitor of insulin receptor signaling. Increased TNFα may be associated with insulin-dependent production of the anti-apoptotic factor, Akt. Since the effects occurred in vivo under normal glucose conditions, we postulate that aspects of the diabetic retinopathy phenotype might be triggered by loss of β2-adrenergic receptor signaling. PMID:23894672

  14. Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

    PubMed

    Viswanathan, Karthik; Koh, Xiaoying; Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M; Sasisekharan, Ram

    2010-10-29

    The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.

  15. Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

    PubMed Central

    Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M.; Sasisekharan, Ram

    2010-01-01

    The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA. PMID:21060797

  16. The effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand directed chiral recognition

    PubMed Central

    Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

    2011-01-01

    The β2 adrenergic receptor2-AR) is a model system for studying the ligand recognition process in G-protein coupled receptors. Fenoterol (FEN) is a β2-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van’t Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x’)-isomers is almost entirely enthalpy controlled whereas binding of (R,x’)-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R’)-FEN was shown to selectively activate Gs protein signaling while the (S,R’)- isomer activated both Gi and Gs protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site and the global mechanism of β2-AR activation. Differences in thermodynamic parameters and non-uniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x’)-FEN stereoisomers with a different receptor conformation than the one with which the (S,x’)-isomer interacts. PMID:21618615

  17. Spectroscopy Study of Ar + CO2 Plasmas in ASTRAL.

    NASA Astrophysics Data System (ADS)

    Munoz, Jorge; Boivin, Robert; Kamar, Ola; Loch, Stuart; Ballance, Connor

    2006-10-01

    A spectroscopy study of the ASTRAL (Auburn Steady sTate Research fAciLity) helicon plasma source running Ar + CO2 gas mix is presented. ASTRAL produces Ar plasmas: ne = 10^10 to 10^13 cm-3, Te = 2 to 10 eV and Ti = 0.03 to 0.5 eV. A series of 7 large coils produce an axial magnetic field up to 1.3 kGauss. A fractional helix antenna is used to introduce rf power up to 2 kWatt. A spectrometer which features a 0.33 m Criss-Cross monochromator and a CCD camera is used for this study. Very different plasmas are produced following the relative importance of CO2 in the gas mixture. At low CO2 concentration, the plasmas are similar to those obtained with pure Ar with weak CO2, CO2^+, CO and CO^+ bands. The usual blue plasma core associated with intense Ar II transitions is observed with however a significant white glow coming from the outer plasma regions. At higher CO2 concentration, the plasma becomes essentially molecular and can be described as an intense white plasma column. Molecular dissociative processes associated with the production of strong C and O atomic lines are observed under specific plasma conditions. The atomic spectral lines are compared with ADAS modeling results. This study indicates the possible advantages of using a helicon source to control the CO2 plasma chemistry for industrial applications.

  18. Beta2- and beta3-adrenergic receptor polymorphisms and exercise hemodynamics in postmenopausal women.

    PubMed

    McCole, Steve D; Shuldiner, Alan R; Brown, Michael D; Moore, Geoffrey E; Ferrell, Robert E; Wilund, Kenneth R; Huberty, Andrea; Douglass, Larry W; Hagberg, James M

    2004-02-01

    We sought to determine whether common genetic variations at the beta2 (beta2-AR, Gln27Glu) and beta3 (beta3-AR, Trp64Arg) adrenergic receptor gene loci were associated with cardiovascular (CV) hemodynamics during maximal and submaximal exercise. CV hemodynamics were assessed in 62 healthy postmenopausal women (20 sedentary, 22 physically active, and 20 endurance athletes) during treadmill exercise at 40, 60, 80, and 100% maximal O2 uptake using acetylene rebreathing to quantify cardiac output. The beta2-AR genotype and habitual physical activity (PA) levels interacted to significantly associate with arteriovenous O2 difference (a-vDO2) during submaximal exercise (P = 0.05), with the highest submaximal exercise a-vDO2 in sedentary women homozygous for the beta2-AR Gln allele and no genotype-dependent differences in submaximal exercise a-vDO2 in physically active and athletic women. The beta2-AR genotype also was independently associated with a-vDO2 during submaximal (P = 0.004) and approximately 100% maximal O2 uptake exercise (P = 0.006), with a 1.2-2 ml/100 ml greater a-vDO2 in the Gln/Gln than in the Glu/Glu genotype women. The beta3-AR genotype, independently or interacting with habitual PA levels, was not significantly associated with any CV hemodynamic variables during submaximal or maximal exercise. Thus it appears that the beta2-AR genotype, both independently and interacting with habitual PA levels, is significantly associated with a-vDO2 during exercise in postmenopausal women, whereas the beta3-AR genotype does not appear to be associated with any maximal or submaximal exercise CV hemodynamic responses in postmenopausal women.

  19. 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.

  20. Neonatal stimulation of 5-HT(2) receptors reduces androgen receptor expression in the rat anteroventral periventricular nucleus and sexually dimorphic preoptic area.

    PubMed

    Dakin, C L; Wilson, C A; Kalló, I; Coen, C W; Davies, D C

    2008-05-01

    Masculinization of the brain is dependent upon a perinatal surge in testosterone. It also requires a transient decrease in hypothalamic 5-HT concentration and turnover and an increase in androgen receptor (AR) expression during the second postnatal week. We have previously shown that increasing 5-HT activity over this period in male or androgenized female rats feminizes their adult behaviour and also feminizes the size of their anteroventral periventricular nucleus (AVPV) and sexually dimorphic nucleus of the preoptic area (SDN-POA). To investigate the role of 5-HT in sexual differentiation of the brain, 5-HT activity was raised over postnatal days 8-16 in male, female and androgenized female rats by daily administration of the 5-HT(2) receptor agonist (-)[2,5 dimethoxy-4-iodophenyl]-2-amino propane hydrochloride [(-)DOI]. By postnatal day 18, the size of the AVPV and SDN-POA was sexually dimorphic; their sizes were feminized by (-)DOI treatment. In the absence of (-)DOI treatment, there were significantly more AR-immunoreactive cells in the AVPV of males, and in the SDN-POA of males and androgenized females, than in those of females on postnatal day 18. (-)DOI treatment reduced the number of AR-immunoreactive cells in the AVPV and SDN-POA of males and androgenized females, but not of females, by postnatal day 18. These results suggest that 5-HT(2) receptor activation can influence sexual differentiation of the brain by controlling AR expression.

  1. Sustained βAR Stimulation Mediates Cardiac Insulin Resistance in a PKA-Dependent Manner

    PubMed Central

    Denkaew, Tananat; Phosri, Sarawuth; Pinthong, Darawan; Parichatikanond, Warisara; Shimauchi, Tsukasa; Nishida, Motohiro

    2016-01-01

    Insulin resistance is a condition in which cells are defective in response to the actions of insulin in tissue glucose uptake. Overstimulation of β-adrenergic receptorsARs) leads to the development of heart failure and is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which sustained βAR stimulation affects insulin resistance in the heart are incompletely understood. In this study, we demonstrate that sustained βAR stimulation resulted in the inhibition of insulin-induced glucose uptake, and a reduction of insulin induced glucose transporter (GLUT)4 expression that were mediated by the β2AR subtype in cardiomyocytes and heart tissue. Overstimulation of β2AR inhibited the insulin-induced translocation of GLUT4 to the plasma membrane of cardiomyocytes. Additionally, βAR mediated cardiac insulin resistance by reducing glucose uptake and GLUT4 expression via the cAMP-dependent and protein kinase A-dependent pathways. Treatment with β-blockers, including propranolol and metoprolol antagonized isoproterenol-mediated insulin resistance in the heart. The data in this present study confirm a critical role for protein kinase A in βAR-mediated insulin resistance. PMID:26652903

  2. 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.

  3. 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

  4. Anatomical relationships between serotonin 5-HT2A and dopamine D2 receptors in living human brain.

    PubMed

    Ishii, Tatsuya; Kimura, Yasuyuki; Ichise, Masanori; Takahata, Keisuke; Kitamura, Soichiro; Moriguchi, Sho; Kubota, Manabu; Zhang, Ming-Rong; Yamada, Makiko; Higuchi, Makoto; Okubo, Yoshinori; Suhara, Tetsuya

    2017-01-01

    Seven healthy volunteers underwent PET scans with [18F]altanserin and [11C]FLB 457 for 5-HT2A and D2 receptors, respectively. As a measure of receptor density, a binding potential (BP) was calculated from PET data for 76 cerebral cortical regions. A correlation matrix was calculated between the binding potentials of [18F]altanserin and [11C]FLB 457 for those regions. The regional relationships were investigated using a bicluster analysis of the correlation matrix with an iterative signature algorithm. We identified two clusters of regions. The first cluster identified a distinct profile of correlation coefficients between 5-HT2A and D2 receptors, with the former in regions related to sensorimotor integration (supplementary motor area, superior parietal gyrus, and paracentral lobule) and the latter in most cortical regions. The second cluster identified another distinct profile of correlation coefficients between 5-HT2A receptors in the bilateral hippocampi and D2 receptors in most cortical regions. The observation of two distinct clusters in the correlation matrix suggests regional interactions between 5-HT2A and D2 receptors in sensorimotor integration and hippocampal function. A bicluster analysis of the correlation matrix of these neuroreceptors may be beneficial in understanding molecular networks in the human brain.

  5. Clozapine Reverses Phencyclidine-Induced Desynchronization of Prefrontal Cortex through a 5-HT1A Receptor-Dependent Mechanism

    PubMed Central

    Kargieman, Lucila; Riga, Maurizio S; Artigas, Francesc; Celada, Pau

    2012-01-01

    The non-competitive NMDA receptor (NMDA-R) antagonist phencyclidine (PCP)—used as a pharmacological model of schizophrenia—disrupts prefrontal cortex (PFC) activity. PCP markedly increased the discharge rate of pyramidal neurons and reduced slow cortical oscillations (SCO; 0.15–4 Hz) in rat PFC. Both effects were reversed by classical (haloperidol) and atypical (clozapine) antipsychotic drugs. Here we extended these observations to mice brain and examined the potential involvement of 5-HT2A and 5-HT1A receptors (5-HT2AR and 5-HT1AR, respectively) in the reversal by clozapine of PCP actions. Clozapine shows high in vitro affinity for 5-HT2AR and behaves as partial agonist in vivo at 5-HT1AR. We used wild-type (WT) mice and 5-HT1AR and 5-HT2AR knockout mice of the same background (C57BL/6) (KO-1A and KO-2A, respectively). Local field potentials (LFPs) were recorded in the PFC of WT, KO-1A, and KO-2A mice. PCP (10 mg/kg, intraperitoneally) reduced SCO equally in WT, KO-2A, and KO-1A mice (58±4%, 42±7%, and 63±7% of pre-drug values, n=23, 13, 11, respectively; p<0.0003). Clozapine (0.5 mg/kg, intraperitoneally) significantly reversed PCP effect in WT and KO-2A mice, but not in KO-1A mice nor in WT mice pretreated with the selective 5-HT1AR antagonist WAY-100635.The PCP-induced disorganization of PFC activity does not appear to depend on serotonergic function. However, the lack of effect of clozapine in KO-1A mice and the prevention by WAY-100635 indicates that its therapeutic action involves 5-HT1AR activation without the need to block 5-HT2AR, as observed with clozapine-induced cortical dopamine release. PMID:22012474

  6. Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

    PubMed

    Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

    2006-10-01

    To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

  7. 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.

  8. A Sphingosine 1-phosphate receptor 2 selective allosteric agonist

    PubMed Central

    Satsu, Hideo; Schaeffer, Marie-Therese; Guerrero, Miguel; Saldana, Adrian; Eberhart, Christina; Hodder, Peter; Cayanan, Charmagne; Schürer, Stephan; Bhhatarai, Barun; Roberts, Ed; Rosen, Hugh; Brown, Steven J.

    2013-01-01

    Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound. PMID:23849205

  9. 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.

  10. G protein, phosphorylated-GATA4 and VEGF expression in the hearts of transgenic mice overexpressing β1- and β2-adrenergic receptors

    PubMed Central

    Tae, Hyun-Jin; Petrashevskaya, Natalia; Kim, In Hye; Park, Joon Ha; Lee, Jae-Chul; Won, Moo-Ho; Kim, Yang Hee; Ahn, Ji Hyeon; Park, Jinseu; Choi, Soo Young; Jeon, Yong Hwan

    2017-01-01

    β1- and β2-adrenergic receptors (ARs) regulate cardiac contractility, calcium handling and protein phosphorylation. The present study aimed to examine the expression levels of vascular endothelial growth factor A (VEGF-A) and several G proteins, and the phosphorylation of transcription factor GATA binding protein 4 (GATA4), by western blot analysis, using isolated hearts from 6 month-old transgenic (TG) mice that overexpress β1AR or β2AR. Cardiac contractility/relaxation and heart rate was increased in both β1AR TG and β2AR TG mouse hearts compared with wild type; however, no significant differences were observed between the β1- and β2AR TG mouse hearts. Protein expression levels of inhibitory guanine nucleotide-binding protein (Gi) 2, Gi3 and G-protein-coupled receptor kinase 2 were upregulated in both TG mice, although the upregulation of Gi2 was more prominent in the β2AR TG mice. VEGF-A expression levels were also increased in both TG mice, and were highest in the β1AR TG mice. In addition, the levels of phosphorylated-GATA4 expression were increased in β1- and β2AR TG mice. In conclusion, the present study demonstrated that cardiac contractility/relaxation and heart rate is increased in β1AR TG and β2AR TG mice, and indicated that this increase may be related to the overexpression of G proteins and G-protein-associated proteins. PMID:28487987

  11. 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

  12. Efficiencies of state and velocity-changing collisions of superthermal CN A(2)Pi with He, Ar, N(2) and O(2).

    PubMed

    Alagappan, Azhagammai; Ballingall, Iain; Costen, Matthew L; McKendrick, Kenneth G; Paterson, Grant

    2007-02-14

    Polarized laser photolysis of ICN is combined with saturated optical pumping to prepare state-selected CN Alpha(2)Pi (nu' = 4, J = 0.5, F(2), f) with a well-defined anisotropic superthermal speed distribution. The collisional evolution of the prepared state is observed by Doppler-resolved Frequency Modulated (FM) spectroscopy via stimulated emission on the CN Alpha(2)Pi-Chi(2)Sigma(+) (4,2) band. The phenomenological rate constants for removal of the prepared state in collisions with He, Ar, N(2) and O(2) are reported. The observed collision cross-sections are consistent with attractive forces contributing significantly for all the colliders with the exception of He. The collisional evolution of the prepared velocity distribution demonstrates that no significant back-transfer into the prepared level occurs, and that any elastic scattering is strongly in the forward hemisphere.

  13. Inductively coupled Cl2/Ar plasma: Experimental investigation and modeling

    NASA Astrophysics Data System (ADS)

    Efremov, A. M.; Kim, Dong-Pyo; Kim, Chang-Il

    2003-07-01

    Electrophysical and kinetic characteristics of Cl2/Ar plasma were investigated to understand the influence of the addition of Ar on the volume densities and fluxes of active particles, both neutral and charged. Our analysis combined both experimental methods and plasma modeling. It was found that addition of Ar to Cl2 leads to deformation of the electron energy distribution function and an increase of the electron mean energy due to the ``transparency'' effect. Direct electron impact dissociation of Cl2 molecules represents the main source of chlorine atoms in the plasma volume. The contributions of stepwise dissociation and ionization involving Ar metastable atoms were found to be negligible. Addition of Ar to Cl2 causes the decrease of both electron and ion densities due to a decrease in the total ionization rate and the acceleration of heterogeneous decay of charged particles.

  14. A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

    PubMed

    Arruda, Maria Augusta; Stoddart, Leigh A; Gherbi, Karolina; Briddon, Stephen J; Kellam, Barrie; Hill, Stephen J

    2017-01-01

    Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A 3 receptor (A 3 AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A 1 receptor, and β 1 and β 2 adrenoceptors (β 1 AR and β 2 AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A 3 AR for a range of classical adenosine receptor antagonists were consistent with A 3 AR pharmacology and correlated well ( R 2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β 1 AR was potently inhibited by low concentrations of the β 1 -selective antagonist CGP 20712A (pK i 9.68) but not by the β 2 -selective antagonist ICI 118551(pK i 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β 2 AR this affinity order was reversed with ICI 118551 showing the highest affinity (pK i 8.73) and CGP20712A (pK i 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A 3 AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A 3 AR (pK i ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected

  15. The effects and possible mechanism of β2AR gene expression in cardiocytes of canines with heart failure.

    PubMed

    Gong, Haibin; San, Yu; Wang, Lei; Lv, Qian; Chen, Libin

    2017-07-01

    The objective of the present study was to observe the changes of β 2 -adrenergic receptor2 AR) protein expression in a canine model of heart failure (HF), and the function of cardiocytes after transfection with Adv-β 2 AR. The canine model of chronic HF was induced by rapid right ventricular pacing and cardiocytes were isolated with collagenase II. Cardiocytes were transfected with Adv-β 2 AR to observe contractile function with a motion edge-detection system of single cells. Expression of β 2 AR protein in cardiocytes was measured by immunoblotting and the levels of intracellular cAMP were measured by ELISA. Compared with the control group (the sham group), the expression of β 2 AR protein in HF cardiocytes did not change, but the basal (1 mM Ca 2+ ) contraction amplitude percentage (1.809±0.922 vs. 1.120±0.432%, P<0.05), the maximum contraction amplitude percentage (14.855±2.377 vs. 10.784±2.675%, P<0.01) and the basal levels of intracellular cAMP (9.39±2.54 vs. 5.26±0.95 pmol/ml, n=6, P<0.05) of HF cardiocytes were significantly decreased. However, when HF cardiocytes were transfected with Adv-β 2 AR and cultured for 48 h, compared with the non-transfected group, the basal contraction amplitude percentage (0.851±0.324 vs. 1.629±0.522%, P<0.05), the maximum contraction amplitude percentage (9.260±2.208% vs. 12.205±1.437%, P<0.01) and the basal levels of intracellular cAMP (5.26±0.95 vs. 9.03±1.03 pmol/ml, n=6, P<0.05) of cardiocytes in the transfected group were significantly increased. In conclusion, the expression of β 2 AR protein in HF cardiocytes did not change, but contraction function was impaired. The moderate overexpression of β 2 AR gene in the HF cardiocytes increased the levels of intracellular cAMP and improved contraction function.

  16. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling.

    PubMed

    Beautrait, Alexandre; Paradis, Justine S; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M; Khoury, Etienne; Audet, Martin; Roux, Philippe P; Veprintsev, Dmitry B; Laporte, Stéphane A; Bouvier, Michel

    2017-04-18

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

  17. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    NASA Astrophysics Data System (ADS)

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

    2017-04-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

  18. Biological and physical controls on O2/Ar, Ar and pCO2 variability at the Western Antarctic Peninsula and in the Drake Passage

    NASA Astrophysics Data System (ADS)

    Eveleth, R.; Cassar, N.; Doney, S. C.; Munro, D. R.; Sweeney, C.

    2017-05-01

    Using simultaneous sub-kilometer resolution underway measurements of surface O2/Ar, total O2 and pCO2 from annual austral summer surveys in 2012, 2013 and 2014, we explore the impacts of biological and physical processes on the O2 and pCO2 system spatial and interannual variability at the Western Antarctic Peninsula (WAP). In the WAP, mean O2/Ar supersaturation was (7.6±9.1)% and mean pCO2 supersaturation was (-28±22)%. We see substantial spatial variability in O2 and pCO2 including sub-mesoscale/mesoscale variability with decorrelation length scales of 4.5 km, consistent with the regional Rossby radius. This variability is embedded within onshore-offshore gradients. O2 in the LTER grid region is driven primarily by biological processes as seen by the median ratio of the magnitude of biological oxygen (O2/Ar) to physical oxygen (Ar) supersaturation anomalies (%) relative to atmospheric equilibrium (2.6), however physical processes have a more pronounced influence in the southern onshore region of the grid where we see active sea-ice melting. Total O2 measurements should be interpreted with caution in regions of significant sea-ice formation and melt and glacial meltwater input. pCO2 undersaturation predominantly reflects biological processes in the LTER grid. In contrast we compare these results to the Drake Passage where gas supersaturations vary by smaller magnitudes and decorrelate at length scales of 12 km, in line with latitudinal changes in the regional Rossby radius. Here biological processes induce smaller O2/Ar supersaturations (mean (0.14±1.3)%) and pCO2 undersaturations (mean (-2.8±3.9)%) than in the WAP, and pressure changes, bubble and gas exchange fluxes drive stable Ar supersaturations.

  19. Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance.

    PubMed

    Kohli, Manish; Ho, Yeung; Hillman, David W; Van Etten, Jamie L; Henzler, Christine; Yang, Rendong; Sperger, Jamie M; Li, Yingming; Tseng, Elizabeth; Hon, Ting; Clark, Tyson; Tan, Winston; Carlson, Rachel E; Wang, Liguo; Sicotte, Hugues; Thai, Ho; Jimenez, Rafael; Huang, Haojie; Vedell, Peter T; Eckloff, Bruce W; Quevedo, Jorge F; Pitot, Henry C; Costello, Brian A; Jen, Jin; Wieben, Eric D; Silverstein, Kevin A T; Lang, Joshua M; Wang, Liewei; Dehm, Scott M

    2017-08-15

    Purpose: Androgen receptor (AR) variant AR-V7 is a ligand-independent transcription factor that promotes prostate cancer resistance to AR-targeted therapies. Accordingly, efforts are under way to develop strategies for monitoring and inhibiting AR-V7 in castration-resistant prostate cancer (CRPC). The purpose of this study was to understand whether other AR variants may be coexpressed with AR-V7 and promote resistance to AR-targeted therapies. Experimental Design: We utilized complementary short- and long-read sequencing of intact AR mRNA isoforms to characterize AR expression in CRPC models. Coexpression of AR-V7 and AR-V9 mRNA in CRPC metastases and circulating tumor cells was assessed by RNA-seq and RT-PCR, respectively. Expression of AR-V9 protein in CRPC models was evaluated with polyclonal antisera. Multivariate analysis was performed to test whether AR variant mRNA expression in metastatic tissues was associated with a 12-week progression-free survival endpoint in a prospective clinical trial of 78 CRPC-stage patients initiating therapy with the androgen synthesis inhibitor, abiraterone acetate. Results: AR-V9 was frequently coexpressed with AR-V7. Both AR variant species were found to share a common 3' terminal cryptic exon, which rendered AR-V9 susceptible to experimental manipulations that were previously thought to target AR-V7 uniquely. AR-V9 promoted ligand-independent growth of prostate cancer cells. High AR-V9 mRNA expression in CRPC metastases was predictive of primary resistance to abiraterone acetate (HR = 4.0; 95% confidence interval, 1.31-12.2; P = 0.02). Conclusions: AR-V9 may be an important component of therapeutic resistance in CRPC. Clin Cancer Res; 23(16); 4704-15. ©2017 AACR . ©2017 American Association for Cancer Research.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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

  1. The potential of AR-V7 as a therapeutic target.

    PubMed

    Uo, Takuma; Plymate, Stephen R; Sprenger, Cynthia C

    2018-03-01

    The androgen receptor variant AR-V7 is gaining attention as a potential predictive marker for as well as one of the resistance mechanisms to the most current anti-androgen receptor (AR) therapies in castration-resistant prostate cancer (CRPC). Accordingly, development of next-generation drugs that directly or indirectly target AR-V7 signaling is urgently needed. Areas covered: We review proposed mechanisms of drug resistance in relation to AR-V7 status, the mechanisms of generation of AR-V7, and its transcriptome, cistrome, and interactome. Pharmacological agents that interfere with these processes are being developed to counteract pan AR and AR-V7-specific signaling. Also, we address the current status of the preclinical and clinical studies targeting AR-V7 signaling. Expert opinion: AR-V7 is considered a true therapeutic target, however, it remains to be determined if AR-V7 is a principal driver or merely a bystander requiring heterodimerization with co-expressed full-length AR or other variants to drive CRPC progression. While untangling AR-V7 biology, multiple strategies are being developed to counteract drug resistance, including selective blockade of AR-V7 signaling as well as inhibition of pan-AR signaling. Ideally anti-AR therapies will be combined with agents preventing activation and enrichment of AR negative tumor cells that are otherwise depressed by AR activity axis.

  2. 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.

  3. Selective Vasopressin-1a receptor antagonist prevents brain edema, reduces astrocytic cell swelling and GFAP, V1aR and AQP4 expression after focal traumatic brain injury

    PubMed Central

    Marmarou, Christina R.; Liang, Xiuyin; Abidi, Naqeeb H.; Parveen, Shanaz; Taya, Keisuke; Henderson, Scott C.; Young, Harold F.; Filippidis, Aristotelis S.; Baumgarten, Clive M.

    2014-01-01

    A secondary and often lethal consequence of traumatic brain injury is cellular edema that we posit is due to astrocytic swelling caused by transmembrane water fluxes augmented by vasopressin-regulated aquaporin-4 (AQP4). We therefore tested whether vasopressin 1a receptor (V1aR) inhibition would suppress astrocyte AQP4, reduce astrocytic edema, and thereby diminish TBI-induced edematous changes. V1aR inhibition by SR49059 significantly reduced brain edema after cortical contusion injury (CCI) in rat 5 h post-injury. Injured-hemisphere brain water content (n=6 animals/group) and astrocytic area (n=3/group) were significantly higher in CCI-vehicle (80.5±0.3%; 18.0±1.4 µm2) versus sham groups (78.3±0.1%; 9.5±0.9 µm2), and SR49059 blunted CCI-induced increases in brain edema (79.0±0.2%; 9.4±0.8 µm2). CCI significantly up-regulated GFAP, V1aR and AQP4 protein levels and SR49059 suppressed injury induced up regulation (n=6/group). In CCI-vehicle, sham and CCI-SR49059 groups, GFAP was 1.58±0.04, 0.47±0.02, and 0.81±0.03, respectively; V1aR was 1.00±0.06, 0.45±0.05, and 0.46±0.09; and AQP4 was 2.03± 0.34, 0.49±0.04, and 0.92±0.22. Confocal immunohistochemistry gave analogous results. In CCI-vehicle, sham and CCI-SR49059 groups, fluorescence intensity of GFAP was 349±38, 56±5, and 244±30, respectively, V1aR was 601±71, 117.8±14, and 390±76, and AQP4 was 818±117, 158±5, and 458±55 (n=3/group). The results support that edema was predominantly cellular following CCI and documented that V1aR inhibition with SR49059 suppressed injury-induced up regulation of GFAP, V1A and AQP4, blunting edematous changes. Our findings suggest V1aR inhibitors may be potential therapeutic tools to prevent cellular swelling and provide treatment for post-traumatic brain edema. PMID:24933327

  4. Selective vasopressin-1a receptor antagonist prevents brain edema, reduces astrocytic cell swelling and GFAP, V1aR and AQP4 expression after focal traumatic brain injury.

    PubMed

    Marmarou, Christina R; Liang, Xiuyin; Abidi, Naqeeb H; Parveen, Shanaz; Taya, Keisuke; Henderson, Scott C; Young, Harold F; Filippidis, Aristotelis S; Baumgarten, Clive M

    2014-09-18

    A secondary and often lethal consequence of traumatic brain injury is cellular edema that we posit is due to astrocytic swelling caused by transmembrane water fluxes augmented by vasopressin-regulated aquaporin-4 (AQP4). We therefore tested whether vasopressin 1a receptor (V1aR) inhibition would suppress astrocyte AQP4, reduce astrocytic edema, and thereby diminish TBI-induced edematous changes. V1aR inhibition by SR49059 significantly reduced brain edema after cortical contusion injury (CCI) in rat 5h post-injury. Injured-hemisphere brain water content (n=6 animals/group) and astrocytic area (n=3/group) were significantly higher in CCI-vehicle (80.5±0.3%; 18.0±1.4 µm(2)) versus sham groups (78.3±0.1%; 9.5±0.9 µm(2)), and SR49059 blunted CCI-induced increases in brain edema (79.0±0.2%; 9.4±0.8µm(2)). CCI significantly up-regulated GFAP, V1aR and AQP4 protein levels and SR49059 suppressed injury induced up regulation (n=6/group). In CCI-vehicle, sham and CCI-SR49059 groups, GFAP was 1.58±0.04, 0.47±0.02, and 0.81±0.03, respectively; V1aR was 1.00±0.06, 0.45±0.05, and 0.46±0.09; and AQP4 was 2.03±0.34, 0.49±0.04, and 0.92±0.22. Confocal immunohistochemistry gave analogous results. In CCI-vehicle, sham and CCI-SR49059 groups, fluorescence intensity of GFAP was 349±38, 56±5, and 244±30, respectively, V1aR was 601±71, 117.8±14, and 390±76, and AQP4 was 818±117, 158±5, and 458±55 (n=3/group). The results support that edema was predominantly cellular following CCI and documented that V1aR inhibition with SR49059 suppressed injury-induced up regulation of GFAP, V1A and AQP4, blunting edematous changes. Our findings suggest V1aR inhibitors may be potential therapeutic tools to prevent cellular swelling and provide treatment for post-traumatic brain edema. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Suppression of osteoclastogenesis via α2-adrenergic receptors

    PubMed Central

    Hamajima, Kosuke; Hamamura, Kazunori; Chen, Andy; Yokota, Hiroki; Mori, Hironori; Yo, Shoyoku; Kondo, Hisataka; Tanaka, Kenjiro; Ishizuka, Kyoko; Kodama, Daisuke; Hirai, Takao; Miyazawa, Ken; Goto, Shigemi; Togari, Akifumi

    2018-01-01

    The sympathetic nervous system is known to regulate osteoclast development. However, the involvement of α2-adrenergic receptors2-ARs) in osteoclastogenesis is not well understood. In the present study, their potential role in osteoclastogenesis was investigated. Guanabenz, clonidine and xylazine were used as agonists of α2-ARs, while yohimbine and idazoxan were employed as antagonists. Using RAW264.7 pre-osteoclast and primary bone marrow cells, the mRNA expression of the osteoclast-related genes nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), tartrate-resistant acid phosphatase (TRAP) and cathepsin K was evaluated following induction with receptor activator of nuclear factor κB ligand (RANKL). TRAP staining was also conducted to assess effects on osteoclastogenesis in mouse bone marrow cells in vitro. Administration of 5–20 µM guanabenz (P<0.01, for RANKL-only treatment), 20 µM clonidine (P<0.05, for RANKL-only treatment) and 20 µM xylazine (P<0.05, for RANKL-only treatment) attenuated RANKL-induced upregulation of NFATc1, TRAP and cathepsin K mRNA. Furthermore, the reductions in these mRNAs by 10 µM guanabenz and 20 µM clonidine in the presence of RANKL were attenuated by 20 µM yohimbine or idazoxan (P<0.05). The administration of 5–20 µM guanabenz (P<0.01, for RANKL-only treatment) and 10–20 µM clonidine (P<0.05, for RANKL-only treatment) also decreased the number of TRAP-positive multi-nucleated osteoclasts. Collectively, the present study demonstrates that α2-ARs may be involved in the regulation of osteoclastogenesis. PMID:29725523

  6. Androgen receptor (AR) suppresses miRNA-145 to promote renal cell carcinoma (RCC) progression independent of VHL status

    PubMed Central

    Chen, Yuan; Sun, Yin; Rao, Qun; Xu, Hua; Li, Lei; Chang, Chawnshang

    2015-01-01

    Mutational inactivation of the VHL tumor suppressor plays key roles in the development of renal cell carcinoma (RCC), and mutated VHL-mediated VEGF induction has become the main target for the current RCC therapy. Here we identified a signal pathway of VEGF induction by androgen receptor (AR)/miRNA-145 as a new target to suppress RCC progression. Mechanism dissection revealed that AR might function through binding to the androgen receptor element (ARE) located on the promoter region of miRNA-145 to suppress p53's ability to induce expression of miRNA-145 that normally suppresses expression of HIF2α/VEGF/MMP9/CCND1. Suppressing AR with AR-shRNA or introducing exogenous miRNA-145 mimic can attenuate RCC progression independent of VHL status. MiR-145 mimic in preclinical RCC orthotopic xenograft mouse model revealed its efficacy in suppression of RCC progression. These results together identified signals by AR-suppressed miRNA-145 as a key player in the RCC progression via regulating HIF2α/VEGF/MMP9/CCND1 expression levels. Blockade of the newly identified signal by AR inhibition or miRNA-145 mimics has promising therapeutic benefit to suppress RCC progression. PMID:26304926

  7. Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes

    PubMed Central

    Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K.

    2017-01-01

    Aims Cardiac β-adrenergic receptorAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Methods and results Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. Conclusions These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous

  8. Similarity- and Substructure-Based Development of β2-Adrenergic Receptor Ligands Based on Unusual Scaffolds

    PubMed Central

    2017-01-01

    The β2-adrenergic receptor2AR) is a G protein-coupled receptor (GPCR) and a well-explored target. Here, we report the discovery of 13 ligands, ten of which are novel, of this particular GPCR. They have been identified by similarity- and substructure-based searches using multiple ligands, which were described in an earlier study, as starting points. Of note, two of the molecules used as queries here distinguish themselves from other β2AR antagonists by their unique scaffold. The molecules described in this work allow us to explore the ligand space around the previously reported molecules in greater detail, leading to insights into their structure–activity relationship. We also report experimental binding and selectivity data and putative binding modes for the novel molecules. PMID:28523097

  9. P2X7 Receptor as a Therapeutic Target.

    PubMed

    De Marchi, Elena; Orioli, Elisa; Dal Ben, Diego; Adinolfi, Elena

    2016-01-01

    P2X7 receptor is an ATP-gated cation channel that upon agonist interaction leads to cellular influx of Na(+) and Ca(2+) and efflux of K(+). P2X7 is expressed by a wide variety of cells and its activation mediates a large number of biological processes like inflammation, neuromodulation, cell death or cell proliferation and it has been associated to related pathological conditions including infectious, inflammatory, autoimmune, neurological, and musculoskeletal disorders and, in the last years, to cancer. This chapter describes structural features of P2X7, chemical properties of its agonist, antagonist, and allosteric modulators and summarizes recent advances on P2X7 receptor as therapeutic target in the aforementioned diseases. We also give an overview on recent literature suggesting that P2X7 single-nucleotide polymorphisms could be exploited as diagnostic biomarkers for the development of tailored therapies. © 2016 Elsevier Inc. All rights reserved.

  10. Reduction of lymphocyte G protein-coupled receptor kinase-2 (GRK2) after exercise training predicts survival in patients with heart failure.

    PubMed

    Rengo, Giuseppe; Galasso, Gennaro; Femminella, Grazia D; Parisi, Valentina; Zincarelli, Carmela; Pagano, Gennaro; De Lucia, Claudio; Cannavo, Alessandro; Liccardo, Daniela; Marciano, Caterina; Vigorito, Carlo; Giallauria, Francesco; Ferrara, Nicola; Furgi, Giuseppe; Filardi, Pasquale Perrone; Koch, Walter J; Leosco, Dario

    2014-01-01

    Increased cardiac G protein-coupled receptor kinase-2 (GRK2) expression has a pivotal role at inducing heart failure (HF)-related β-adrenergic receptorAR) dysfunction. Importantly, abnormalities of βAR signalling in the failing heart, including GRK2 overexpression, are mirrored in circulating lymphocytes and correlate with HF severity. Exercise training has been shown to exert several beneficial effects on the failing heart, including normalization of cardiac βAR function and GRK2 protein levels. In the present study, we evaluated whether lymphocyte GRK2 levels and short-term changes of this kinase after an exercise training programme can predict long-term survival in HF patients. For this purpose, we prospectively studied 193 HF patients who underwent a 3-month exercise training programme. Lymphocyte GRK2 protein levels, plasma N-terminal pro-brain natriuretic peptide, and norepinephrine were measured at baseline and after training along with clinical and functional parameters (left ventricular ejection fraction, NYHA class, and peak-VO2). Cardiac-related mortality was evaluated during a mean follow-up period of 37 ± 20 months. Exercise was associated with a significant reduction of lymphocyte GRK2 protein levels (from 1.29 ± 0.52 to 1.16 ± 0.65 densitometric units, p < 0.0001). Importantly, exercise related changes of GRK2 (delta values) robustly predicted survival in our study population. Interestingly, HF patients who did not show reduced lymphocyte GRK2 protein levels after training presented the poorest outcome. Our data offer the first demonstration that changes of lymphocyte GRK2 after exercise training can strongly predict outcome in advanced HF.

  11. Reduction of high-affinity beta2-adrenergic receptor binding by hyperforin and hyperoside on rat C6 glioblastoma cells measured by fluorescence correlation spectroscopy.

    PubMed

    Prenner, Lars; Sieben, Anne; Zeller, Karin; Weiser, Dieter; Häberlein, Hanns

    2007-05-01

    Beta-adrenergic receptors (beta-AR) are potential targets for antidepressants. Desensitization and downregulation of beta-AR are discussed as possible modes of action for antidepressants. We have investigated the effects of hyperforin and hyperoside, compounds with potentially antidepressant activity from St. John's Wort, on the binding behavior and dynamics of beta2-AR in living rat C6 glioblastoma cells, compared to desipramine (desmethylimipramine; DMI) by means of fluorescence correlation spectroscopy (FCS) and fluorescence microscopy. FCS-binding studies with the fluorescently labeled ligand Alexa532-noradrenaline (Alexa532-NA) binding to beta2-AR of C6 cells showed a significant reduction in total beta2-AR binding after preincubation with hyperforin and hyperoside for 3 days, respectively, which was also found for DMI. This was mainly observed in high-affinity receptor-ligand complexes with hindered lateral mobility (D2 = 1.1 (+/-0.4) microm2/s) in the biomembrane. However, internalization of beta2-AR was found neither in z-scans of these C6 cells nor in HEK 293 cells stably transfected with GFP-tagged beta2-adrenergic receptors (beta2AR-GFP) after incubation up to 6 days with either DMI, hyperforin, or hyperoside. Thus, under these conditions reduction of beta2-AR binding was not mediated by receptor internalization. Additionally, preincubation of C6 cells with DMI, hyperforin, and hyperoside led to a loss of second messenger cAMP after beta2-adrenergic stimulating conditions with terbutaline. Our current results indicate that hyperforin and hyperoside from St. John's Wort, as well as DMI, reduce beta2-adrenergic sensitivity in C6 cells, emphasizing the potential usefulness of St. John's Wort dry extracts in clinical treatment of depressive symptoms.

  12. Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

    PubMed Central

    Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

    2011-01-01

    Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

  13. 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

  14. An Anti-Parkinson’s Disease Drug via Targeting Adenosine A2A Receptor Enhances Amyloid-β Generation and γ-Secretase Activity

    PubMed Central

    Li, Xiaohang; Wang, Xin; Zhou, Yue; Yang, Wenjuan; Chen, Ming; Zhao, Jian; Pei, Gang

    2016-01-01

    γ-secretase mediates the intramembranous proteolysis of amyloid precursor protein (APP) and determines the generation of Aβ which is associated with Alzheimer’s disease (AD). Here we identified that an anti-Parkinson’s disease drug, Istradefylline, could enhance Aβ generation in various cell lines and primary neuronal cells of APP/PS1 mouse. Moreover, the increased generation of Aβ42 was detected in the cortex of APP/PS1 mouse after chronic treatment with Istradefylline. Istradefylline promoted the activity of γ-secretase which could lead to increased Aβ production. These effects of Istradefylline were reduced by the knockdown of A2AR but independent of A2AR-mediated G protein- or β-arrestin-dependent signal pathway. We further observed that A2AR colocalized with γ-secretase in endosomes and physically interacted with the catalytic subunit presenilin-1 (PS1). Interestingly, Istradefylline attenuated the interaction in time- and dosage-dependent manners. Moreover the knockdown of A2AR which in theory would release PS1 potentiated both Aβ generation and γ-secretase activity. Thus, our study implies that the association of A2AR could modulate γ-secretase activity. Istradefylline enhance Aβ generation and γ-secretase activity possibly via modulating the interaction between A2AR and γ-secretase, which may bring some undesired effects in the central nervous system (CNS). PMID:27835671

  15. AmeriFlux US-AR2 ARM USDA UNL OSU Woodward Switchgrass 2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Billesbach, Dave; Bradford, James

    This is the AmeriFlux version of the carbon flux data for the site US-AR2 ARM USDA UNL OSU Woodward Switchgrass 2. Site Description - The ARM USDA UNL OSU Woodward Switchgrass 2 tower is located on public land owned by the USDA-ARS Southern Plains Range Research Station in Woodward, Oklahoma. The site is on a former wheat field that is in the process of changing to switchgrass. A companion site (ARM USDA UNL OSU Woodward Switchgrass 1) is on a former native prairie. Previous wheat was planted in Fall 2008. In Spring 2009, herbicide was applied to kill the wheatmore » prior to switchgrass planting. Later in the year, the site was sprayed with post-emergence herbicide. In 2010, fertilization occurred before herbicide was sprayed for broadleaf control.« less

  16. Atomic alignment effect on reactivity and on product alignment in the energy-transfer reaction of oriented Ar (3P2, 4s [3/2]2, M(J) = 2) + Kr (4p6, 1S0) → Ar (3p6, 1S0) + Kr (5p [3/2]2).

    PubMed

    Ohoyama, H

    2015-03-12

    Steric effect for the formation of Kr (5p [3/2]₂) in the energy transfer reaction of Ar (³P₂, 4s [3/2]₂) + Kr has been studied by using an oriented Ar (³P₂, 4s [3/2]₂, M(J) = 2) beam at a collision energy of ∼0.09 eV. The emission intensity of Kr (5p [3/2]₂) is ca. 2 times enhanced when the angular momentum (J(Ar)) of Ar (³P₂) is aligned perpendicular to the relative velocity vector (v(R)). In addition, the Kr (5p [3/2]₂) emission is highly polarized parallel to v(R) (I(∥)/I(⊥) ∼ 1.2) when JAr is aligned perpendicular to v(R). The observed polarization moments indicate that the alignment of the unpaired Ar (3p) orbital of Ar (³P₂) to v(R), (Σ (|L′| = 0), Π (|L′| = 1)), dominates the energy transfer probability (σ(Π)(∥): σ(Σ)(∥): σ(Π)(⊥): σ(Σ)(⊥) = 0.49:1.33:0.55:1.23) and also the alignment of the Kr (5p) orbital of Kr (5p [3/2]₂) to v(R): the Σ-configuration of the Ar (3p) orbital leads to the parallel alignment (Σ-configuration) of the Kr(5p) orbital to v(R), conversely, the Π-configuration of Ar (3p) orbital leads to the perpendicular alignment (Π-configuration) of the Kr(5p) orbital. In addition, the selectivity of the alignment of the Kr (5p) orbital turns out to vary from perpendicular to parallel as the collision energy increases after a threshold down to 0.03 eV.

  17. Expression and localization of the AT1 and AT2 angiotensin II receptors and α1A and α1D adrenergic receptors in aorta of hypertensive and diabetic rats.

    PubMed

    Rodríguez, Jessica Edith; Romero-Nava, Rodrigo; Reséndiz-Albor, Aldo Arturo; Rosales-Cruz, Erika; Hong, Enrique; Huang, Fengyang; Villafaña, Santiago

    2017-01-01

    Hypertension and diabetes are multifactorial diseases that frequently coexist and exacerbate each another. During the development of diabetes, the impairment of noradrenergic and renin-angiotensin systems has been reported in the response mediated by α 1 -AR and AT 1 receptors. Although their participation in the development of cardiovascular complications is still controversial, some studies have found increased or diminished response to the vasoconstrictive effect of noradrenaline or angiotensin II in a time-dependent manner of diabetes. Thus, the aim of this work was to investigate the possible changes in the expression or localization of α 1 -AR (α 1A and α 1D ) and angiotensin II receptors (AT 1 and AT 2 ) in aorta of rats after 4 weeks of the onset of diabetes. In order to be able to examine the expression of these receptors, immunofluorescence procedure was performed in tunica intima and tunica media of histological sections of aorta. Fluorescence was detected by a confocal microscopy. Our results showed that the receptors are expressed in both tunics, where adrenergic receptors have a higher density in tunica intima and tunica media of SHR compared with WKY; meanwhile, the expression of angiotensin II receptors is not modified in both groups of rats. On the other hand, the results showed that diabetes produced an increase or a decrease in the expression of receptors that is not associated to a specific type of receptor, vascular region, or strain of rat. In conclusion, diabetes and hypertension modify the expression of the receptors in tunica intima and tunica media of aorta in a different way.

  18. Agonist-induced modulation of inverse agonist efficacy at the beta 2-adrenergic receptor.

    PubMed

    Chidiac, P; Nouet, S; Bouvier, M

    1996-09-01

    Sustained stimulation of several G protein-coupled receptors is known to lead to a reduction in the signaling efficacy. This phenomenon, named agonist-induced desensitization, has been best studied for the beta 2-adrenergic receptor (AR) and is characterized by a decreased efficacy of beta-adrenergic agonists to stimulate the adenylyl cyclase activity. Recently, several beta-adrenergic ligands were found to inhibit the spontaneous agonist-independent activity of the beta 2AR. These compounds, termed inverse agonists, have different inhibitory efficacies, ranging from almost neutral antagonists to full inverse agonists. The current study was undertaken to determine whether, as is the case for agonists, desensitization can affect the efficacies of inverse agonists. Agonist-promoted desensitization of the human beta 2AR expressed in Sf9 cells potentiated the inhibitory actions of the inverse agonists, with the extent of the potentiation being inversely proportional to their intrinsic activity. For example, desensitization increased the inhibitory action of the weak inverse agonist labetalol by 29%, whereas inhibition of the spontaneous activity by the strong inverse agonist timolol was not enhanced by the desensitizing stimuli. Interestingly, dichloroisoproterenol acted stochastically as either a weak partial agonist or a weak inverse agonist in control conditions but always behaved as an inverse agonist after desensitization. These data demonstrate that like for agonists, the efficacies of inverse agonists can be modulated by a desensitizing treatment. Also, the data show that the initial state of the receptor can determine whether a ligand behaves as a partial agonist or an inverse agonist.

  19. 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

  20. The Sigma-22) Receptor: A Novel Protein for the Imaging and Treatment of Cancer

    PubMed Central

    Mach, Robert H.; Zeng, Chenbo; Hawkins, William G.

    2014-01-01

    The sigma-22) receptor is an important target for the development of molecular probes in oncology because of its 10-fold higher density in proliferating tumor cells than in quiescent tumor cells, and the observation that σ2 receptor agonists are able to kill tumor cells via apoptotic and non-apoptotic mechanisms. Although recent evidence indicates the σ2 receptor binding site is localized within the progesterone receptor membrane component 1 (PGRMC1), most information regarding this protein has been obtained using either radiolabeled or fluorescent receptor-based probes, and from biochemical analysis of the effect of σ2 selective ligands on cells grown in culture. This article reviews the development of σ2 receptor ligands, and presents an overview of how they have been used in vitro and in vivo to increase our understanding of the role of the σ2 receptor in cancer and proliferation. PMID:23734634

  1. Lack of β2-AR Increases Anxiety-Like Behaviors and Rewarding Properties of Cocaine.

    PubMed

    Zhu, Huiwen; Liu, Zhiyuan; Zhou, Yiming; Yin, Xuming; Xu, Bo; Ma, Lan; Liu, Xing

    2017-01-01

    It is well known that β-adrenoceptors (β-ARs) play a critical role in emotional arousal and stressful events, but the specific contributions of the β2-AR subtype to the psychological disorders are largely unknown. To investigate whether β2-AR are involved in anxiety-like behavior and reward to addictive drugs, we conducted a series of behavioral tests on β2-AR knock-out (KO) mice. β2-AR KO mice exhibited increased preference for the dark compartment and closed arm in tests of Light/Dark box and elevated plus maze, indicating that β2-AR deletion elevates level of anxiety or innate fear. β2-AR KO mice also showed decreased immobility in tail suspension test (TST), suggesting that β2-AR deletion inhibits depression-like behavior. Interestingly, β2-AR ablation did not change basal locomotion but significantly increased locomotor activity induced by acute cocaine administration. β2-AR KO mice showed enhanced place preference for cocaine, which could be attenuated by β1-selective AR antagonist betaxolol. Consistently, β2-AR agonist suppressed cocaine-conditioned place preference (CPP). These data indicate that β2-AR deletion enhances acute response and reward to cocaine. Our results suggest that β2-AR regulates anxiety level, depression-like behavior and hedonic properties of cocaine, implicating that β2-AR are the potential targets for the treatment of emotional disorders and cocaine addiction.

  2. A disease-associated mutation in the adhesion GPCR BAI2 (ADGRB2) increases receptor signaling activity.

    PubMed

    Purcell, Ryan H; Toro, Camilo; Gahl, William A; Hall, Randy A

    2017-12-01

    Mutations in G protein-coupled receptors (GPCRs) that increase constitutive signaling activity can cause human disease. A de novo C-terminal mutation (R1465W) in the adhesion GPCR BAI2 (also known as ADGRB2) was identified in a patient suffering from progressive spastic paraparesis and other neurological symptoms. In vitro studies revealed that this mutation strongly increases the constitutive signaling activity of an N-terminally cleaved form of BAI2, which represents the activated form of the receptor. Further studies dissecting the mechanism(s) underling this effect revealed that wild-type BAI2 primarily couples to Gα z , with the R1465W mutation conferring increased coupling to Gα i . The R1465W mutation also increases the total and surface expression of BAI2. The mutation has no effect on receptor binding to β-arrestins, but does perturb binding to the endocytic protein endophilin A1, identified here as a novel interacting partner for BAI2. These studies provide new insights into the signaling capabilities of the adhesion GPCR BAI2/ADGRB2 and shed light on how an apparent gain-of-function mutation to the receptor's C-terminus may lead to human disease. © 2017 Wiley Periodicals, Inc.

  3. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2-8) in Normal and Cancerous Breast and Prostate Cells.

    PubMed

    Hu, Dong Gui; McKinnon, Ross A; Hulin, Julie-Ann; Mackenzie, Peter I; Meech, Robyn

    2016-12-27

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2-8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer ( PCGEM1 , PEG3 , EPHA3 , and EFNB2 ) or other types of human cancers ( TOX3 , ST8SIA4 , and SLITRK3 ), and genes that are diagnostic/prognostic biomarkers of prostate cancer ( GRINA3 , and BCHE ).

  4. The Elusive Palladium-Diazo Adduct Captured: Synthesis, Isolation and Structural Characterization of [(ArNHC-PPh2 )Pd(η2 -N2 C(Ph)CO2 Et)].

    PubMed

    Rull, Silvia G; Álvarez, Eleuterio; Fructos, Manuel R; Belderrain, Tomás R; Pérez, Pedro J

    2017-06-07

    The first example of a diazo palladium adduct is reported. The complexes [(ArNHC-PPh 2 )M(η 2 -N 2 C(Ph)CO 2 Et)] (M=Ni, 3; M=Pd, 4; ArNHC-PPh 2 =3-(2,6-diisopropylphenyl)-1-[(diphenylphosphino)ethyl]imidazol-2-ylidene) were prepared by ligand exchange with styrene-coordinated precursors [(ArNHC-PPh 2 )M(styrene)] (M=Ni, 1; M=Pd, 2). Complex 4 was fully characterized, including X-ray analyses; this constitutes the first example of a diazo adduct compound with palladium, thereby closing the gap between Groups 8 and 10 regarding this type of compounds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. TNF Receptor 2 Makes Tumor Necrosis Factor a Friend of Tumors

    PubMed Central

    Sheng, Yuqiao; Li, Feng; Qin, Zhihai

    2018-01-01

    Tumor necrosis factor (TNF) is widely accepted as a tumor-suppressive cytokine via its ubiquitous receptor TNF receptor 1 (TNFR1). The other receptor, TNFR2, is not only expressed on some tumor cells but also on suppressive immune cells, including regulatory T cells and myeloid-derived suppressor cells. In contrast to TNFR1, TNFR2 diverts the tumor-inhibiting TNF into a tumor-advocating factor. TNFR2 directly promotes the proliferation of some kinds of tumor cells. Also activating immunosuppressive cells, it supports immune escape and tumor development. Hence, TNFR2 may represent a potential target of cancer therapy. Here, we focus on expression and role of TNFR2 in the tumor microenvironment. We summarize the recent progress in understanding how TNFR2-dependent mechanisms promote carcinogenesis and tumor growth and discuss the potential value of TNFR2 in cancer treatment. PMID:29892300

  6. MT1 and MT2 Melatonin Receptors: A Therapeutic Perspective

    PubMed Central

    Liu, Jiabei; Clough, Shannon J.; Hutchinson, Anthony J.; Adamah-Biassi, Ekue B.; Popovska-Gorevski, Marina; Dubocovich, Margarita L.

    2016-01-01

    Melatonin, or 5-methoxy-N-acetyltryptamine, is synthesized and released by the pineal gland and locally in the retina following a circadian rhythm, with low levels during the day and elevated levels at night. Melatonin activates two high-affinity G protein–coupled receptors, termed MT1 and MT2, to exert beneficial actions in sleep and circadian abnormality, mood disorders, learning and memory, neuroprotection, drug abuse, and cancer. Progress in understanding the role of melatonin receptors in the modulation of sleep and circadian rhythms has led to the discovery of a novel class of melatonin agonists for treating insomnia, circadian rhythms, mood disorders, and cancer. This review describes the pharmacological properties of a slow-release melatonin preparation (i.e., Circadin®) and synthetic ligands (i.e., agomelatine, ramelteon, tasimelteon), with emphasis on identifying specific therapeutic effects mediated through MT1 and MT2 receptor activation. Discovery of selective ligands targeting the MT1 or the MT2 melatonin receptors may promote the development of novel and more efficacious therapeutic agents. PMID:26514204

  7. Ar + CO2 and He + CO2 Plasmas in ASTRAL

    NASA Astrophysics Data System (ADS)

    Boivin, R. F.; Gardner, A.; Munoz, J.; Kamar, O.; Loch, S.

    2007-11-01

    Spectroscopy study of the ASTRAL helicon plasma source running Ar + CO2 and He + CO2 gas mixes is presented. ASTRAL produces plasmas with the following parameters: ne = 10^10 - 10^13 cm-3, Te = 2 - 10 eV and Ti = 0.03 - 0.5 eV, B-field <= 1.3 kGauss, rf power <= 2 kWatt. A 0.33 m scanning monochromator is used for this study. Using Ar + CO2 gas mixes, very different plasmas are observed as the concentration of CO2 is changed. At low CO2 concentration, the bluish plasma is essentially atomic and argon transitions dominate the spectra. Weak C I and O I lines are present in the 750 - 1000 nm range. At higher CO2 concentration, the plasma becomes essentially molecular and is characterized by intense, white plasma columns. Here, spectra are filled with molecular bands (CO2, CO2^+, CO and CO^+). Limited molecular dissociative excitation processes associated with the production of C I and O I emission are also observed. On the other hand, He + CO2 plasmas are different. Here, rf matches are only possible at low CO2 concentration. Under these conditions, the spectra are characterized by strong C I and O I transitions with little or no molecular bands. Strong dissociative processes observed in these plasmas can be link to the high Te associated with He plasmas. An analysis of the spectra with possible scientific and industrial applications will be presented.

  8. Alpha-2 adrenoceptors and imidazoline receptors in cardiomyocytes mediate counterbalancing effect of agmatine on NO synthesis and intracellular calcium handling.

    PubMed

    Maltsev, Alexander V; Kokoz, Yuri M; Evdokimovskii, Edward V; Pimenov, Oleg Y; Reyes, Santiago; Alekseev, Alexey E

    2014-03-01

    Evidence suggests that intracellular Ca(2+) levels and contractility of cardiomyocytes can be modulated by targeting receptors other than already identified adrenergic or non-adrenergic sarcolemmal receptors. This study uncovers the presence in myocardial cells of adrenergic α22-AR) and imidazoline I1 (I1R) receptors. In isolated left ventricular myocytes generating stationary spontaneous Ca(2+) transients in the absence of triggered action potentials, the prototypic agonist of both receptors agmatine can activate corresponding signaling cascades with opposing outcomes on nitric oxide (NO) synthesis and intracellular Ca(2+) handling. Specifically, activation of α2-AR signaling through PI3 kinase and Akt/protein kinase B stimulates NO production and abolishes Ca(2+) transients, while targeting of I1R signaling via phosphatidylcholine-specific phospholipase C (PC-PLC) and protein kinase C (PKC) suppresses NO synthesis and elevates averaged intracellular Ca(2+). We identified that endothelial NO synthase (eNOS) is a major effector for both signaling cascades. According to the established eNOS transitions between active (Akt-dependent) and inactive (PKC-dependent) conformations, we suggest that balance between α2-AR and I1R signaling pathways sets eNOS activity, which by defining operational states of myocellular sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) can adjust Ca(2+) re-uptake and thereby cardiac inotropy. These results indicate that the conventional catalog of cardiomyocyte sarcolemmal receptors should be expanded by the α2-AR and I1R populations, unveiling previously unrecognized targets for endogenous ligands as well as for existing and potential pharmacological agents in cardiovascular medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Endomorphin-2: A Biased Agonist at the μ-Opioid Receptor

    PubMed Central

    Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J.; McArdle, Craig A.; Rosethorne, Elizabeth M.; Charlton, Steven J.; Krasel, Cornelius; Bailey, Christopher P.; Henderson, Graeme

    2012-01-01

    Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K+ current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K+ current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins. PMID:22553358

  10. Endomorphin-2: a biased agonist at the μ-opioid receptor.

    PubMed

    Rivero, Guadalupe; Llorente, Javier; McPherson, Jamie; Cooke, Alex; Mundell, Stuart J; McArdle, Craig A; Rosethorne, Elizabeth M; Charlton, Steven J; Krasel, Cornelius; Bailey, Christopher P; Henderson, Graeme; Kelly, Eamonn

    2012-08-01

    Previously we correlated the efficacy for G protein activation with that for arrestin recruitment for a number of agonists at the μ-opioid receptor (MOPr) stably expressed in HEK293 cells. We suggested that the endomorphins (endomorphin-1 and -2) might be biased toward arrestin recruitment. In the present study, we investigated this phenomenon in more detail for endomorphin-2, using endogenous MOPr in rat brain as well as MOPr stably expressed in HEK293 cells. For MOPr in neurons in brainstem locus ceruleus slices, the peptide agonists [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and endomorphin-2 activated inwardly rectifying K(+) current in a concentration-dependent manner. Analysis of these responses with the operational model of pharmacological agonism confirmed that endomorphin-2 had a much lower operational efficacy for G protein-mediated responses than did DAMGO at native MOPr in mature neurons. However, endomorphin-2 induced faster desensitization of the K(+) current than did DAMGO. In addition, in HEK293 cells stably expressing MOPr, the ability of endomorphin-2 to induce phosphorylation of Ser375 in the COOH terminus of the receptor, to induce association of arrestin with the receptor, and to induce cell surface loss of receptors was much more efficient than would be predicted from its efficacy for G protein-mediated signaling. Together, these results indicate that endomorphin-2 is an arrestin-biased agonist at MOPr and the reason for this is likely to be the ability of endomorphin-2 to induce greater phosphorylation of MOPr than would be expected from its ability to activate MOPr and to induce activation of G proteins.

  11. 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.

  12. Pharmacological profiles of cloned mammalian P2Y-receptor subtypes.

    PubMed

    von Kügelgen, Ivar

    2006-06-01

    it is blocked by suramin and reactive blue 2 (RB2). The P2Y12-receptor plays a crucial role in platelet aggregation as well as in inhibition of neuronal cells. It is activated by ADP and very potently by 2-methylthio-ADP. Nucleotide antagonists including N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (=cangrelor; AR-C69931MX), the nucleoside analogue AZD6140, as well as active metabolites of the thienopyridine compounds clopidogrel and prasugrel block the receptor. These P2Y12-antagonists are used in pharmacotherapy to inhibit platelet aggregation. The P2Y13-receptor is expressed in immunocytes and neuronal cells and is again activated by ADP and 2-methylthio-ADP. The 2-chloro-5-nitro pyridoxal-phosphate analogue 6-(2'-chloro-5'-nitro-azophenyl)-pyridoxal-alpha5-phosphate (MRS2211) is a selective antagonist. mRNA encoding for the human P2Y14-receptor is found in many tissues. However, a physiological role of the receptor has not yet been established. UDP-glucose and related analogues act as agonists; antagonists are not known. Finally, UDP has been reported to act on receptors for cysteinyl leukotrienes as an additional agonist--indicating a dual agonist specificity of these receptors.

  13. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    PubMed Central

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

    2017-01-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways. PMID:28416805

  14. 40 CFR 721.1240 - Benzene, (2-bromoethyl)-, ar-bromo derivatives.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Benzene, (2-bromoethyl)-, ar-bromo... Specific Chemical Substances § 721.1240 Benzene, (2-bromoethyl)-, ar-bromo derivatives. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzene...

  15. 40 CFR 721.1240 - Benzene, (2-bromoethyl)-, ar-bromo derivatives.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Benzene, (2-bromoethyl)-, ar-bromo... Specific Chemical Substances § 721.1240 Benzene, (2-bromoethyl)-, ar-bromo derivatives. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzene...

  16. 40 CFR 721.1240 - Benzene, (2-bromoethyl)-, ar-bromo derivatives.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Benzene, (2-bromoethyl)-, ar-bromo... Specific Chemical Substances § 721.1240 Benzene, (2-bromoethyl)-, ar-bromo derivatives. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzene...

  17. 40 CFR 721.1240 - Benzene, (2-bromoethyl)-, ar-bromo derivatives.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Benzene, (2-bromoethyl)-, ar-bromo... Specific Chemical Substances § 721.1240 Benzene, (2-bromoethyl)-, ar-bromo derivatives. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzene...

  18. 40 CFR 721.1240 - Benzene, (2-bromoethyl)-, ar-bromo derivatives.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Benzene, (2-bromoethyl)-, ar-bromo... Specific Chemical Substances § 721.1240 Benzene, (2-bromoethyl)-, ar-bromo derivatives. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified as benzene...

  19. 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

  20. A1 adenosine receptor-induced phosphorylation and modulation of transglutaminase 2 activity in H9c2 cells: A role in cell survival.

    PubMed

    Vyas, Falguni S; Hargreaves, Alan J; Bonner, Philip L R; Boocock, David J; Coveney, Clare; Dickenson, John M

    2016-05-01

    The regulation of tissue transglutaminase (TG2) activity by the GPCR family is poorly understood. In this study, we investigated the modulation of TG2 activity by the A1 adenosine receptor in cardiomyocyte-like H9c2 cells. H9c2 cells were lysed following stimulation with the A1 adenosine receptor agonist N(6)-cyclopentyladenosine (CPA). Transglutaminase activity was determined using an amine incorporating and a protein cross linking assay. TG2 phosphorylation was assessed via immunoprecipitation and Western blotting. The role of TG2 in A1 adenosine receptor-induced cytoprotection was investigated by monitoring hypoxia-induced cell death. CPA induced time and concentration-dependent increases in amine incorporating and protein crosslinking activity of TG2. CPA-induced increases in TG2 activity were attenuated by the TG2 inhibitors Z-DON and R283. Responses to CPA were blocked by PKC (Ro 31-8220), MEK1/2 (PD 98059), p38 MAPK (SB 203580) and JNK1/2 (SP 600125) inhibitors and by removal of extracellular Ca(2+). CPA triggered robust increases in the levels of TG2-associated phosphoserine and phosphothreonine, which were attenuated by PKC, MEK1/2 and JNK1/2 inhibitors. Fluorescence microscopy revealed TG2-mediated biotin-X-cadaverine incorporation into proteins and proteomic analysis identified known (Histone H4) and novel (Hexokinase 1) protein substrates for TG2. CPA pre-treatment reversed hypoxia-induced LDH release and decreases in MTT reduction. TG2 inhibitors R283 and Z-DON attenuated A1 adenosine receptor-induced cytoprotection. TG2 activity was stimulated by the A1 adenosine receptor in H9c2 cells via a multi protein kinase dependent pathway. These results suggest a role for TG2 in A1 adenosine receptor-induced cytoprotection. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Draft Genome Sequence of Achromobacter sp. Strain AR476-2, Isolated from a Cellulolytic Consortium

    PubMed Central

    Kurth, Daniel; Romero, Cintia M.; Fernandez, Pablo M.; Ferrero, Marcela A.

    2016-01-01

    Achromobacter sp. AR476-2 is a noncellulolytic strain previously isolated from a cellulolytic consortium selected from samples of insect gut. Its genome sequence could contribute to the unraveling of the complex interaction of microorganisms and enzymes involved in the biodegradation of lignocellulosic biomass in nature. PMID:27340069

  2. Cocaine Self-Administration Produces a Persistent Increase in Dopamine D2High Receptors

    PubMed Central

    Briand, Lisa A.; Flagel, Shelly B.; Seeman, Philip; Robinson, Terry E.

    2008-01-01

    Cocaine addicts are reported to have decreased numbers of striatal dopamine D2 receptors. However, in rodents, repeated cocaine administration consistently produces hypersensitivity to the psychomotor activating effects of both indirect dopamine agonists, such as cocaine itself, and importantly, to direct-acting D2 receptor agonists. The current study reports a possible resolution to this long-standing paradox. The dopamine D2 receptor exists in both a low and a high affinity state, and dopamine exerts its effects via the more functionally relevant high-affinity D2 receptor (D2High). We report here that cocaine self-administration experience produces a large (approximately 150%) increase in the proportion of D2High receptors in the striatum with no change in the total number of D2 receptors, and this effect is evident both 3 and 30 days after the discontinuation of cocaine self-administration. Changes in D2High receptors would not be evident with the probes used in human (and non-human primate) imaging studies. We suggest, therefore, that cocaine addicts and animals previously treated with cocaine may be hyper-responsive to dopaminergic drugs in part because an increase in D2High receptors results in dopamine supersensitivity. This may also help explain why stimuli that increase dopamine neurotransmission, including drugs themselves, are so effective in producing relapse in individuals with a history of exposure to cocaine. PMID:18284941

  3. 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.

  4. Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2f as a clinical candidate.

    PubMed

    Aikawa, Katsuji; Asano, Moriteru; Ono, Koji; Habuka, Noriyuki; Yano, Jason; Wilson, Keith; Fujita, Hisashi; Kandori, Hitoshi; Hara, Takahito; Morimoto, Megumi; Santou, Takashi; Yamaoka, Masuo; Nakayama, Masaharu; Hasuoka, Atsushi

    2017-07-01

    We previously reported that 4-(pyrrolidin-1-yl)benzonitrile derivative 1b was a selective androgen receptor modulator (SARM) that exhibited anabolic effects on organs such as muscles and the central nervous system (CNS), but neutral effects on the prostate. From further modification, we identified that 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2a showed strong AR binding affinity with improved metabolic stabilities. Based on these results, we tried to enhance the AR agonistic activities by modifying the substituents of the 5-oxopyrrolidine ring. As a consequence, we found that 4-[(2S,3S)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl]-2-(trifluoromethyl)benzonitrile (2f) had ideal SARM profiles in Hershberger assay and sexual behavior induction assay. Furthermore, 2f showed good pharmacokinetic profiles in rats, dogs, monkeys, excellent nuclear selectivity and acceptable toxicological profiles. We also determined its binding mode by obtaining the co-crystal structures with AR. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. 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

  6. Heterologous desensitization of cardiac β-adrenergic signal via hormone-induced βAR/arrestin/PDE4 complexes.

    PubMed

    Shi, Qian; Li, Minghui; Mika, Delphine; Fu, Qin; Kim, Sungjin; Phan, Jason; Shen, Ao; Vandecasteele, Gregoire; Xiang, Yang K

    2017-05-01

    Cardiac β-adrenergic receptorAR) signalling is susceptible to heterologous desensitization by different neurohormonal stimuli in clinical conditions associated with heart failure. We aim to examine the underlying mechanism of cross talk between βARs and a set of G-protein coupled receptors (GPCRs) activated by hormones/agonists. Rat ventricular cardiomyocytes were used to determine heterologous phosphorylation of βARs under a series of GPCR agonists. Activation of Gs-coupled dopamine receptor, adenosine receptor, relaxin receptor and prostaglandin E2 receptor, and Gq-coupled α1 adrenergic receptor and angiotensin II type 1 receptor promotes phosphorylation of β1AR and β2AR at putative protein kinase A (PKA) phosphorylation sites; but activation of Gi-coupled α2 adrenergic receptor and activation of protease-activated receptor does not. The GPCR agonists that promote β2AR phosphorylation effectively inhibit βAR agonist isoproterenol-induced PKA phosphorylation of phospholamban and contractile function in ventricular cardiomyocytes. Heterologous GPCR stimuli have minimal to small effect on isoproterenol-induced β2AR activation and G-protein coupling for cyclic adenosine monophosphate (cAMP) production. However, these GPCR stimuli significantly promote phosphorylation of phosphodiesterase 4D (PDE4D), and recruit PDE4D to the phosphorylated β2AR in a β-arrestin 2 dependent manner without promoting β2AR endocytosis. The increased binding between β2AR and PDE4D effectively hydrolyzes cAMP signal generated by subsequent stimulation with isoproterenol. Mutation of PKA phosphorylation sites in β2AR, inhibition of PDE4, or genetic ablation of PDE4D or β-arrestin 2 abolishes this heterologous inhibitory effect. Ablation of β-arrestin 2 or PDE4D gene also rescues β-adrenergic stimuli-induced myocyte contractile function. These data reveal essential roles of β-arrestin 2 and PDE4D in a common mechanism for heterologous desensitization of cardiac βARs

  7. Positive association between a DNA sequence variant in the serotonin 2A receptor gene and schizophrenia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Inayama, Y.; Yoneda, H.; Sakai, T.

    Sixty-two patients with schizophrenia and 96 normal controls were investigated for genetic association with restriction fragment length polymorphisms (RFLPs) in the serotonin receptor genes. A positive association between the serotonin 2A receptor gene (HTR2A) and schizophrenia was found, but not between schizophrenia and the serotonin 1A receptor gene. The positive association we report here would suggest that the DNA region with susceptibility to schizophrenia lies in the HTR2A on the long arm of chromosome 13. 15 refs., 2 tabs.

  8. Isolation and characterization of vB_ArS-ArV2 - first Arthrobacter sp. infecting bacteriophage with completely sequenced genome.

    PubMed

    Šimoliūnas, Eugenijus; Kaliniene, Laura; Stasilo, Miroslav; Truncaitė, Lidija; Zajančkauskaitė, Aurelija; Staniulis, Juozas; Nainys, Juozas; Kaupinis, Algirdas; Valius, Mindaugas; Meškys, Rolandas

    2014-01-01

    This is the first report on a complete genome sequence and biological characterization of the phage that infects Arthrobacter. A novel virus vB_ArS-ArV2 (ArV2) was isolated from soil using Arthrobacter sp. 68b strain for phage propagation. Based on transmission electron microscopy, ArV2 belongs to the family Siphoviridae and has an isometric head (∼63 nm in diameter) with a non-contractile flexible tail (∼194×10 nm) and six short tail fibers. ArV2 possesses a linear, double-stranded DNA genome (37,372 bp) with a G+C content of 62.73%. The genome contains 68 ORFs yet encodes no tRNA genes. A total of 28 ArV2 ORFs have no known functions and lack any reliable database matches. Proteomic analysis led to the experimental identification of 14 virion proteins, including 9 that were predicted by bioinformatics approaches. Comparative phylogenetic analysis, based on the amino acid sequence alignment of conserved proteins, set ArV2 apart from other siphoviruses. The data presented here will help to advance our understanding of Arthrobacter phage population and will extend our knowledge about the interaction between this particular host and its phages.

  9. α7 Nicotinic Agonist AR-R17779 Protects Mice against 2,4,6-Trinitrobenzene Sulfonic Acid-Induced Colitis in a Spleen-Dependent Way.

    PubMed

    Grandi, Andrea; Zini, Irene; Flammini, Lisa; Cantoni, Anna M; Vivo, Valentina; Ballabeni, Vigilio; Barocelli, Elisabetta; Bertoni, Simona

    2017-01-01

    The existence of a cholinergic anti-inflammatory pathway negatively modulating the inflammatory and immune responses in various clinical conditions and experimental models has long been postulated. In particular, the protective involvement of the vagus nerve and of nicotinic Ach receptors (nAChRs) has been proposed in intestinal inflammation and repeatedly investigated in DSS- and TNBS-induced colitis. However, the role of α 7 nAChRs stimulation is still controversial and the potential contribution of α 4 β 2 nAChRs has never been explored in this experimental condition. Our aims were therefore to pharmacologically investigate the role played by both α 7 and α 4 β 2 nAChRs in the modulation of the local and systemic inflammatory responses activated in TNBS-induced colitis in mice and to assess the involvement of the spleen in nicotinic responses. To this end, TNBS-exposed mice were sub-acutely treated with various subcutaneous doses of highly selective agonists (AR-R17779 and TC-2403) and antagonists (methyllycaconitine and dihydro-β-erythroidine) of α 7 and α 4 β 2 nAChRs, respectively, or with sulfasalazine 50 mg/kg per os and clinical and inflammatory responses were evaluated by means of biochemical, histological and flow cytometry assays. α 4 β 2 ligands evoked weak and contradictory effects, while α 7 nAChR agonist AR-R17779 emerged as the most beneficial treatment, able to attenuate several local markers of colitis severity and to revert the rise in splenic T-cells and in colonic inflammatory cytokines levels induced by haptenization. After splenectomy, AR-R17779 lost its protective effects, demonstrating for the first time that, in TNBS-model of experimental colitis, the anti-inflammatory effect of exogenous α 7 nAChR stimulation is strictly spleen-dependent. Our findings showed that the selective α 7 nAChRs agonist AR-R17779 exerted beneficial effects in a model of intestinal inflammation characterized by activation of the adaptive immune

  10. Troglitazone stimulates {beta}-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1{sub A} receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tilley, Douglas G., E-mail: douglas.tilley@jefferson.edu; Center for Translational Medicine, Thomas Jefferson University; Nguyen, Anny D.

    2010-06-11

    Peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPAR{gamma}-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPAR{gamma} activity, thus we hypothesized that a PPAR{gamma} agonist may exert physiologic effects via the angiotensin II type 1{sub A} receptor (AT1{sub A}R). In AT1{sub A}R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPAR{gamma} agonist troglitazone (Trog) enhanced AT1{sub A}R internalization and recruitment of endogenous {beta}-arrestin1/2 ({beta}arr1/2) to the AT1{sub A}R. A fluorescence assay to measure diacylglycerolmore » (DAG) accumulation showed that although Ang II induced AT1{sub A}R-G{sub q} protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of {beta}arr1/2 was selective to AT1{sub A}R as the response was prevented by an ARB- and Trog-mediated {beta}arr1/2 recruitment to {beta}1-adrenergic receptor ({beta}1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be {beta}arr2-dependent, as cardiomyocytes isolated from {beta}arr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPAR{gamma} agonist Trog acts at the AT1{sub A}R to simultaneously block G{sub q} protein activation and induce the recruitment of {beta}arr1/2, which leads to an increase in cardiomyocyte contractility.« less

  11. Nucleoporin 62 and Ca(2+)/calmodulin dependent kinase kinase 2 regulate androgen receptor activity in castrate resistant prostate cancer cells.

    PubMed

    Karacosta, Loukia G; Kuroski, Laura A; Hofmann, Wilma A; Azabdaftari, Gissou; Mastri, Michalis; Gocher, Angela M; Dai, Shuhang; Hoste, Allen J; Edelman, Arthur M

    2016-02-15

    Re-activation of the transcriptional activity of the androgen receptor (AR) is an important factor mediating progression from androgen-responsive to castrate-resistant prostate cancer (CRPC). However, the mechanisms regulating AR activity in CRPC remain incompletely understood. Ca(2+) /calmodulin-dependent kinase kinase (CaMKK) 2 was previously shown to regulate AR activity in androgen-responsive prostate cancer cells. Our objective was to further explore the basis of this regulation in CRPC cells. The abundance of CaMKK2 in nuclear fractions of androgen-responsive prostate cancer and CRPC, cells were determined by subcellular fractionation and Western blotting. CaMKK2 association with nuclear pore complexes (NPCs) and nucleoporins (Nups) including Nup62, were imaged by structured illumination and super-resolution fluorescence microscopy and co-immunoprecipitation, respectively. The abundance and subcellular localization of CaMKK2 and Nup62 in human clinical specimens of prostate cancer was visualized by immunohistochemistry. The role of Nups in the growth and viability of CRPC cells was assessed by RNA interference and cell counting. The involvement of CaMKK2 and Nup62 in regulating AR transcriptional activity was addressed by RNA interference, chromatin immunoprecipitation, androgen response element reporter assay, and Western blotting. CaMKK2 was expressed at higher levels in the nuclear fraction of CPRC C4-2 cells, than in that of androgen-responsive LNCaP cells. In C4-2 cells, CaMKK2 associated with NPCs of the nuclear envelope and physically interacted with Nup62. CaMKK2 and Nup62 demonstrated pronounced, and similar increases in both expression and perinuclear/nuclear localization in human clinical specimens of advanced prostate cancer relative to normal prostate. Knockdown of Nup62, but not of Nups, 98 or 88, reduced growth and viability of C4-2 cells. Knockdown of Nup62 produced a greater reduction of the growth and viability of C4-2 cells than of non

  12. Spectral studies of SiCl4 + N2O + Ar and SiH4 + Ar mixtures in a shock tube in 160-550 nm range

    NASA Technical Reports Server (NTRS)

    Park, C.; Fujiwara, T.

    1978-01-01

    Gases containing SiO, SiO2, SiH, and Si2 were produced in the reflected-shock region of a shock tube by heating SiCl4 + N2O + Ar and SiH4 + Ar mixtures with shock waves. Spectral absorption characteristics were measured in the 160-550 nm wavelength range and in the 2800-3600 K temperature range and compared to calculated values. The sums of the squares of electronic transition moments at equilibrium separation were derived. It was found that absorption by SiO2 and other known bands of SiO, SiH, and Si2 were too weak to be measured. The cross section of absorption by a continuum, believed due to SiH, varied from 2.5 x 10 to the -17th sq cm at 280 nm to 1.6 x 10 to the -18th sq cm at 440 nm.

  13. Adenosine 2A receptor occupancy by tozadenant and preladenant in rhesus monkeys.

    PubMed

    Barret, Olivier; Hannestad, Jonas; Alagille, David; Vala, Christine; Tavares, Adriana; Papin, Caroline; Morley, Thomas; Fowles, Krista; Lee, Hsiaoju; Seibyl, John; Tytgat, Dominique; Laruelle, Marc; Tamagnan, Gilles

    2014-10-01

    Motor symptoms in Parkinson disease (PD) are caused by a loss of dopamine input from the substantia nigra to the striatum. Blockade of adenosine 2A (A(2A)) receptors facilitates dopamine D(2) receptor function. In phase 2 clinical trials, A(2A) antagonists (istradefylline, preladenant, and tozadenant) improved motor function in PD. We developed a new A(2A) PET radiotracer, (18)F-MNI-444, and used it to investigate the relationship between plasma levels and A(2A) occupancy by preladenant and tozadenant in nonhuman primates (NHP). A series of 20 PET experiments was conducted in 5 adult rhesus macaques. PET data were analyzed with both plasma-input (Logan graphical analysis) and reference-region-based (simplified reference tissue model and noninvasive Logan graphical analysis) methods. Whole-body PET images were acquired for radiation dosimetry estimates. Human pharmacokinetic parameters for tozadenant and preladenant were used to predict A(2A) occupancy in humans, based on median effective concentration (EC(50)) values estimated from the NHP PET measurements. (18)F-MNI-444 regional uptake was consistent with A(2A) receptor distribution in the brain. Selectivity was demonstrated by dose-dependent blocking by tozadenant and preladenant. The specific-to-nonspecific ratio was superior to that of other A(2A) PET radiotracers. Pharmacokinetic modeling predicted that tozadenant and preladenant may have different profiles of A(2A) receptor occupancy in humans. (18)F-MNI-444 appears to be a better PET radiotracer for A(2A) imaging than currently available radiotracers. Assuming that EC(50) in humans is similar to that in NHP, it appears that tozadenant will provide a more sustained A(2A) receptor occupancy than preladenant in humans at clinically tested doses. © 2014 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  14. Draft Genome Sequence of Achromobacter sp. Strain AR476-2, Isolated from a Cellulolytic Consortium.

    PubMed

    Kurth, Daniel; Romero, Cintia M; Fernandez, Pablo M; Ferrero, Marcela A; Martinez, M Alejandra

    2016-06-23

    Achromobacter sp. AR476-2 is a noncellulolytic strain previously isolated from a cellulolytic consortium selected from samples of insect gut. Its genome sequence could contribute to the unraveling of the complex interaction of microorganisms and enzymes involved in the biodegradation of lignocellulosic biomass in nature. Copyright © 2016 Kurth et al.

  15. PGE2 maintains the tone of the guinea pig trachea through a balance between activation of contractile EP1 receptors and relaxant EP2 receptors

    PubMed Central

    Säfholm, J; Dahlén, S-E; Delin, I; Maxey, K; Stark, K; Cardell, L-O; Adner, M

    2013-01-01

    Background and Purpose The guinea pig trachea (GPT) is commonly used in airway pharmacology. The aim of this study was to define the expression and function of EP receptors for PGE2 in GPT as there has been ambiguity concerning their role. Experimental Approach Expression of mRNA for EP receptors and key enzymes in the PGE2 pathway were assessed by real-time PCR using species-specific primers. Functional studies of GPT were performed in tissue organ baths. Key Results Expression of mRNA for the four EP receptors was found in airway smooth muscle. PGE2 displayed a bell-shaped concentration–response curve, where the initial contraction was inhibited by the EP1 receptor antagonist ONO-8130 and the subsequent relaxation by the EP2 receptor antagonist PF-04418948. Neither EP3 (ONO-AE5-599) nor EP4 (ONO-AE3-208) selective receptor antagonists affected the response to PGE2. Expression of COX-2 was greater than COX-1 in GPT, and the spontaneous tone was most effectively abolished by selective COX-2 inhibitors. Furthermore, ONO-8130 and a specific PGE2 antibody eliminated the spontaneous tone, whereas the EP2 antagonist PF-04418948 increased it. Antagonists of other prostanoid receptors had no effect on basal tension. The relaxant EP2 response to PGE2 was maintained after long-term culture, whereas the contractile EP1 response showed homologous desensitization to PGE2, which was prevented by COX-inhibitors. Conclusions and Implications Endogenous PGE2, synthesized predominantly by COX-2, maintains the spontaneous tone of GPT by a balance between contractile EP1 receptors and relaxant EP2 receptors. The model may be used to study interactions between EP receptors. PMID:22934927

  16. Blockade of human P2X7 receptor function with a monoclonal antibody.

    PubMed

    Buell, G; Chessell, I P; Michel, A D; Collo, G; Salazzo, M; Herren, S; Gretener, D; Grahames, C; Kaur, R; Kosco-Vilbois, M H; Humphrey, P P

    1998-11-15

    A monoclonal antibody (MoAb) specific for the human P2X7 receptor was generated in mice. As assessed by flow cytometry, the MoAb labeled human blood-derived macrophage cells natively expressing P2X7 receptors and cells transfected with human P2X7 but not other P2X receptor types. The MoAb was used to immunoprecipitate the human P2X7 receptor protein, and in immunohistochemical studies on human lymphoid tissue, P2X7 receptor labeling was observed within discrete areas of the marginal zone of human tonsil sections. The antibody also acted as a selective antagonist of human P2X7 receptors in several functional studies. Thus, whole cell currents, elicited by the brief application of 2',3'-(4-benzoyl)-benzoyl-ATP in cells expressing human P2X7, were reduced in amplitude by the presence of the MoAb. Furthermore, preincubation of human monocytic THP-1 cells with the MoAb antagonized the ability of P2X7 agonists to induce the release of interleukin-1beta.

  17. Is lipid signaling through cannabinoid 2 receptors part of a protective system?

    PubMed Central

    Pacher, P.; Mechoulam, R.

    2011-01-01

    The mammalian body has a highly developed immune system which guards against continuous invading protein attacks and aims at preventing, attenuating or repairing the inflicted damage. It is conceivable that through evolution analogous biological protective systems have been evolved against non-protein attacks. There is emerging evidence that lipid endocannabinoid signaling through cannabinoid 2 (CB2) receptors may represent an example/part of such a protective system/armamentarium. Inflammation/tissue injury triggers rapid elevations in local endocannabinoid levels, which in turn regulate signaling responses in immune and other cells modulating their critical functions. Changes in endocannabinoid levels and/or CB2 receptor expressions have been reported in almost all diseases affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, auto-immune, lung disorders to pain and cancer, and modulating CB2 receptor activity holds tremendous therapeutic potential in these pathologies. While CB2 receptor activation in general mediates immunosuppressive effects, which limit inflammation and associated tissue injury in large number of pathological conditions, in some disease states activation of the CB2 receptor may enhance or even trigger tissue damage, which will also be discussed alongside the protective actions of the CB2 receptor stimulation with endocannabinoids or synthetic agonists, and the possible biological mechanisms involved in these effects. PMID:21295074

  18. Novel Nine-Exon AR Transcripts (Exon 1/Exon 1b/Exons 2–8) in Normal and Cancerous Breast and Prostate Cells

    PubMed Central

    Hu, Dong Gui; McKinnon, Ross A.; Hulin, Julie-Ann; Mackenzie, Peter I.; Meech, Robyn

    2016-01-01

    Nearly 20 different transcripts of the human androgen receptor (AR) are reported with two currently listed as Refseq isoforms in the NCBI database. Isoform 1 encodes wild-type AR (type 1 AR) and isoform 2 encodes the variant AR45 (type 2 AR). Both variants contain eight exons: they share common exons 2–8 but differ in exon 1 with the canonical exon 1 in isoform 1 and the variant exon 1b in isoform 2. Splicing of exon 1 or exon 1b is reported to be mutually exclusive. In this study, we identified a novel exon 1b (1b/TAG) that contains an additional TAG trinucleotide upstream of exon 1b. Moreover, we identified AR transcripts in both normal and cancerous breast and prostate cells that contained either exon 1b or 1b/TAG spliced between the canonical exon 1 and exon 2, generating nine-exon AR transcripts that we have named isoforms 3a and 3b. The proteins encoded by these new AR variants could regulate androgen-responsive reporters in breast and prostate cancer cells under androgen-depleted conditions. Analysis of type 3 AR-GFP fusion proteins showed partial nuclear localization in PC3 cells under androgen-depleted conditions, supporting androgen-independent activation of the AR. Type 3 AR proteins inhibited androgen-induced growth of LNCaP cells. Microarray analysis identified a small set of type 3a AR target genes in LNCaP cells, including genes known to modulate growth and proliferation of prostate cancer (PCGEM1, PEG3, EPHA3, and EFNB2) or other types of human cancers (TOX3, ST8SIA4, and SLITRK3), and genes that are diagnostic/prognostic biomarkers of prostate cancer (GRINA3, and BCHE). PMID:28035996

  19. Metabotropic Glutamate2 Receptors Play a Key Role in Modulating Head Twitches Induced by a Serotonergic Hallucinogen in Mice

    PubMed Central

    Benvenga, Mark J.; Chaney, Stephen F.; Baez, Melvyn; Britton, Thomas C.; Hornback, William J.; Monn, James A.; Marek, Gerard J.

    2018-01-01

    There is substantial evidence that glutamate can modulate the effects of 5-hydroxytryptamine2A (5-HT2A) receptor activation through stimulation of metabotropic glutamate2/3 (mGlu2/3) receptors in the prefrontal cortex. Here we show that constitutive deletion of the mGlu2 gene profoundly attenuates an effect of 5-HT2A receptor activation using the mouse head twitch response (HTR). MGlu2 and mGlu3 receptor knockout (KO) as well as age-matched ICR (CD-1) wild type (WT) mice were administered (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and observed for head twitch activity. DOI failed to produce significant head twitches in mGlu2 receptor KO mice at a dose 10-fold higher than the peak effective dose in WT or mGlu3 receptor KO mice. In addition, the mGlu2/3 receptor agonist LY379268, and the mGlu2 receptor positive allosteric modulator (PAM) CBiPES, potently blocked the HTR to DOI in WT and mGlu3 receptor KO mice. Conversely, the mGlu2/3 receptor antagonist LY341495 (10 mg/kg) increased the HTR produced by DOI (3 mg/kg) in mGlu3 receptor KO mice. Finally, the mGlu2 receptor potentiator CBiPES was able to attenuate the increase in the HTR produced by LY341495 in mGlu3 receptor KO mice. Taken together, all of these results are consistent with the hypothesis that that DOI-induced head twitches are modulated by mGlu2 receptor activation. These results also are in keeping with a critical autoreceptor function for mGlu2 receptors in the prefrontal cortex with differential effects of acute vs. chronic perturbation (e.g., constitutive mGlu2 receptor KO mice). The robust attenuation of DOI-induced head twitches in the mGlu2 receptor KO mice appears to reflect the critical role of glutamate in ongoing regulation of 5-HT2A receptors in the prefrontal cortex. Future experiments with inducible knockouts for the mGlu2 receptor and/or selective mGlu3 receptor agonists/PAMs/antagonists could provide an important tools in understanding glutamatergic modulation of prefrontal

  20. The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma.

    PubMed

    Gurd, J W; Bissoon, N

    1997-08-01

    The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.

  1. Beta2-adrenergic receptor homodimers: Role of transmembrane domain 1 and helix 8 in dimerization and cell surface expression.

    PubMed

    Parmar, Vikas K; Grinde, Ellinor; Mazurkiewicz, Joseph E; Herrick-Davis, Katharine

    2017-09-01

    Even though there are hundreds of reports in the published literature supporting the hypothesis that G protein-coupled receptors (GPCR) form and function as dimers this remains a highly controversial area of research and mechanisms governing homodimer formation are poorly understood. Crystal structures revealing homodimers have been reported for many different GPCR. For adrenergic receptors, a potential dimer interface involving transmembrane domain 1 (TMD1) and helix 8 (H8) was identified in crystal structures of the beta 1 -adrenergic (β 1 -AR) and β 2 -AR. The purpose of this study was to investigate a potential role for TMD1 and H8 in dimerization and plasma membrane expression of functional β 2 -AR. Charged residues at the base of TMD1 and in the distal portion of H8 were replaced, singly and in combination, with non-polar residues or residues of opposite charge. Wild type and mutant β 2 -AR, tagged with YFP and expressed in HEK293 cells, were evaluated for plasma membrane expression and function. Homodimer formation was evaluated using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and fluorescence correlation spectroscopy. Amino acid substitutions at the base of TMD1 and in the distal portion of H8 disrupted homodimer formation and caused receptors to be retained in the endoplasmic reticulum. Mutations in the proximal region of H8 did not disrupt dimerization but did interfere with plasma membrane expression. This study provides biophysical evidence linking a potential TMD1/H8 interface with ER export and the expression of functional β 2 -AR on the plasma membrane. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Developmental Changes is Expression of Beta-Adrenergic Receptors in Cultures of C2C12 Skeletal Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, K. Y.; Vaughn, J. R.

    2000-01-01

    beta-Adrenergic receptor (bAR) agonists have been reported to modulate growth in several mammalian and avian species, and bAR agonists presumably exert their physiological action on skeletal muscle cells through this receptor. Because of the importance of bAR regulation on muscle protein metabolism in muscle cells, the objectives of this study were to determine the developmental expression pattern of the bAR population in C2C12 skeletal muscle cells, and to analyze changes in both the quantity and isoform expression of the major muscle protein, myosin. The number of bAR in mononucleated C2C12 cells was approximately 8,000 bAR per cell, which is comparable with the population reported in several other nonmuscle cell types. However, the bar population increased after myoblast fusion to greater than 50,000 bAR per muscle cell equivalent. The reasons for this apparent over-expression of bAR in C2C12 cells is not known. The quantity of myosin also increased after C2C12 myoblast fusion, but the quantity of myosin was less than that reported in primary muscle cell cultures. Finally, at least five different isoforms of myosin heavy chain could be resolved in C2C12 cells, and three of these exhibited either increased or decreased developmental regulation relative to the others. Thus, C2C12 myoblasts undergo developmental regulation of bAR population and myosin heavy chain isoform expression.

  3. Male hormones activate EphA2 to facilitate Kaposi’s sarcoma-associated herpesvirus infection: Implications for gender disparity in Kaposi’s sarcoma

    PubMed Central

    Deng, Zhaohui; Liang, Deguang; Zhou, Xin; Sun, Rui

    2017-01-01

    There is increasing consensus that males are more vulnerable than females to infection by several pathogens. However, the underlying mechanism needs further investigation. Here, it was showed that knockdown of androgen receptor (AR) expression or pre-treatment with 5α-dihydrotestosterone, the AR agonist, led to a considerably dysregulated Kaposi’s sarcoma-associated herpesvirus (KSHV) infection. In endothelial cells, membrane-localized AR promoted the endocytosis and nuclear trafficking of KSHV. The AR interacted with ephrin receptor A2 (EphA2) and increased its phosphorylation at residue Ser897, which was specifically upregulated upon KSHV infection. This phosphorylation resulted from the AR-mediated recruitment of Src, which resulted in the activation of p90 ribosomal S6 kinase 1 (RSK1), which directly phosphorylates EphA2 at Ser897. Finally, the EphA2-mediated entry of KSHV was abolished in a Ser897Asn EphA2 mutant. Taken together, membrane-localized AR was identified as a KSHV entry factor that cooperatively activates Src/RSK1/EphA2 signaling, which subsequently promotes KSHV infection of both endothelial and epithelial cells. PMID:28957431

  4. Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.

    PubMed

    Scott, G; Deng, A; Rodriguez-Burford, C; Seiberg, M; Han, R; Babiarz, L; Grizzle, W; Bell, W; Pentland, A

    2001-12-01

    Previous studies have shown that the protease-activated receptor 2 is involved in skin pigmentation through increased phagocytosis of melanosomes by keratinocytes. Ultraviolet irradiation is a potent stimulus for melanosome transfer. We show that protease-activated receptor 2 expression in human skin is upregulated by ultraviolet irradiation. Subjects with skin type I, II, or III were exposed to two or three minimal erythema doses of irradiation from a solar simulator. Biopsies were taken from nonexposed and irradiated skin 24 and 96 h after irradiation and protease-activated receptor 2 expression was detected using immunohistochemical staining. In nonirradiated skin, protease-activated receptor 2 expression was confined to keratinocytes in the lower one-third of the epidermis. After ultraviolet irradiation protease-activated receptor 2 expression was observed in keratinocytes in the upper two-thirds of the epidermis or the entire epidermis at both time points studied. Subjects with skin type I showed delayed upregulation of protease-activated receptor 2 expression, however, compared with subjects with skin types II and III. Irradiated cultured human keratinocytes showed upregulation in protease-activated receptor 2 expression as determined by immunofluorescence microscopy and Western blotting. Cell culture supernatants from irradiated keratinocytes also exhibited a dose-dependent increase in protease-activated receptor-2 cleavage activity. These results suggest an important role for protease-activated receptor-2 in pigmentation in vivo. Differences in protease-activated receptor 2 regulation in type I skin compared with skin types II and III suggest a potential mechanism for differences in tanning in subjects with different skin types.

  5. Investigation of a van der Waals complex with C 1 symmetry: the free-jet rotational spectrum of 1,2-difluoroethane-Ar

    NASA Astrophysics Data System (ADS)

    Melandri, Sonia; Velino, Biagio; Favero, Paolo G.; Dell'Erba, Adele; Caminati, Walther

    2000-04-01

    The van der Waals complex between Ar and 1,2-difluoroethane has been investigated by free-jet absorption millimeter-wave spectroscopy in the frequency range 60-78 GHz. The analysis of the spectroscopic constants derived from the rotational spectrum allowed the determination of the dimer's structure. 1,2-Difluoroethane is in the gauche conformation and the Ar atom is in a position stabilized by the interaction with one fluorine and the two carbon atoms. The distance between Ar and the center of mass (CM) of the monomer is 3.968 Å, the angle between the Ar-CM line and the C-C bond is 65° and the dihedral angle Ar-CM-C-C is 99°. From centrifugal distortion effects the dissociation energy of the complex has been estimated to be 2.1 kJ/mol.

  6. Sox2 Is an Androgen Receptor-Repressed Gene That Promotes Castration-Resistant Prostate Cancer

    PubMed Central

    Kregel, Steven; Kiriluk, Kyle J.; Rosen, Alex M.; Cai, Yi; Reyes, Edwin E.; Otto, Kristen B.; Tom, Westin; Paner, Gladell P.; Szmulewitz, Russell Z.; Vander Griend, Donald J.

    2013-01-01

    Despite advances in detection and therapy, castration-resistant prostate cancer continues to be a major clinical problem. The aberrant activity of stem cell pathways, and their regulation by the Androgen Receptor (AR), has the potential to provide insight into novel mechanisms and pathways to prevent and treat advanced, castrate-resistant prostate cancers. To this end, we investigated the role of the embryonic stem cell regulator Sox2 [SRY (sex determining region Y)-box 2] in normal and malignant prostate epithelial cells. In the normal prostate, Sox2 is expressed in a portion of basal epithelial cells. Prostate tumors were either Sox2-positive or Sox2-negative, with the percentage of Sox2-positive tumors increasing with Gleason Score and metastases. In the castration-resistant prostate cancer cell line CWR-R1, endogenous expression of Sox2 was repressed by AR signaling, and AR chromatin-IP shows that AR binds the enhancer element within the Sox2 promoter. Likewise, in normal prostate epithelial cells and human embryonic stem cells, increased AR signaling also decreases Sox2 expression. Resistance to the anti-androgen MDV3100 results in a marked increase in Sox2 expression within three prostate cancer cell lines, and in the castration-sensitive LAPC-4 prostate cancer cell line ectopic expression of Sox2 was sufficient to promote castration-resistant tumor formation. Loss of Sox2 expression in the castration-resistant CWR-R1 prostate cancer cell line inhibited cell growth. Up-regulation of Sox2 was not associated with increased CD133 expression but was associated with increased FGF5 (Fibroblast Growth Factor 5) expression. These data propose a model of elevated Sox2 expression due to loss of AR-mediated repression during castration, and consequent castration-resistance via mechanisms not involving induction of canonical embryonic stem cell pathways. PMID:23326489

  7. 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

  8. 8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

    PubMed

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

    2013-01-01

    Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  9. NWA 7034 Martian breccia: Ar/Ar ages of ca. 1.2 to 1.4 Ga

    NASA Astrophysics Data System (ADS)

    Cohen, B. E.; Mark, D. F.; Cassata, W.; Lee, M. R.; Smith, C. L.

    2015-12-01

    NWA 7034 and its paired stones are some of the oldest and most diverse of the Martian meteorites. They are complex polymict breccias of impact, igneous, and sedimentary clasts set in a dark grey matrix [1; 2]. The rock also contains angular mineral fragments, including K-feldspar, plagioclase feldspar, and pyroxene [1; 2]. Mineral fragments are often > 1 mm wide, and clasts can be > 1 cm. This diverse breccia assemblage indicates formation via repeated impact events, supported by Rb-Sr, Sm-Nd and U-Pb ages ranging from 1.3 to 4.4 Ga [1, 2, and references therein]. In this study we investigate the distribution of ages yielded by Ar/Ar, with nine aliquots analyzed to date, and additional analyses planned. In order to analyze only single phases, chips of matrix/clasts were restricted to visibly monomict fragments < 1 mm diameter, while mineral separates were analyzed as single crystals. Cosmogenic Ar corrections are from [3]. Analyses were undertaken at SUERC and Lawrence Livermore National Laboratory, and the results pooled. The bulk of aliquots (n = 8) yielded ages of ca. 1.2-1.4 Ga indicating a major thermal event occurred at around the same time as crystallization of the Nakhlite group of meteorites. Select step ages are considerably older (> 2 Ga), supporting results of other chronometers that much older material is present in this sample. These results also demonstrate that some older fragments retained Ar during breccia formation. [1] Wittmann A. et al. (2015) Meteoritics & Planet. Sci., 50, 326-352. [2] Santos A. R. et al. (2015) GCA, 157, 56-85. [3] Cassata W. S., and Borg L. E. (2015) 46th LPSC, Abstract #2742.

  10. [2.2.2]Paracyclophane as a receptor for the cesium cation in the gas phase

    NASA Astrophysics Data System (ADS)

    Makrlík, Emanuel; Sýkora, David; Böhm, Stanislav; Vaňura, Petr

    2017-10-01

    By using electrospray ionisation mass spectrometry, it was proven experimentally that the cesium cation (Cs+) forms with [2.2.2]paracyclophane (C24H24) the cationic complex [Cs(C24H24)]+. Further, applying quantum chemical calculations, the most probable structure of the [Cs(C24H24)]+ complex was derived. In the resulting complex with a symmetry very close to C3, the 'central' cation Cs+, fully located in the cavity of the parent [2.2.2]paracyclophane ligand, is bound to all three benzene rings of [2.2.2]paracyclophane via cation-π interaction. Finally, the interaction energy, E(int), of the considered cation-π complex [Cs(C24H24)]+ was found to be -73.2 kJ/mol, confirming the formation of this fascinating complex species as well. This means that [2.2.2]paracyclophane can be considered as a receptor for the Cs+ cation in the gas phase.

  11. LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation

    PubMed Central

    Kraehenmann, Rainer; Pokorny, Dan; Aicher, Helena; Preller, Katrin H.; Pokorny, Thomas; Bosch, Oliver G.; Seifritz, Erich; Vollenweider, Franz X.

    2017-01-01

    Rationale: Stimulation of serotonin 2A (5-HT2A) receptors by lysergic acid diethylamide (LSD) and related compounds such as psilocybin has previously been shown to increase primary process thinking – an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). The main outcome variable in this study was primary index (PI), a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) rating scale. Results: LSD, compared with placebo, significantly increased primary index (p < 0.001, Bonferroni-corrected). The LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment (p < 0.05, Bonferroni-corrected), and blissful state (p < 0.05, Bonferroni-corrected) on the 5D-ASC. Both LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin. Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and psychedelic

  12. LSD Increases Primary Process Thinking via Serotonin 2A Receptor Activation.

    PubMed

    Kraehenmann, Rainer; Pokorny, Dan; Aicher, Helena; Preller, Katrin H; Pokorny, Thomas; Bosch, Oliver G; Seifritz, Erich; Vollenweider, Franz X

    2017-01-01

    Rationale: Stimulation of serotonin 2A (5-HT2A) receptors by lysergic acid diethylamide (LSD) and related compounds such as psilocybin has previously been shown to increase primary process thinking - an ontologically and evolutionary early, implicit, associative, and automatic mode of thinking which is typically occurring during altered states of consciousness such as dreaming. However, it is still largely unknown whether LSD induces primary process thinking under placebo-controlled, standardized experimental conditions and whether these effects are related to subjective experience and 5-HT2A receptor activation. Therefore, this study aimed to test the hypotheses that LSD increases primary process thinking and that primary process thinking depends on 5-HT2A receptor activation and is related to subjective drug effects. Methods: Twenty-five healthy subjects performed an audio-recorded mental imagery task 7 h after drug administration during three drug conditions: placebo, LSD (100 mcg orally) and LSD together with the 5-HT2A receptor antagonist ketanserin (40 mg orally). The main outcome variable in this study was primary index (PI), a formal measure of primary process thinking in the imagery reports. State of consciousness was evaluated using the Altered State of Consciousness (5D-ASC) rating scale. Results: LSD, compared with placebo, significantly increased primary index ( p < 0.001, Bonferroni-corrected). The LSD-induced increase in primary index was positively correlated with LSD-induced disembodiment ( p < 0.05, Bonferroni-corrected), and blissful state ( p < 0.05, Bonferroni-corrected) on the 5D-ASC. Both LSD-induced increases in primary index and changes in state of consciousness were fully blocked by ketanserin. Conclusion: LSD induces primary process thinking via activation of 5-HT2A receptors and in relation to disembodiment and blissful state. Primary process thinking appears to crucially organize inner experiences during both dreams and psychedelic

  13. 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).

  14. Fenoterol, a beta(2)-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through beta-arrestin-2 in THP-1 cell line.

    PubMed

    Wang, Wei; Xu, Ming; Zhang, You-yi; He, Bei

    2009-11-01

    To investigate the molecular mechanism and signaling pathway by which fenoterol, a beta(2)-adrenergic receptor (beta(2)-AR) agonist, produces anti-inflammatory effects. THP-1, a monocytic cell line, was used to explore the mechanism of beta(2)-AR stimulation in LPS-induced secretion of inflammatory cytokines and changes of toll-like receptors (TLRs). We labeled TLR4 and CD14 using monoclonal anti-TLR4 PE-conjugated and anti-CD14 FITC-conjugated antibodies in THP-1 cells stimulated by beta(2)-AR in the presence or absence of lipopolysaccharide (LPS) and small, interfering RNA (siRNA)-mediated knockdown of beta-arrestin-2, and then analyzed their changes in distribution by flow cytometry, Western blotting and confocal analysis. LPS-induced membrane-bound CD14, TLR4/CD14 complex levels and elevation of inflammatory cytokines were all significantly reduced by pre-incubation of fenoterol (P<0.05). However, the total level of CD14 and TLR4 was not significantly changed. Interestingly, confocal microscopy revealed redistribution of CD14 and TLR4/CD14 complex under beta(2)-AR stimulation. Furthermore, siRNA-mediated knockdown of beta-arrestin-2 eliminated the anti-inflammatory effects and redistribution of CD14 and TLR4/CD14 complex stimulated by beta(2)-AR. beta(2)-AR agonist exerts its anti-inflammatory effects by down-regulating TLR signaling in THP-1 cells, potentially resulting from beta-arrestin-2 mediated redistribution of CD14 and TLR14/CD14 complex.

  15. LeEix1 functions as a decoy receptor to attenuate LeEix2 signaling.

    PubMed

    Bar, Maya; Sharfman, Miya; Avni, Adi

    2011-03-01

    The receptors for the fungal elicitor EIX (LeEix1 and LeEix2) belong to a class of leucine-rich repeat cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Both receptors are able to bind the EIX elicitor while only the LeEix2 receptor mediates defense responses. We show that LeEix1 acts as a decoy receptor and attenuates EIX induced internalization and signaling of the LeEix2 receptor. We demonstrate that BAK1 binds LeEix1 but not LeEix2. In plants where BAK1 was silenced, LeEix1 was no longer able to attenuate plant responses to EIX, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires the kinase activity of BAK1.

  16. Human PIRH2 Enhances Androgen Receptor Signaling through Inhibition of Histone Deacetylase 1 and Is Overexpressed in Prostate Cancer

    PubMed Central

    Logan, Ian R.; Gaughan, Luke; McCracken, Stuart R. C.; Sapountzi, Vasileia; Leung, Hing Y.; Robson, Craig N.

    2006-01-01

    The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer. PMID:16914734

  17. Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase 1 and is overexpressed in prostate cancer.

    PubMed

    Logan, Ian R; Gaughan, Luke; McCracken, Stuart R C; Sapountzi, Vasileia; Leung, Hing Y; Robson, Craig N

    2006-09-01

    The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer.

  18. Targeting Sulfotransferase (SULT) 2B1b as a regulator of Cholesterol Metabolism in Prostate Cancer

    DTIC Science & Technology

    2016-10-01

    Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and...and PCa cell lines and that genetic knock down suppresses LNCaP growth and diminishes androgen receptor ( AR ) activity. It is hypothesized that SULT2B1b...knock down suppresses LNCaP growth and diminishes androgen receptor ( AR ) activity. It is hypothesized that SULT2B1b modulates PCa growth and

  19. Branched Chain RNA In Situ Hybridization for Androgen Receptor Splice Variant AR-V7 as a Prognostic Biomarker for Metastatic Castration-Sensitive Prostate Cancer.

    PubMed

    Saylor, Philip J; Lee, Richard J; Arora, Kshitij S; Deshpande, Vikram; Hu, Rong; Olivier, Kara; Meneely, Erika; Rivera, Miguel N; Ting, David T; Wu, Chin-Lee; Miyamoto, David T

    2017-01-15

    The androgen receptor (AR) mRNA splice variant AR-V7 has emerged as a predictive biomarker for response to AR-targeted therapies. There are currently no commercially available assays to detect AR splice variants. The branched chain RNA in situ hybridization (ISH) platform enables the highly sensitive detection of RNA transcripts in formalin-fixed, paraffin-embedded (FFPE) tissues. We designed a branched chain RNA ISH probe to target the unique cryptic exon CE3 of AR-V7 using multiple tiling probes. This automated ISH assay was applied to tumor tissue from two distinct clinical cohorts that we hypothesized would differ in AR-V7 status. We detected AR-V7 in all tumor samples from men with metastatic castration-resistant prostate cancer with tissue obtained after disease progression despite at least one subsequent line of hormonal therapy (abiraterone, enzalutamide, or bicalutamide; n = 12). We detected AR-V7 in just one tumor from men who had undergone prostatectomy for localized adenocarcinoma (n = 30; Gleason 4 + 5 = 9 in the AR-V7-positive sample). Given the apparent distinction between the above groups by AR-V7 signal, we analyzed pretreatment AR-V7 status as a predictive and prognostic biomarker in men with treatment-naïve metastatic disease. Patients with metastases but without detectable AR-V7 RNA at baseline had significantly longer overall survival (log-rank P = 0.044) and a trend toward superior progression-free survival (log-rank P = 0.055). Within an institutional cohort, the RNA ISH assay identified AR-V7 within FFPE tissue and may have prognostic value in metastatic castration-sensitive prostate cancer. These preliminary findings warrant further study in larger cohorts. Clin Cancer Res; 23(2); 363-9. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. Hydrogen-atom tunneling through a very high barrier; spontaneous thiol → thione conversion in thiourea isolated in low-temperature Ar, Ne, H2 and D2 matrices.

    PubMed

    Rostkowska, Hanna; Lapinski, Leszek; Nowak, Maciej J

    2018-05-23

    Spontaneous thiol → thione hydrogen-atom transfer has been investigated for molecules of thiourea trapped in Ar, Ne, normal-H2 (n-H2) and normal-D2 (n-D2) low-temperature matrices. The most stable thione isomer was the only form of the compound present in the matrices after their deposition. According to MP2/6-311++G(2d,p) calculations, the thiol tautomer should be higher in energy by 62.5 kJ mol-1. This less stable thiol form of the compound was photochemically generated in a thione → thiol process, occurring upon UV irradiation of the matrix. Subsequently, a very slow spontaneous conversion of the thiol tautomer into the thione form was observed for the molecules isolated in Ar, Ne, n-H2 and n-D2 matrices kept at 3.5 K and in the dark. Since the thiol → thione transformation in thiourea is a process involving the dissociation of a chemical bond, the barrier for this hydrogen-atom transfer is very high (104-181 kJ mol-1). Crossing such a high potential-energy barrier at a temperature as low as 3.5 K, is possible only by hydrogen-atom tunneling. The experimentally measured time constants of this tunneling process: 52 h (Ar), 76 h (Ne), 94 h (n-H2) and 94 h (n-D2), do not differ much from one another. Hence, the dependence of the tunneling rate on the matrix environment is not drastic. The progress of the thiol → thione conversion was also monitored for Ar matrices at different temperature: 3.5 K, 9 K and 15 K. For this temperature range, the experiments revealed no detectable temperature dependence of the rate of the tunneling process.

  1. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  2. The effect of polymorphisms of the beta(2)-adrenergic receptor on the response to regular use of albuterol in asthma.

    PubMed

    Israel, E; Drazen, J M; Liggett, S B; Boushey, H A; Cherniack, R M; Chinchilli, V M; Cooper, D M; Fahy, J V; Fish, J E; Ford, J G; Kraft, M; Kunselman, S; Lazarus, S C; Lemanske, R F; Martin, R J; McLean, D E; Peters, S P; Silverman, E K; Sorkness, C A; Szefler, S J; Weiss, S T; Yandava, C N

    2000-07-01

    Inhaled beta-adrenergic agonists are the most commonly used medications for the treatment of asthma although there is evidence that regular use may produce adverse effects in some patients. Polymorphisms of the beta(2)-adrenergic receptor (beta(2)-AR) can affect regulation of the receptor. Smaller studies examining the effects of such polymorphisms on the response to beta-agonist therapy have produced inconsistent results. We examined whether polymorphisms at codon 16 (beta(2)-AR-16) and codon 27 (beta(2)-AR-27) of the beta(2)-AR might affect the response to regular versus as-needed use of albuterol by genotyping the 190 asthmatics who had participated in a trial examining the effects of regular versus as needed albuterol use. During the 16-wk treatment period there was a small decline in morning peak expiratory flow in patients homozygous for arginine at B(2)-AR-16 (Arg/Arg) who used albuterol regularly. This effect was magnified during a 4-wk run out period, during which all patients returned to using as-needed albuterol, so that by the end of the study Arg Arg patients who had regularly used albuterol had a morning peak expiratory flow 30. 5 +/- 12.1 L/min lower (p = 0.012) than Arg/Arg patients who had used albuterol on an as needed basis. There was no decline in peak flow with regular use of albuterol in patients who were homozygous for glycine at beta(2)-AR-16. Evening peak expiratory flow also declined in the Arg/Arg patients who used albuterol regularly but not in those who used albuterol on an as-needed basis. No significant differences in outcomes between regular and as-needed treatment were associated with polymorphisms at position 27 of the beta(2)-AR. No other differences in asthma outcomes that we investigated occurred in relation to these beta(2)-AR polymorphisms. Polymorphisms of the beta(2)-AR may influence airway responses to regular inhaled beta-agonist treatment.

  3. A simple and highly selective 2,2-diferrocenylpropane-based multi-channel ion pair receptor for Pb(2+) and HSO4(-).

    PubMed

    Wan, Qian; Zhuo, Ji-Bin; Wang, Xiao-Xue; Lin, Cai-Xia; Yuan, Yao-Feng

    2015-03-28

    A structurally simple, 2,2-diferrocenylpropane-based ion pair receptor 1 was synthesized and characterized by (1)H NMR, (13)C NMR, HRMS, elemental analyses, and single-crystal X-ray diffraction. The ion pair receptor 1 showed excellent selectivity and sensitivity towards Pb(2+) with multi-channel responses: a fluorescence enhancement (more than 42-fold), a notable color change from yellow to red, redox anodic shift (ΔE1/2 = 151 mV), while HSO4(-) promoted fluorescence enhancement when Pb(2+) or Zn(2+) was bonded to the cation binding-site. (1)H NMR titration and density functional theory were performed to reveal the sensing mechanism based on photo-induced electron transfer (PET).

  4. Stereodynamics of Ne(3P2) reacting with Ar, Kr, Xe, and N2

    NASA Astrophysics Data System (ADS)

    Zou, Junwen; Gordon, Sean D. S.; Tanteri, Silvia; Osterwalder, Andreas

    2018-04-01

    Stereodynamics experiments of Ne(3P2) reacting with Ar, Kr, Xe, and N2 leading to Penning and associative ionization have been performed in a crossed molecular beam apparatus. A curved magnetic hexapole was used to state-select and polarize Ne(3P2) atoms which were then oriented in a rotatable magnetic field and crossed with a beam of Ar, Kr, Xe, or N2. The ratio of associative to Penning ionization was recorded as a function of the magnetic field direction for collision energies between 320 cm-1 and 500 cm-1. Reactivities are obtained for individual states that differ only in Ω, the projection of the neon total angular momentum vector on the inter-particle axis. The results are rationalized on the basis of a model involving a long-range and a short-range reaction mechanism. Substantially lower probability for associative ionization was observed for N2, suggesting that predissociation plays a critical role in the overall reaction pathway.

  5. Gain in Transcriptional Activity by Primate-specific Coevolution of Melanoma Antigen-A11 and Its Interaction Site in Androgen Receptor*

    PubMed Central

    Liu, Qiang; Su, Shifeng; Blackwelder, Amanda J.; Minges, John T.; Wilson, Elizabeth M.

    2011-01-01

    Male sex development and growth occur in response to high affinity androgen binding to the androgen receptor (AR). In contrast to complete amino acid sequence conservation in the AR DNA and ligand binding domains among mammals, a primate-specific difference in the AR NH2-terminal region that regulates the NH2- and carboxyl-terminal (N/C) interaction enables direct binding to melanoma antigen-A11 (MAGE-11), an AR coregulator that is also primate-specific. Human, mouse, and rat AR share the same NH2-terminal 23FQNLF27 sequence that mediates the androgen-dependent N/C interaction. However, the mouse and rat AR FXXLF motif is flanked by Ala33 that evolved to Val33 in primates. Human AR Val33 was required to interact directly with MAGE-11 and for the inhibitory effect of the AR N/C interaction on activation function 2 that was relieved by MAGE-11. The functional importance of MAGE-11 was indicated by decreased human AR regulation of an androgen-dependent endogenous gene using lentivirus short hairpin RNAs and by the greater transcriptional strength of human compared with mouse AR. MAGE-11 increased progesterone and glucocorticoid receptor activity independently of binding an FXXLF motif by interacting with p300 and p160 coactivators. We conclude that the coevolution of the AR NH2-terminal sequence and MAGE-11 expression among primates provides increased regulatory control over activation domain dominance. Primate-specific expression of MAGE-11 results in greater steroid receptor transcriptional activity through direct interactions with the human AR FXXLF motif region and indirectly through steroid receptor-associated p300 and p160 coactivators. PMID:21730049

  6. Modeling Truncated AR Expression in a Natural Androgen Responsive Environment and Identification of RHOB as a Direct Transcriptional Target

    PubMed Central

    Tsai, Hui-Chi; Boucher, David L.; Martinez, Anthony; Tepper, Clifford G.; Kung, Hsing-Jien

    2012-01-01

    Recent studies identifying putative truncated androgen receptor isoforms with ligand-independent activity have shed new light on the acquisition of androgen depletion independent (ADI) growth of prostate cancer. In this study, we present a model system in which a C-terminally truncated variant of androgen receptor (TC-AR) is inducibly expressed in LNCaP, an androgen-dependent cell line, which expresses little truncated receptor. We observed that when TC-AR is overexpressed, the endogenous full length receptor (FL-AR) is transcriptionally downmodulated. This in essence allows us to “replace” FL-AR with TC-AR and compare their individual properties in exactly the same genetic and cellular background, which has not been performed before. We show that the TC-AR translocates to the nucleus, activates transcription of AR target genes in the absence of DHT and is sufficient to confer ADI growth to the normally androgen dependent LNCaP line. We also show that while there is significant overlap in the genes regulated by FL- and TC-AR there are also differences in the respective suites of target genes with each AR form regulating genes that the other does not. Among the genes uniquely activated by TC-AR is RHOB which is shown to be involved in the increased migration and morphological changes observed in LN/TC-AR, suggesting a role of RHOB in the regulation of androgen-independent behavior of prostate cancer cells. PMID:23209612

  7. Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis

    PubMed Central

    Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L. M.; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana

    2017-01-01

    The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT. PMID:28704421

  8. Transcriptional role of androgen receptor in the expression of long non-coding RNA Sox2OT in neurogenesis.

    PubMed

    Tosetti, Valentina; Sassone, Jenny; Ferri, Anna L M; Taiana, Michela; Bedini, Gloria; Nava, Sara; Brenna, Greta; Di Resta, Chiara; Pareyson, Davide; Di Giulio, Anna Maria; Carelli, Stephana; Parati, Eugenio A; Gorio, Alfredo

    2017-01-01

    The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT.

  9. Effects of serotonin 2A/1A receptor stimulation on social exclusion processing

    PubMed Central

    Preller, Katrin H.; Pokorny, Thomas; Hock, Andreas; Kraehenmann, Rainer; Stämpfli, Philipp; Seifritz, Erich; Scheidegger, Milan; Vollenweider, Franz X.

    2016-01-01

    Social ties are crucial for physical and mental health. However, psychiatric patients frequently encounter social rejection. Moreover, an increased reactivity to social exclusion influences the development, progression, and treatment of various psychiatric disorders. Nevertheless, the neuromodulatory substrates of rejection experiences are largely unknown. The preferential serotonin (5-HT) 2A/1A receptor agonist, psilocybin (Psi), reduces the processing of negative stimuli, but whether 5-HT2A/1A receptor stimulation modulates the processing of negative social interactions remains unclear. Therefore, this double-blind, randomized, counterbalanced, cross-over study assessed the neural response to social exclusion after the acute administration of Psi (0.215 mg/kg) or placebo (Pla) in 21 healthy volunteers by using functional magnetic resonance imaging (fMRI) and resting-state magnetic resonance spectroscopy (MRS). Participants reported a reduced feeling of social exclusion after Psi vs. Pla administration, and the neural response to social exclusion was decreased in the dorsal anterior cingulate cortex (dACC) and the middle frontal gyrus, key regions for social pain processing. The reduced neural response in the dACC was significantly correlated with Psi-induced changes in self-processing and decreased aspartate (Asp) content. In conclusion, 5-HT2A/1A receptor stimulation with psilocybin seems to reduce social pain processing in association with changes in self-experience. These findings may be relevant to the normalization of negative social interaction processing in psychiatric disorders characterized by increased rejection sensitivity. The current results also emphasize the importance of 5-HT2A/1A receptor subtypes and the Asp system in the control of social functioning, and as prospective targets in the treatment of sociocognitive impairments in psychiatric illnesses. PMID:27091970

  10. The serotonergic hallucinogen 5-methoxy-N,N-dimethyltryptamine disrupts cortical activity in a regionally-selective manner via 5-HT(1A) and 5-HT(2A) receptors.

    PubMed

    Riga, Maurizio S; Bortolozzi, Analia; Campa, Letizia; Artigas, Francesc; Celada, Pau

    2016-02-01

    5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a natural hallucinogen, acting as a non-selective serotonin 5-HT(1A)/5-HT(2A)-R agonist. Psychotomimetic agents such as the non-competitive NMDA-R antagonist phencyclidine and serotonergic hallucinogens (DOI and 5-MeO-DMT) disrupt cortical synchrony in the low frequency range (<4 Hz) in rat prefrontal cortex (PFC), an effect reversed by antipsychotic drugs. Here we extend these observations by examining the effect of 5-MeO-DMT on low frequency cortical oscillations (LFCO, <4 Hz) in PFC, visual (V1), somatosensory (S1) and auditory (Au1) cortices, as well as the dependence of these effects on 5-HT(1A)-R and 5-HT(2A)-R, using wild type (WT) and 5-HT(2A)-R knockout (KO2A) anesthetized mice. 5-MeO-DMT reduced LFCO in the PFC of WT and KO2A mice. The effect in KO2A mice was fully prevented by the 5-HT(1A)-R antagonist WAY-100635. Systemic and local 5-MeO-DMT reduced 5-HT release in PFC mainly via 5-HT(1A)-R. Moreover, 5-MeO-DMT reduced LFCO in S1, Au1 and V1 of WT mice and only in V1 of KO2A mice, suggesting the involvement of 5-HT(1A)-R activation in the 5-MeO-DMT-induced disruption of V1 activity. In addition, antipsychotic drugs reversed 5-MeO-DMT effects in WT mice. The present results suggest that the hallucinogen action of 5-MeO-DMT is mediated by simultaneous alterations of the activity of sensory (S1, Au1, V1) and associative (PFC) cortical areas, also supporting a role of 5-HT(1A)-R stimulation in V1 and PFC, in addition to the well-known action on 5-HT(2A)-R. Moreover, the reversal by antipsychotic drugs of 5-MeO-DMT effects adds to previous literature supporting the usefulness of the present model in antipsychotic drug development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. β2-AR activation induces chemoresistance by modulating p53 acetylation through upregulating Sirt1 in cervical cancer cells.

    PubMed

    Chen, Hongyu; Zhang, Wei; Cheng, Xiang; Guo, Liang; Xie, Shuai; Ma, Yuanfang; Guo, Ning; Shi, Ming

    2017-07-01

    It has been suggested that β2-adrenergic receptor2-AR)-mediated signaling induced by catecholamines regulates the degradation of p53. However, the underlying molecular mechanisms were not known. In the present study, we demonstrated that catecholamines upregulated the expression of silent information regulator 1 (Sirt1) through activating β2-AR-mediated signaling pathway, since selective β2-AR antagonist ICI 118, 551 and non-selective β-blocker proprenolol effectively repressed isoproterenol (ISO)-induced Sirt1 expression. Catecholamines inhibited doxorubicin (DOX)-induced p53 acetylation and transcription-activation activities by inducing the expression of Sirt1. Knockdown of the Sirt1 expression by the specific siRNA remarkably blocked the inhibitory effects of ISO on DOX-induced p53 acetylation. In addition, we demonstrated that catecholamines induced resistance of cervical cancer cells to chemotherapeutics both in vitro and in vivo and that β2-AR was overexpressed in cervical cancer tissues. Our data suggest that the p53-dependent, chemotherapeutics-induced cytotoxicity in cervical cancer cells may be compromised by catecholamines-induced upregulation of the Sirt1 expression through activating the β2-AR signaling. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  12. The truncated metabolite GLP-2 (3-33) interacts with the GLP-2 receptor as a partial agonist.

    PubMed

    Thulesen, Jesper; Knudsen, Lotte Bjerre; Hartmann, Bolette; Hastrup, Sven; Kissow, Hannelouise; Jeppesen, Palle Bekker; Ørskov, Cathrine; Holst, Jens Juul; Poulsen, Steen Seier

    2002-01-15

    The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.

  13. Interaction with beta-arrestin determines the difference in internalization behavor between beta1- and beta2-adrenergic receptors.

    PubMed

    Shiina, T; Kawasaki, A; Nagao, T; Kurose, H

    2000-09-15

    The beta(1)-adrenergic receptor (beta(1)AR) shows the resistance to agonist-induced internalization. As beta-arrestin is important for internalization, we examine the interaction of beta-arrestin with beta(1)AR with three different methods: intracellular trafficking of beta-arrestin, binding of in vitro translated beta-arrestin to intracellular domains of beta(1)- and beta(2)ARs, and inhibition of betaAR-stimulated adenylyl cyclase activities by beta-arrestin. The green fluorescent protein-tagged beta-arrestin 2 translocates to and stays at the plasma membrane by beta(2)AR stimulation. Although green fluorescent protein-tagged beta-arrestin 2 also translocates to the plasma membrane, it returns to the cytoplasm 10-30 min after beta(1)AR stimulation. The binding of in vitro translated beta-arrestin 1 and beta-arrestin 2 to the third intracellular loop and the carboxyl tail of beta(1)AR is lower than that of beta(2)AR. The fusion protein of beta-arrestin 1 with glutathione S-transferase inhibits the beta(1)- and beta(2)AR-stimulated adenylyl cyclase activities, although inhibition of the beta(1)AR-stimulated activity requires a higher concentration of the fusion protein than that of the beta(2)AR-stimulated activity. These results suggest that weak interaction of beta(1)AR with beta-arrestins explains the resistance to agonist-induced internalization. This is further supported by the finding that beta-arrestin can induce internalization of beta(1)AR when beta-arrestin 1 does not dissociate from beta(1)AR by fusing to the carboxyl tail of beta(1)AR.

  14. β3 adrenergic receptor in the kidney may be a new player in sympathetic regulation of renal function.

    PubMed

    Procino, Giuseppe; Carmosino, Monica; Milano, Serena; Dal Monte, Massimo; Schena, Giorgia; Mastrodonato, Maria; Gerbino, Andrea; Bagnoli, Paola; Svelto, Maria

    2016-09-01

    To date, the study of the sympathetic regulation of renal function has been restricted to the important contribution of β1- and β2-adrenergic receptors (ARs). Here we investigate the expression and the possible physiologic role of β3-adrenergic receptor (β3-AR) in mouse kidney. The β3-AR is expressed in most of the nephron segments that also express the type 2 vasopressin receptor (AVPR2), including the thick ascending limb and the cortical and outer medullary collecting duct. Ex vivo experiments in mouse kidney tubules showed that β3-AR stimulation with the selective agonist BRL37344 increased intracellular cAMP levels and promoted 2 key processes in the urine concentrating mechanism. These are accumulation of the water channel aquaporin 2 at the apical plasma membrane in the collecting duct and activation of the Na-K-2Cl symporter in the thick ascending limb. Both effects were prevented by the β3-AR antagonist L748,337 or by the protein kinase A inhibitor H89. Interestingly, genetic inactivation of β3-AR in mice was associated with significantly increased urine excretion of water, sodium, potassium, and chloride. Stimulation of β3-AR significantly reduced urine excretion of water and the same electrolytes. Moreover, BRL37344 promoted a potent antidiuretic effect in AVPR2-null mice. Thus, our findings are of potential physiologic importance as they uncover the antidiuretic effect of β3-AR stimulation in the kidney. Hence, β3-AR agonism might be useful to bypass AVPR2-inactivating mutations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Dorsomedial pontine neurons with descending projections to the medullary reticular formation express orexin-1 and adrenergic alpha2A receptor mRNA.

    PubMed

    Volgin, Denys V; Malinowska, Monika; Kubin, Leszek

    2009-08-14

    Neurons located in the dorsomedial pontine rapid eye movement (REM) sleep-triggering region send axons to the medial medullary reticular formation (mMRF). This pathway is believed to be important for the generation of REM sleep motor atonia, but other than that they are glutamatergic little is known about neurochemical signatures of these pontine neurons important for REM sleep. We used single-cell reverse transcription and polymerase chain reaction (RT-PCR) to determine whether dorsomedial pontine cells with projections to the mMRF express mRNA for selected membrane receptors that mediate modulatory influences on REM sleep. Fluorescein (FITC)-labeled latex microspheres were microinjected into the mMRF of 26-34-day-old rats under pentobarbital anesthesia. After 5-6 days, rats were sacrificed, pontine slices were obtained and neurons were dissociated from 400 to 600 microm micropunches extracted from dorsomedial pontine reticular formation. We found that 32 out of 51 FITC-labeled cells tested (63+/-7% (SE)) contained the orexin type 1 receptor (ORX1r) mRNA, 27 out of 73 (37+/-6%) contained the adrenergic alpha(2A) receptor (alpha(2A)r) RNA, and 6 out of 31 (19+/-7%) contained both mRNAs. The percentage of cells positive for the ORX1r mRNA was significantly lower (p<0.04) for the dorsomedial pontine cells that were not retrogradely labeled from the mMRF (32+/-11%), whereas alpha(2A)r mRNA was present in a similar percentage of FITC-labeled and unlabeled neurons. Our data suggest that ORX and adrenergic pathways converge on a subpopulation of cells of the pontine REM sleep-triggering region that have descending projections to the medullary region important for the motor control during REM sleep.

  16. Molecular modeling and docking studies of human 5-hydroxytryptamine 2A (5-HT2A) receptor for the identification of hotspots for ligand binding.

    PubMed

    Kanagarajadurai, Karuppiah; Malini, Manoharan; Bhattacharya, Aditi; Panicker, Mitradas M; Sowdhamini, Ramanathan

    2009-12-01

    The serotonergic system has been implicated in emotional and cognitive function. In particular, 5-HT(2A) (5-hydroxytrytamine receptor 2A) is attributed to a number of disorders like schizophrenia, depression, eating disorders and anxiety. 5-HT(2A), being a GPCR (G-protein coupled receptor), is important in the pharmaceutical industry as a proven target for these disorders. Despite their extensive clinical importance, the structural studies of this protein is lacking due to difficulties in determining its crystal structure. We have performed sequence analysis and molecular modeling of 5-HT(2A) that has revealed a set of conserved residues and motifs considered to play an important role in maintaining structural integrity and function of the receptor. The analysis also revealed a set of residues specific to the receptor which distinguishes them from other members of the subclass and their orthologs. Further, starting from the model structure of human 5-HT(2A) receptor, docking studies were attempted to envisage how it might interact with eight of its ligands (such as serotonin, dopamine, DOI, LSD, haloperidol, ketanserin, risperidone and clozapine). The binding studies of dopamine to 5-HT(2A) receptor can bring up better understanding in the etiology of a number of neurological disorders involving both these two receptors. Our sequence analysis and study of interactions of this receptor with other ligands reveal additional residue hotspots such as Asn 363 and Tyr 370. The function of these residues can be further analyzed by rational design of site-directed mutagenesis. Two distinct binding sites are identified which could play important roles in ligand binding and signaling.

  17. MDMA Increases Excitability in the Dentate Gyrus: Role of 5HT2A Receptor Induced PGE2 Signaling

    PubMed Central

    Collins, Stuart A.; Huff, Courtney; Chiaia, Nicolas; Gudelsky, Gary A.; Yamamoto, Bryan K.

    2015-01-01

    MDMA is a widely abused psychostimulant which causes release of serotonin in various forebrain regions. Recently, we reported that MDMA increases extracellular glutamate concentrations in the dentate gyrus, via activation of 5HT2A receptors. We examined the role of prostaglandin signaling in mediating the effects of 5HT2A receptor activation on the increases in extracellular glutamate and the subsequent long-term loss of parvalbumin interneurons in the dentate gyrus caused by MDMA. Administration of MDMA into the dentate gyrus of rats increased PGE2 concentrations which was prevented by coadministration of MDL100907, a 5HT2A receptor antagonist. MDMA-induced increases in extracellular glutamate were inhibited by local administration of SC-51089, an inhibitor of the EP1 prostaglandin receptor. Systemic administration of SC-51089 during injections of MDMA prevented the decreases in parvalbumin interneurons observed 10 days later. The loss of parvalbumin immunoreactivity after MDMA exposure coincided with a decrease in paired-pulse inhibition and afterdischarge threshold in the dentate gyrus. These changes were prevented by inhibition of EP1 and 5HT2A receptors during MDMA. Additional experiments revealed an increased susceptibility to kainic acid-induced seizures in MDMA treated rats which could be prevented with SC51089 treatments during MDMA exposure. Overall, these findings suggest that 5HT2A receptors mediate MDMA-induced PGE2 signaling and subsequent increases in glutamate. This signaling mediates parvalbumin cell losses as well as physiologic changes in the dentate gyrus, suggesting that the lack of the inhibition provided by these neurons increases the excitability within the dentate gyrus of MDMA treated rats. PMID:26670377

  18. Comparative Exploration of the Structure-Activity Space of Cloned α-Like Octopamine Receptors from a Marine and a Terrestrial Arthropod.

    PubMed

    Dalwadi, Dhwanil A; Schetz, John A

    2017-09-01

    The α -like octopamine receptors (OctR) are believed to be the evolutionary precursor to the vertebrate α 2 -adrenergic receptors ( α 2 -ARs) based upon sequence similarity and the ability to interact with norepinephrine and a number of compounds that bind with high affinity to α 2 -ARs. Barnacles and fruit flies are two prominent model marine and terrestrial representatives of the Arthropoda phylum, and although α -like OctRs have been cloned from Balanus improvisus (BiOctR) and Drosophila melanogaster (DmOctR), little is known about the structure-activity space for these important species. A diverse panel of 22 probes spanning different structural classes were employed to interrogate the structure-activity of the BiOctR and DmOctR. While BiOctR and DmOctR exhibited similar functional profiles for mammalian biogenic amine G protein-coupled receptor agonists and antagonists, some ligands had dramatically different mechanisms of action. For instance, significant differences in the efficacy for some agonists were observed, including that vertebrate biogenic amines structurally related to octopamine acted as superagonists at the DmOctR but partial agonists at the BiOctR, and the two species diverged in their sensitivities to the α 2 -AR antagonist [ 3 H]rauwolscine. Furthermore, sodium enhanced [ 3 H]rauwolscine's interactions with the BiOctR, but not at a vertebrate α 2 -AR. Molecular mechanistic studies indicate that rauwolscine interacts with the BiOctR, DmOctR, and α 2C -adrenergic receptor at an allosteric site. In addition, compounds that acted as agonists at a cloned α -like BiOctR also induced a hyperactivity response in Balanus cyprids mediated by the α -like OctR, suggesting that the receptor may serve as a higher throughput proxy for discovering compounds with potential cyprid deterrent properties. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  19. 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

  20. Single-channel activations and concentration jumps: comparison of recombinant NR1a/NR2A and NR1a/NR2D NMDA receptors

    PubMed Central

    Wyllie, David J A; Béhé, Philippe; Colquhoun, David

    1998-01-01

    We have expressed recombinant NR1a/NR2A and NR1a/NR2D N-methyl-D-aspartate (NMDA) receptor channels in Xenopus oocytes and made recordings of single-channel and macroscopic currents in outside-out membrane patches. For each receptor type we measured (a) the individual single-channel activations evoked by low glutamate concentrations in steady-state recordings, and (b) the macroscopic responses elicited by brief concentration jumps with high agonist concentrations, and we explore the relationship between these two sorts of observation. Low concentration (5–100 nM) steady-state recordings of NR1a/NR2A and NR1a/NR2D single-channel activity generated shut-time distributions that were best fitted with a mixture of five and six exponential components, respectively. Individual activations of either receptor type were resolved as bursts of openings, which we refer to as ‘super-clusters’. During a single activation, NR1a/NR2A receptors were open for 36 % of the time, but NR1a/NR2D receptors were open for only 4 % of the time. For both, distributions of super-cluster durations were best fitted with a mixture of six exponential components. Their overall mean durations were 35.8 and 1602 ms, respectively. Steady-state super-clusters were aligned on their first openings and averaged. The average was well fitted by a sum of exponentials with time constants taken from fits to super-cluster length distributions. It is shown that this is what would be expected for a channel that shows simple Markovian behaviour. The current through NR1a/NR2A channels following a concentration jump from zero to 1 mM glutamate for 1 ms was well fitted by three exponential components with time constants of 13 ms (rising phase), 70 ms and 350 ms (decaying phase). Similar concentration jumps on NR1a/NR2D channels were well fitted by two exponentials with means of 45 ms (rising phase) and 4408 ms (decaying phase) components. During prolonged exposure to glutamate, NR1a/NR2A channels desensitized

  1. A comparative study on the frequency effects of the electrical characteristics of the pulsed dielectric barrier discharge in He/O{sub 2} and in Ar/O{sub 2} at atmospheric pressure

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, Guangsheng; Tan, Zhenyu, E-mail: tzy@sdu.edu.cn; Pan, Jie

    In this work, a comparative study on the frequency effects of the electrical characteristics of the pulsed dielectric barrier discharges in He/O{sub 2} and in Ar/O{sub 2} at atmospheric pressure has been performed by means of the numerical simulation based on a 1-D fluid model at frequencies below 100 kHz. The frequency dependences of the characteristic quantities of the discharges in the two gases have been systematically calculated and analyzed under the oxygen concentrations below 2%. The characteristic quantities include the discharge current density, the averaged electron density, the electric field, and the averaged electron temperature. Especially, the frequency effects onmore » the averaged particle densities of the reactive species have also been calculated. This work gives the following significant results. For the two gases, there are two bipolar discharges in one period of applied voltage pulse under the considered frequency range and oxygen concentrations, as occurred in the pure noble gases. The frequency affects the two discharges in He/O{sub 2}, but in Ar/O{sub 2}, it induces a strong effect only on the first discharge. For the first discharge in each gas, there is a characteristic frequency at which the characteristic quantities reach their respective minimum, and this frequency appears earlier for Ar/O{sub 2}. For the second discharge in Ar/O{sub 2}, the averaged electron density presents a slight variation with the frequency. In addition, the discharge in Ar/O{sub 2} is strong and the averaged electron temperature is low, compared to those in He/O{sub 2.} The total averaged particle density of the reactive species in Ar/O{sub 2} is larger than those in He/O{sub 2} by about one order of magnitude.« less

  2. 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.

  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. Hypothyroidism Affects D2 Receptor-mediated Breathing without altering D2 Receptor Expression

    PubMed Central

    Schlenker, Evelyn H.; Rio, Rodrigo Del; Schultz, Harold D.

    2015-01-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age- matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. PMID:24434437

  5. Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

    PubMed

    Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

    2014-03-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. 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

  7. Stereoselective binding of agonists to the β2-adrenergic receptor: insights into molecular details and thermodynamics from molecular dynamics simulations.

    PubMed

    Plazinska, Anita; Plazinski, Wojciech

    2017-05-02

    The β 2 -adrenergic receptor2 -AR) is one of the most studied G-protein-coupled receptors. When interacting with ligand molecules, it exhibits a binding characteristic that is strongly dependent on ligand stereoconfiguration. In particular, many experimental and theoretical studies confirmed that stereoisomers of an important β 2 -AR agonist, fenoterol, are associated with diverse mechanisms of binding and activation of β 2 -AR. The objective of the present study was to explore the stereoselective binding of fenoterol to β 2 -AR through the application of an advanced computational methodology based on enhanced-sampling molecular dynamics simulations and potentials of interactions tailored to investigate the stereorecognition effects. The results remain in very good, quantitative agreement with the experimental data (measured in the context of ligand-receptor affinities and their dependence on the temperature), which provides an additional validation for the applied computational protocols. Additionally, our results contribute to the understanding of stereoselective agonist binding by β 2 -AR. Although the significant role of the N293 6.55 residue is confirmed, we additionally show that stereorecognition does not depend solely on the N293-ligand interactions; the stereoselective effects rely on the co-operation of several residues located on both the 6th and 7th transmembrane domains and on extracellular loops. The magnitude and character of the contributions of these residues may be very diverse and result in either enhancing or reducing the stereoselective effects. The same is true when considering the enthalpic and entropic contributions to the binding free energies, which also are dependent on the ligand stereoconfiguration.

  8. Agonist-biased trafficking of somatostatin receptor 2A in enteric neurons.

    PubMed

    Zhao, Peishen; Canals, Meritxell; Murphy, Jane E; Klingler, Diana; Eriksson, Emily M; Pelayo, Juan-Carlos; Hardt, Markus; Bunnett, Nigel W; Poole, Daniel P

    2013-09-06

    Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions.

  9. Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons*

    PubMed Central

    Zhao, Peishen; Canals, Meritxell; Murphy, Jane E.; Klingler, Diana; Eriksson, Emily M.; Pelayo, Juan-Carlos; Hardt, Markus; Bunnett, Nigel W.; Poole, Daniel P.

    2013-01-01

    Somatostatin (SST) 14 and SST 28 activate somatostatin 2A receptors (SSTR2A) on enteric neurons to control gut functions. SST analogs are treatments of neuroendocrine and bleeding disorders, cancer, and diarrhea, with gastrointestinal side effects of constipation, abdominal pain, and nausea. How endogenous agonists and drugs differentially regulate neuronal SSTR2A is unexplored. We evaluated SSTR2A trafficking in murine myenteric neurons and neuroendocrine AtT-20 cells by microscopy and determined whether agonist degradation by endosomal endothelin-converting enzyme 1 (ECE-1) controls SSTR2A trafficking and association with β-arrestins, key regulators of receptors. SST-14, SST-28, and peptide analogs (octreotide, lanreotide, and vapreotide) stimulated clathrin- and dynamin-mediated internalization of SSTR2A, which colocalized with ECE-1 in endosomes and the Golgi. After incubation with SST-14, SSTR2A recycled to the plasma membrane, which required active ECE-1 and an intact Golgi. SSTR2A activated by SST-28, octreotide, lanreotide, or vapreotide was retained within the Golgi and did not recycle. Although ECE-1 rapidly degraded SST-14, SST-28 was resistant to degradation, and ECE-1 did not degrade SST analogs. SST-14 and SST-28 induced transient interactions between SSTR2A and β-arrestins that were stabilized by an ECE-1 inhibitor. Octreotide induced sustained SSTR2A/β-arrestin interactions that were not regulated by ECE-1. Thus, when activated by SST-14, SSTR2A internalizes and recycles via the Golgi, which requires ECE-1 degradation of SST-14 and receptor dissociation from β-arrestins. After activation by ECE-1-resistant SST-28 and analogs, SSTR2A remains in endosomes because of sustained β-arrestin interactions. Therapeutic SST analogs are ECE-1-resistant and retain SSTR2A in endosomes, which may explain their long-lasting actions. PMID:23913690

  10. Dopamine D2 heteroreceptor complexes and their receptor-receptor interactions in ventral striatum: novel targets for antipsychotic drugs.

    PubMed

    Fuxe, Kjell; Borroto-Escuela, Dasiel O; Tarakanov, Alexander O; Romero-Fernandez, Wilber; Ferraro, Luca; Tanganelli, Sergio; Perez-Alea, Mileidys; Di Palma, Michael; Agnati, Luigi F

    2014-01-01

    This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-and-clasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptor-receptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing β-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex in the ventral striatum are proposed as one molecular mechanism for the potential antipsychotic effects of NT. Indications were obtained that the psychotic actions of the 5-HT2AR hallucinogens LSD and DOI can involve enhancement of D2R protomer signaling via a biased agonist action at the 5-HT2A protomer in the D2-5-HT2A heteroreceptor complex in the ventral striatum. Facilitatory allosteric D2likeR-OTR interactions in heteroreceptor complexes in nucleus accumbens may have a role in social and emotional behaviors. By blocking the D2 protomers of these heteroreceptor complexes, antipsychotics can fail to reduce the negative symptoms of schizophrenia. The discovery of different types of D2 heteroreceptor complexes gives an increased understanding of molecular mechanisms involved in causing schizophrenia and new strategies for its treatment and understanding the side effects of antipsychotics. © 2014 Elsevier B.V. All rights reserved.

  11. Measurements of Enthalpy of Sublimation of Ne, N2, O2, Ar, CO2, Kr, Xe, and H2O using a Double Paddle Oscillator.

    PubMed

    Shakeel, Hamza; Wei, Haoyan; Pomeroy, Joshua M

    2018-03-01

    We report precise experimental values of the enthalpy of sublimation (Δ H s ) of quenched condensed films of neon (Ne), nitrogen (N 2 ), oxygen (O 2 ), argon (Ar), carbon dioxide (CO 2 ), krypton (Kr), xenon (Xe), and water (H 2 O) vapor using a single consistent measurement platform. The experiments are performed well below the triple point temperature of each gas and fall in the temperature range where existing experimental data is very limited. A 6 cm 2 and 400 µm thick double paddle oscillator (DPO) with high quality factor (Q ≈ 4 × 10 5 at 298K) and high frequency stability (33 parts per billion) is utilized for the measurements. The enthalpies of sublimation are derived by measuring the rate of mass loss during temperature programmed desorption. The mass change is detected due to change in the resonance frequency of the self-tracking oscillator. Our measurements typically remain within 10% of the available literature, theory, and National Institute of Standards and Technology (NIST) Web Thermo Tables ( WTT ) values, but are performed using an internally consistent method across different gases.

  12. 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

  13. Comparative molecular field analysis of the binding of the stereoisomers of fenoterol and fenoterol derivatives to the beta2 adrenergic receptor.

    PubMed

    Jozwiak, Krzysztof; Khalid, Chakir; Tanga, Mary J; Berzetei-Gurske, Ilona; Jimenez, Lucita; Kozocas, Joseph A; Woo, Anthony; Zhu, Weizhong; Xiao, Rui-Ping; Abernethy, Darrell R; Wainer, Irving W

    2007-06-14

    Stereoisomers of fenoterol and six fenoterol derivatives have been synthesized and their binding affinities for the beta2 adrenergic receptor (Kibeta2-AR), the subtype selectivity relative to the beta1-AR (Kibeta1-AR/Kibeta2-AR) and their functional activities were determined. Of the 26 compounds synthesized in the study, submicromolar binding affinities were observed for (R,R)-fenoterol, the (R,R)-isomer of the p-methoxy, and (R,R)- and (R,S)-isomers of 1-naphthyl derivatives and all of these compounds were active at submicromolar concentrations in cardiomyocyte contractility tests. The Kibeta1-AR/Kibeta2-AR ratios were >40 for (R,R)-fenoterol and the (R,R)-p-methoxy and (R,S)-1-naphthyl derivatives and 14 for the (R,R)-1-napthyl derivative. The binding data was analyzed using comparative molecular field analysis (CoMFA), and the resulting model indicated that the fenoterol derivatives interacted with two separate binding sites and one steric restricted site on the pseudo-receptor and that the chirality of the second stereogenic center affected Kibeta2 and subtype selectivity.

  14. Production of interleukin-2 (IL-2) and expression of IL-2 receptor in patients with IgA nephropathy.

    PubMed

    Lee, T W; Kim, M J

    1992-01-01

    IL-2 production has been measured in several disease including type I diabetes mellitus, systemic lupus erythematosus, acquired immunodeficiency syndrome and active pulmonary sarcoidosis and its pathogenetic role was suggested. In IgA nephropathy, altered T cell subsets were reported to be associated with increased synthesis of IgA. The altered IL-2 production and the expression of IL-2 receptor might be involved in the pathogenesis of IgA nephropathy. To investigate the role of T cell mediated immunity in the pathogenesis of IgA nephropathy, the immune parameters such as T cell subsets, NK cell activity, interleukin-2 (IL-2) production and IL-2 receptor expression on peripheral blood mononuclear cells (PBMC) were measured before and/or after phytohemagglutinin (PHA) stimulation in 15 patients with IgA nephropathy. Age and sex matched 15 healthy controls and the correlations between the IL-2 production and immune parameters were evaluated. The mean percentages of T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8) and the CD4/CD8 ratio of the patients were not different from those of controls and the proportions of CD8 CD11b cell in the patients (21.0 +/- 3.6%) were significantly lower than those in controls (30.5 +/- 5.3%) (p < 0.005). The production of IL-2 by fresh PBMC of both patients and controls was in undetectable ranges. The production of IL-2 by PHA stimulated PBMC of patients was significantly higher than that of controls (140.03 +/- 43.2 U/ml vs 106.5 +/- 42.1 U/ml, p < 0.05). The proportions of lymphocytes expressing the IL-2 receptor (CD25) before the stimulation with PHA in patients were 1.22 +/- 1.00 percent and were not different from those in controls (1.12 +/- 0.78 percent). The correlations between the production of IL-2 and the concentrations of serum IgA, the degrees of histologic alterations and the proportions of CD8 and CD8CD11b cells were not significant. There was a weak tendency of a positive correlation (p < 0.1) between

  15. Fenoterol, a β2-adrenoceptor agonist, inhibits LPS-induced membrane-bound CD14, TLR4/CD14 complex, and inflammatory cytokines production through β-arrestin-2 in THP-1 cell line

    PubMed Central

    Wang, Wei; Xu, Ming; Zhang, You-yi; He, Bei

    2009-01-01

    Aim: To investigate the molecular mechanism and signaling pathway by which fenoterol, a β2-adrenergic receptor2-AR) agonist, produces anti-inflammatory effects. Methods: THP-1, a monocytic cell line, was used to explore the mechanism of β2-AR stimulation in LPS-induced secretion of inflammatory cytokines and changes of toll-like receptors (TLRs). We labeled TLR4 and CD14 using monoclonal anti-TLR4 PE-conjugated and anti-CD14 FITC-conjugated antibodies in THP-1 cells stimulated by β2-AR in the presence or absence of lipopolysaccharide (LPS) and small, interfering RNA (siRNA)-mediated knockdown of β-arrestin-2, and then analyzed their changes in distribution by flow cytometry, Western blotting and confocal analysis. Results: LPS-induced membrane-bound CD14, TLR4/CD14 complex levels and elevation of inflammatory cytokines were all significantly reduced by pre-incubation of fenoterol (P<0.05). However, the total level of CD14 and TLR4 was not significantly changed. Interestingly, confocal microscopy revealed redistribution of CD14 and TLR4/CD14 complex under β2-AR stimulation. Furthermore, siRNA-mediated knockdown of β-arrestin-2 eliminated the anti-inflammatory effects and redistribution of CD14 and TLR4/CD14 complex stimulated by β2-AR. Conclusion: β2-AR agonist exerts its anti-inflammatory effects by down-regulating TLR signaling in THP-1 cells, potentially resulting from β-arrestin-2 mediated redistribution of CD14 and TLR14/CD14 complex. PMID:19890360

  16. A Study of the X(sup 2) Sigma(sup +) and A(sup 2) Pi States of MgAr(sup +) and MgKr(sup +)

    NASA Technical Reports Server (NTRS)

    Bauschlicher, Charles W., Jr.; Partridge, Harry; Langhoff, Stephen R. (Technical Monitor)

    1995-01-01

    The ground (sup 2)Sigma(sup +) and lowest excited (sup 2)Pi states of MgAr(sup +) and MgKr(sup +) are studied using the singles and doubles configuration interaction (SDCI) approach, in conjunction with large basis sets. The effect of Mg core correlation and core polarization are accounted for using the core-polarization potential (CPP) approach. Franck-Condon factors, oscillator strengths, radiative lifetimes, dissociation energies, bond lengths, and excitation energies are reported. The computed results are in good agreement with the available experimental data.

  17. Etching Characteristics of VO2 Thin Films Using Inductively Coupled Cl2/Ar Plasma

    NASA Astrophysics Data System (ADS)

    Ham, Yong-Hyun; Efremov, Alexander; Min, Nam-Ki; Lee, Hyun Woo; Yun, Sun Jin; Kwon, Kwang-Ho

    2009-08-01

    A study on both etching characteristics and mechanism of VO2 thin films in the Cl2/Ar inductively coupled plasma was carried. The variable parameters were gas pressure (4-10 mTorr) and input power (400-700 W) at fixed bias power of 150 W and initial mixture composition of 25% Cl2 + 75% Ar. It was found that an increase in both gas pressure and input power results in increasing VO2 etch rate while the etch selectivity over photoresist keeps a near to constant values. Plasma diagnostics by Langmuir probes and zero-dimensional plasma model provided the data on plasma parameters, steady-state densities and fluxes of active species on the etched surface. The model-based analysis of the etch mechanism showed that, for the given ranges of operating conditions, the VO2 etch kinetics corresponds to the transitional regime of ion-assisted chemical reaction and is influenced by both neutral and ion fluxes with a higher sensitivity to the neutral flux.

  18. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    PubMed Central

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  19. Evidence for Noncanonical Neurotransmitter Activation: Norepinephrine as a Dopamine D2-Like Receptor Agonist

    PubMed Central

    Sánchez-Soto, Marta; Bonifazi, Alessandro; Cai, Ning Sheng; Ellenberger, Michael P.; Newman, Amy Hauck

    2016-01-01

    The Gαi/o-coupled dopamine D2-like receptor family comprises three subtypes: the D2 receptor (D2R), with short and long isoform variants (D2SR and D2LR), D3 receptor (D3R), and D4 receptor (D4R), with several polymorphic variants. The common overlap of norepinephrine innervation and D2-like receptor expression patterns prompts the question of a possible noncanonical action by norepinephrine. In fact, previous studies have suggested that norepinephrine can functionally interact with D4R. To our knowledge, significant interactions between norepinephrine and D2R or D3R receptors have not been demonstrated. By using radioligand binding and bioluminescent resonance energy transfer (BRET) assays in transfected cells, the present study attempted a careful comparison between dopamine and norepinephrine in their possible activation of all D2-like receptors, including the two D2R isoforms and the most common D4R polymorphic variants. Functional BRET assays included activation of G proteins with all Gαi/o subunits, adenylyl cyclase inhibition, and β arrestin recruitment. Norepinephrine acted as a potent agonist for all D2-like receptor subtypes, with the general rank order of potency of D3R > D4R ≥ D2SR ≥ D2L. However, for both dopamine and norepinephrine, differences depended on the Gαi/o protein subunit involved. The most striking differences were observed with Gαi2, where the rank order of potencies for both dopamine and norepinephrine were D4R > D2SR = D2LR >> D3R. Furthermore the results do not support the existence of differences in the ability of dopamine and norepinephrine to activate different human D4R variants. The potency of norepinephrine for adrenergic α2A receptor was only about 20-fold higher compared with D3R and D4R across the three functional assays. PMID:26843180

  20. Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain.

    PubMed

    Li, Na; Lu, Zhan-ying; Yu, Li-hua; Burnstock, Geoffrey; Deng, Xiao-ming; Ma, Bei

    2014-03-18

    ATP and P2X receptors play important roles in the modulation of trigeminal neuropathic pain, while the role of G protein-coupled P2Y₂ receptors and the underlying mechanisms are less clear. The threshold and frequency of action potentials, fast inactivating transient K+ channels (IA) are important regulators of membrane excitability in sensory neurons because of its vital role in the control of the spike onset. In this study, pain behavior tests, QT-RT-PCR, immunohistochemical staining, and patch-clamp recording, were used to investigate the role of P2Y₂ receptors in pain behaviour. In control rats: 1) UTP, an agonist of P2Y₂/P2Y₄ receptors, caused a significant decrease in the mean threshold intensities for evoking action potentials and a striking increase in the mean number of spikes evoked by TG neurons. 2) UTP significantly inhibited IA and the expression of Kv1.4, Kv3.4 and Kv4.2 subunits in TG neurons, which could be reversed by the P2 receptor antagonist suramin and the ERK antagonist U0126. In ION-CCI (chronic constriction injury of infraorbital nerve) rats: 1) mRNA levels of Kv1.4, Kv3.4 and Kv4.2 subunits were significantly decreased, while the protein level of phosphorylated ERK was significantly increased. 2) When blocking P2Y₂ receptors by suramin or injection of P2Y2R antisense oligodeoxynucleotides both led to a time- and dose-dependent reverse of allodynia in ION-CCI rats. 3) Injection of P2Y₂ receptor antisense oligodeoxynucleotides induced a pronounced decrease in phosphorylated ERK expression and a significant increase in Kv1.4, Kv3.4 and Kv4.2 subunit expression in trigeminal ganglia. Our data suggest that inhibition of P2Y₂ receptors leads to down-regulation of ERK-mediated phosphorylation and increase of the expression of I(A)-related Kv channels in trigeminal ganglion neurons, which might contribute to the clinical treatment of trigeminal neuropathic pain.

  1. Dorsomedial pontine neurons with descending projections to the medullary reticular formation express orexin-1 and adrenergic α2A receptor mRNA

    PubMed Central

    Volgin, Denys V.; Malinowska, Monika; Kubin, Leszek

    2009-01-01

    Neurons located in the dorsomedial pontine rapid eye movement (REM) sleep-triggering region send axons to the medial medullary reticular formation (mMRF). This pathway is believed to be important for the generation of REM sleep motor atonia, but other than that they are glutamatergic little is known about neurochemical signatures of these pontine neurons important for REM sleep. We used single-cell reverse transcription and polymerase chain reaction (RT-PCR) to determine whether dorsomedial pontine cells with projections to the mMRF express mRNA for selected membrane receptors that mediate modulatory influences on REM sleep. Fluorescein (FITC)-labeled latex microspheres were microinjected into the mMRF of 26–34 day-old rats under pentobarbital anesthesia. After 5–6 days, rats were sacrificed, pontine slices were obtained and neurons were dissociated from 400–600 μm micropunches extracted from dorsomedial pontine reticular formation. We found that 32 out of 51 FITC-labeled cells tested (63%±7(SE)) contained the orexin type 1 receptor (ORX1r) mRNA, 27 out of 73 (37%±6) contained the adrenergic α2A receptor2Ar) RNA, and 6 out of 31 (19%±7) contained both mRNAs. The percentage of cells positive for the ORX1r mRNA was significantly lower (p<0.04) for the dorsomedial pontine cells that were not retrogradely labeled from the mMRF (32%±11), whereas α2Ar mRNA was present in a similar percentage of FITC-labeled and unlabeled neurons. Our data suggest that ORX and adrenergic pathways converge on a subpopulation of cells of the pontine REM sleep-triggering region that have descending projections to the medullary region important for the motor control during REM sleep. PMID:19427365

  2. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain.

    PubMed

    Volkow, N D; Wang, G-J; Logan, J; Alexoff, D; Fowler, J S; Thanos, P K; Wong, C; Casado, V; Ferre, S; Tomasi, D

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [(11)C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors.

  3. Caffeine increases striatal dopamine D2/D3 receptor availability in the human brain

    PubMed Central

    Volkow, N D; Wang, G-J; Logan, J; Alexoff, D; Fowler, J S; Thanos, P K; Wong, C; Casado, V; Ferre, S; Tomasi, D

    2015-01-01

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A2A receptors (A2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [11C]raclopride (DA D2/D3 receptor radioligand sensitive to endogenous DA) to assess if caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300 mg p.o.) significantly increased the availability of D2/D3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D2/D3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D2/D3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D2/D3 receptor availability. Instead, we interpret our findings to reflect an increase in D2/D3 receptor levels in striatum with caffeine (or changes in affinity). The association between increases in D2/D3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D2/D3 receptors. PMID:25871974

  4. Caffeine increases striatal dopamine D 2/D 3 receptor availability in the human brain

    DOE PAGES

    Volkow, N. D.; Wang, G. -J.; Logan, J.; ...

    2015-04-14

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A 2A receptors (A 2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [ 11C]raclopride (DA D 2/D 3 receptor radioligand sensitive to endogenous DA) to assess ifmore » caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300mg p.o.) significantly increased the availability of D 2/D 3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D 2/D 3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D 2/D 3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D 2/D 3 receptor availability. Instead, we interpret our findings to reflect an increase in D 2/D 3 receptor levels in striatum with caffeine (or changes in affinity). Furthermore, the association between increases in D 2/D 3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D 2/D 3 receptors.« less

  5. Caffeine increases striatal dopamine D 2/D 3 receptor availability in the human brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Volkow, N. D.; Wang, G. -J.; Logan, J.

    Caffeine, the most widely consumed psychoactive substance in the world, is used to promote wakefulness and enhance alertness. Like other wake-promoting drugs (stimulants and modafinil), caffeine enhances dopamine (DA) signaling in the brain, which it does predominantly by antagonizing adenosine A 2A receptors (A 2AR). However, it is unclear if caffeine, at the doses consumed by humans, increases DA release or whether it modulates the functions of postsynaptic DA receptors through its interaction with adenosine receptors, which modulate them. We used positron emission tomography and [ 11C]raclopride (DA D 2/D 3 receptor radioligand sensitive to endogenous DA) to assess ifmore » caffeine increased DA release in striatum in 20 healthy controls. Caffeine (300mg p.o.) significantly increased the availability of D 2/D 3 receptors in putamen and ventral striatum, but not in caudate, when compared with placebo. In addition, caffeine-induced increases in D 2/D 3 receptor availability in the ventral striatum were associated with caffeine-induced increases in alertness. Our findings indicate that in the human brain, caffeine, at doses typically consumed, increases the availability of DA D 2/D 3 receptors, which indicates that caffeine does not increase DA in the striatum for this would have decreased D 2/D 3 receptor availability. Instead, we interpret our findings to reflect an increase in D 2/D 3 receptor levels in striatum with caffeine (or changes in affinity). Furthermore, the association between increases in D 2/D 3 receptor availability in ventral striatum and alertness suggests that caffeine might enhance arousal, in part, by upregulating D 2/D 3 receptors.« less

  6. BCAS2 promotes prostate cancer cells proliferation by enhancing AR mRNA transcription and protein stability

    PubMed Central

    Kuo, P-C; Huang, C-W; Lee, C-I; Chang, H-W; Hsieh, S-W; Chung, Y-P; Lee, M-S; Huang, C-S; Tsao, L-P; Tsao, Y-P; Chen, S-L

    2015-01-01

    Background: We showed previously that breast carcinoma amplified sequence 2 (BCAS2) functions as a negative regulator of p53. We also found that BCAS2 is a potential AR-associated protein. AR is essential for the growth and survival of prostate carcinoma. Therefore we characterised the correlation between BCAS2 and AR. Methods: Protein interactions were examined by GST pull-down assay and co-immunoprecipitation. Clinical prostate cancer (PCa) specimens were evaluated by immunohistochemical assay. AR transcriptional activity and LNCaP cell growth were assessed by luciferase assay and MTT assay, respectively. Results: BCAS2 expression was significantly increased in PCa. BCAS2 stabilised AR protein through both hormone-dependent and -independent manners. There are at least two mechanisms for BCAS2-mediated AR protein upregulation: One is p53-dependent. The p53 is suppressed by BCAS2 that results in increasing AR mRNA and protein expression. The other is via p53-independent inhibition of proteasome degradation. As BCAS2 can form a complex with AR and HSP90, it may function with HSP90 to stabilise AR protein from being degraded by proteasome. Conclusions: In this study, we show that BCAS2 is a novel AR-interacting protein and characterise the correlation between BCAS2 and PCa. Thus we propose that BCAS2 could be a diagnostic marker and therapeutic target for PCa. PMID:25461807

  7. Heteromultimerization modulates P2X receptor functions through participating extracellular and C-terminal subdomains.

    PubMed

    Koshimizu, Taka-aki; Ueno, Susumu; Tanoue, Akito; Yanagihara, Nobuyuki; Stojilkovic, Stanko S; Tsujimoto, Gozoh

    2002-12-06

    P2X purinergic receptors (P2XRs) differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. Here we show that homomeric receptors having the extracellular domain of the P2X(3) subunit in the P2X(2a)-based backbone (P2X(2a)/X(3)ex) mimicked two intrinsic functions of P2X(3)R, sensitivity to alphabeta-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X(3) extracellular loop, respectively. The chimeric P2X(2a)R/X(3)ex receptors also desensitized with accelerated rates compared with native P2X(2a)R, and the introduction of P2X(2) C-terminal splicing into the chimeric subunit (P2X(2b)/X(3)ex) further increased the rate of desensitization. Physical and functional heteromerization of native P2X(2a) and P2X(2b) subunits was also demonstrated. In heteromeric receptors, the ectodomain of P2X(3) was a structural determinant for ligand selectivity and recovery from desensitization, and the C terminus of P2X(2) was an important factor for the desensitization rate. Furthermore, [gamma-(32)P]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X(3) subunit in homomeric receptors but not in heteromeric P2X(2) + P2X(3)Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.

  8. Expression of alpha-AR subtypes in T lymphocytes and role of the alpha-ARs in mediating modulation of T cell function.

    PubMed

    Bao, Jing-Yin; Huang, Yan; Wang, Feng; Peng, Yu-Ping; Qiu, Yi-Hua

    2007-01-01

    Previous work in our laboratory has shown that alpha-adrenoreceptors (alpha-ARs) and beta-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of alpha-AR subtypes, alpha(1)-AR and alpha(2)-AR mRNAs, in T lymphocytes and explored the roles of the alpha-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of alpha(1)-AR and alpha(2)-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. T cells expressed both alpha(1)-AR and alpha(2)-AR mRNAs. The expression of both alpha(1)-AR and alpha(2)-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective alpha(1)-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-gamma and IL-4 production induced by Con A. However, the selective alpha(2)-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-gamma and IL-4 production. The inhibited lymphocyte proliferation and IFN-gamma and IL-4 production by clonidine were blocked by yohimbine, an alpha(2)-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. T lymphocytes express

  9. The proteinase-activated receptor-2 mediates phagocytosis in a Rho-dependent manner in human keratinocytes.

    PubMed

    Scott, Glynis; Leopardi, Sonya; Parker, Lorelle; Babiarz, Laura; Seiberg, Miri; Han, Rujiing

    2003-09-01

    Recent work shows that the G-protein-coupled receptor proteinase activated receptor-2 activates signals that stimulate melanosome uptake in keratinocytes in vivo and in vitro. The Rho family of GTP-binding proteins is involved in cytoskeletal remodeling during phagocytosis. We show that proteinase-activated receptor-2 mediated phagocytosis in human keratinocytes is Rho dependent and that proteinase-activated receptor-2 signals to activate Rho. In contrast, Rho activity did not affect either proteinase-activated receptor-2 activity or mRNA and protein levels. We explored the signaling mechanisms of proteinase-activated receptor-2 mediated Rho activation in human keratinocytes and show that activation of proteinase-activated receptor-2, either through specific proteinase-activated receptor-2 activating peptides or through trypsinization, elevates cAMP in keratinocytes. Proteinase-activated receptor-2 mediated Rho activation was pertussis toxin insensitive and independent of the protein kinase A signaling pathway. These data are the first to show that proteinase-activated receptor-2 mediated phagocytosis is Rho dependent and that proteinase-activated receptor-2 signals to Rho and cAMP in keratinocytes. Because phagocytosis of melanosomes is recognized as an important mechanism for melanosome transfer to keratinocytes, these results suggest that Rho is a critical signaling intermediate in melanosome uptake in keratinocytes.

  10. Pharmacology of JB-9315, a new selective histamine H2-receptor antagonist.

    PubMed

    Palacios, B; Montero, M J; Sevilla, M A; San Román, L

    1998-02-01

    1. The histamine H2-receptor antagonistic activity and antisecretory and antiulcer effects of JB-9315 were studied in comparison with the standard H2 blocker ranitidine. 2. In vitro, JB-9315 is a competitive antagonist of histamine H2 receptors in the isolated, spontaneously beating guinea-pig right atrium, with a pA2 value of 7.30 relative to a value of 7.36 for ranitidine. JB-9315 was specific for the histamine H2 receptor because, at high concentration, it did not affect histamine- or acetylcholine-induced contractions in guinea-pig isolated ileum or rat isolated duodenum, respectively. 3. JB-9315 dose dependently inhibited histamine-, pentagastrin- or carbachol-stimulated acid secretion and basal secretion in the perfused stomach preparation of the anesthetized rat. In the pylorus-ligated rat after intraperitoneal administration, total acid output over 4 h was inhibited by JB-9315 with an ID50 of 32.8 mg/kg, confirming its H2-receptor antagonist properties. 4. JB-9315 showed antiulcer activity against cold stress plus indomethacin-induced lesions with an ID50 of 6.8 mg/kg. 5. JB-9315, 50 and 100 mg/kg, inhibited macroscopic gastric hemorrhagic lesions induced by ethanol. In contrast, ranitidine (50 mg/kg) failed to reduce these lesions. 6. These results indicate that JB-9315 is a new antiulcer drug that exerts a cytoprotective effect in addition to its gastric antisecretory activity.

  11. AmTAR2: Functional characterization of a honeybee tyramine receptor stimulating adenylyl cyclase activity.

    PubMed

    Reim, Tina; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang; Thamm, Markus; Scheiner, Ricarda

    2017-01-01

    The biogenic monoamines norepinephrine and epinephrine regulate important physiological functions in vertebrates. Insects such as honeybees do not synthesize these neuroactive substances. Instead, they employ octopamine and tyramine for comparable physiological functions. These biogenic amines activate specific guanine nucleotide-binding (G) protein-coupled receptors (GPCRs). Based on pharmacological data obtained on heterologously expressed receptors, α- and β-adrenergic-like octopamine receptors are better activated by octopamine than by tyramine. Conversely, GPCRs forming the type 1 tyramine receptor clade (synonymous to octopamine/tyramine receptors) are better activated by tyramine than by octopamine. More recently, receptors were characterized which are almost exclusively activated by tyramine, thus forming an independent type 2 tyramine receptor clade. Functionally, type 1 tyramine receptors inhibit adenylyl cyclase activity, leading to a decrease in intracellular cAMP concentration ([cAMP] i ). Type 2 tyramine receptors can mediate Ca 2+ signals or both Ca 2+ signals and effects on [cAMP] i . We here provide evidence that the honeybee tyramine receptor 2 (AmTAR2), when heterologously expressed in flpTM cells, exclusively causes an increase in [cAMP] i . The receptor displays a pronounced preference for tyramine over octopamine. Its activity can be blocked by a series of established antagonists, of which mianserin and yohimbine are most efficient. The functional characterization of two tyramine receptors from the honeybee, AmTAR1 (previously named AmTYR1) and AmTAR2, which respond to tyramine by changing cAMP levels in opposite direction, is an important step towards understanding the actions of tyramine in honeybee behavior and physiology, particularly in comparison to the effects of octopamine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Safety and Immunological Efficacy of a DNA Vaccine Encoding the Androgen Receptor Ligand-Binding Domain (AR-LBD).

    PubMed

    Olson, Brian M; Bradley, Eric S; Sawicki, Thomas; Zhong, Weixiong; Ranheim, Erik A; Bloom, Jordan E; Colluru, Viswa T; Johnson, Laura E; Rekoske, Brian T; Eickhoff, Jens C; McNeel, Douglas G

    2017-05-01

    The androgen receptor (AR) is a key oncogenic driver of prostate cancer, and has been the primary focus of prostate cancer treatment for several decades. We have previously demonstrated that the AR is also an immunological target antigen, recognized in patients with prostate cancer, and targetable by means of vaccines in rodent models with delays in prostate tumor growth. The current study was performed to determine the safety and immunological efficacy of a GMP-grade plasmid DNA vaccine encoding the ligand-binding domain (LBD) of the AR, pTVG-AR. Groups of male mice (n = 6-10 per group) were evaluated after four or seven immunizations, using different schedules and inclusion of GM-CSF as a vaccine adjuvant. Animals were assessed for toxicity using gross observations, pathological analysis, and analysis of serum chemistries. Animals were analyzed for evidence of vaccine-augmented immunity by tetramer analysis. Survival studies using different immunization schedules and inclusion of GM-CSF were conducted in an autochthonous genetically engineered mouse model. No significant toxicities were observed in terms of animal weights, histopathology, hematological changes, or changes in serum chemistries, although there was a trend to lower serum glucose in animals treated with the vaccine. There was specifically no evidence of toxicity in other tissues that express AR, including liver, muscle, hematopoietic, and brain. Vaccination was found to elicit AR LBD-specific CD8+ T cells. In a subsequent study of tumor-bearing animals, animals treated with vaccine had prolonged survival compared with control-immunized mice. These studies demonstrate that, in immunocompetent mice expressing the target antigen, immunization with the pTVG-AR vaccine was both safe and effective in eliciting AR-specific cellular immune responses, and prolonged the survival of prostate tumor-bearing mice. These findings support the clinical evaluation of pTVG-AR in patients with recurrent prostate

  13. The antiandrogenic effect of finasteride against a mutant androgen receptor

    PubMed Central

    Chhipa, Rishi Raj; Zhang, Haitao; Ip, Clement

    2011-01-01

    Finasteride is known to inhibit Type 2 5α-reductase and thus block the conversion of testosterone to dihydrotestosterone (DHT). The structural similarity of finasteride to DHT raises the possibility that finasteride may also interfere with the function of the androgen receptor (AR). Experiments were carried out to evaluate the antiandrogenic effect of finasteride in LNCaP, C4-2 and VCaP human prostate cancer cells. Finasteride decreased DHT binding to AR, and DHT-stimulated AR activity and cell growth in LNCaP and C4-2 cells, but not in VCaP cells. LNCaP and C4-2 (derived from castration-resistant LNCaP) cells express the T877A mutant AR, while VCaP cells express the wild-type AR. When PC-3 cells, which are AR-null, were transfected with either the wild-type or the T877A mutant AR, only the mutant AR-expressing cells were sensitive to finasteride inhibition of DHT binding. Peroxiredoxin-1 (Prx1) is a novel endogenous facilitator of AR binding to DHT. In Prx1-rich LNCaP cells, the combination of Prx1 knockdown and finasteride was found to produce a greater inhibitory effect on AR activity and cell growth than either treatment alone. The observation suggests that cells with a low expression of Prx1 are likely to be more responsive to the antiandrogenic effect of finasteride. Additional studies showed that the efficacy of finasteride was comparable to that of bicalutamide (a widely used non-steroidal antiandrogen). The implication of the above findings is discussed in the context of developing strategies to improve the outcome of androgen deprivation therapy. PMID:21386657

  14. Aberrant dopamine D2-like receptor function in a rodent model of schizophrenia.

    PubMed

    Perez, Stephanie M; Lodge, Daniel J

    2012-11-01

    Based on the observation that antipsychotic medications display antagonist properties at dopamine D2-like receptors, aberrant dopamine signaling has been proposed to underlie psychosis in patients with schizophrenia. Thus, it is not surprising that considerable research has been devoted to understanding the mechanisms involved in the antipsychotic action of these compounds. It is important to note that the majority of these studies have been performed in "normal" experimental animals. Given that these animals do not possess the aberrant neuronal information processing typically associated with schizophrenia, the aim of the current study was to examine the dopamine D2 receptor system in a rodent model of schizophrenia. Here, we demonstrate that methylazoxymethanol acetate (MAM)-treated rats display an enhanced effect of quinpirole on dopamine neuron activity and an aberrant locomotor response to D2-like receptor activation, suggesting changes in postsynaptic D2-like receptor function. To better understand the mechanisms underlying the enhanced response to D2-like ligands in MAM-treated rats, we examined the expression of D2, D3, and dopamine transporter mRNA in the nucleus accumbens and ventral tegmental area by quantitative reverse transcription-polymerase chain reaction. MAM-treated rats displayed a significant increase in dopamine D3 receptor mRNA expression in the nucleus accumbens with no significant changes in the expression of the D2 receptor. Taken together, these data demonstrate robust alterations in dopamine D2-like receptor function in a rodent model of schizophrenia and provide evidence that preclinical studies examining the mechanisms of antipsychotic drug action should be performed in animal models that mirror aspects of the abnormal neuronal transmission thought to underlie symptoms of schizophrenia.

  15. Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression*

    PubMed Central

    Coke, Christopher J.; Scarlett, Kisha A.; Chetram, Mahandranauth A.; Jones, Kia J.; Sandifer, Brittney J.; Davis, Ahriea S.; Marcus, Adam I.

    2016-01-01

    The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. PMID:26841863

  16. Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

    PubMed Central

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

    2012-01-01

    The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

  17. Role of detergents in conformational exchange of a G protein-coupled receptor.

    PubMed

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

    2012-10-19

    The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

  18. Discovery of (1R,2S)-2-{[(2,4-Dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006): A Potent and Efficacious Oral Orexin Receptor Antagonist.

    PubMed

    Yoshida, Yu; Naoe, Yoshimitsu; Terauchi, Taro; Ozaki, Fumihiro; Doko, Takashi; Takemura, Ayumi; Tanaka, Toshiaki; Sorimachi, Keiichi; Beuckmann, Carsten T; Suzuki, Michiyuki; Ueno, Takashi; Ozaki, Shunsuke; Yonaga, Masahiro

    2015-06-11

    The orexin/hypocretin receptors are a family of G protein-coupled receptors and consist of orexin-1 (OX1) and orexin-2 (OX2) receptor subtypes. Orexin receptors are expressed throughout the central nervous system and are involved in the regulation of the sleep/wake cycle. Because modulation of these receptors constitutes a promising target for novel treatments of disorders associated with the control of sleep and wakefulness, such as insomnia, the development of orexin receptor antagonists has emerged as an important focus in drug discovery research. Here, we report the design, synthesis, characterization, and structure-activity relationships (SARs) of novel orexin receptor antagonists. Various modifications made to the core structure of a previously developed compound (-)-5, the lead molecule, resulted in compounds with improved chemical and pharmacological profiles. The investigation afforded a potential therapeutic agent, (1R,2S)-2-{[(2,4-dimethylpyrimidin-5-yl)oxy]methyl}-2-(3-fluorophenyl)-N-(5-fluoropyridin-2-yl)cyclopropanecarboxamide (E2006), an orally active, potent orexin antagonist. The efficacy was demonstrated in mice in an in vivo study by using sleep parameter measurements.

  19. Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer.

    PubMed

    Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B; Sikka, Suresh C; Mondal, Debasis

    2017-11-01

    Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5-20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those

  20. Multimodal actions of the phytochemical sulforaphane suppress both AR and AR-V7 in 22Rv1 cells: Advocating a potent pharmaceutical combination against castration-resistant prostate cancer

    PubMed Central

    Khurana, Namrata; Kim, Hogyoung; Chandra, Partha K.; Talwar, Sudha; Sharma, Pankaj; Abdel-Mageed, Asim B.; Sikka, Suresh C.; Mondal, Debasis

    2017-01-01

    Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P<0.05). Immunoblot analyses showed that SFN (5–20 µM) rapidly decreases both AR-FL and AR-V7 levels, and immunofluorescence microscopy (IFM) depicted decreased AR in both cytoplasm and nucleus with SFN treatment. SFN increased both ubiquitination and proteasomal activity in 22Rv1 cells. Studies using a protein synthesis inhibitor (cycloheximide) or a proteasomal inhibitor (MG132) indicated that SFN increases both ubiquitin-mediated aggregation and subsequent proteasomal-degradation of AR proteins. Previous studies reported that SFN inhibits the chaperone activity of heat-shock protein 90 (Hsp90) and induces the nuclear factor erythroid-2-like 2 (Nrf2) transcription factor. Therefore, we investigated whether the Hsp90 inhibitor, ganetespib (G) or the Nrf2 activator, bardoxolone methyl (BM) can similarly suppress AR levels in 22Rv1 cells. Low doses of G and BM, alone or in combination, decreased both AR-FL and AR-V7 levels, and combined exposure to G+BM sensitized 22Rv1 cells to ENZ. Therefore, adjunct treatment with the phytochemical SFN or a safe pharmaceutical combination of G+BM may be effective against CRPC cells, especially those

  1. 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.

  2. In search of selective P2 receptor ligands: interaction of dihydropyridine derivatives at recombinant rat P2X(2) receptors.

    PubMed

    Jacobson, K A; Kim, Y C; King, B F

    2000-07-03

    1,4-Dihydropyridines are regarded as privileged structures for drug design, i.e. they tend to bind to a wide variety of receptor sites. We have shown that upon appropriate manipulation of the substituent groups on a 1,4-dihydropyridine template, high affinity and selectivity for the A(3) subtype of adenosine receptors ('P1 receptors') may be attained. In the present study we have begun to extend this approach to P2 receptors which are activated by ATP and other nucleotides. Nicardipine, a representative dihydropyridine, used otherwise as an L-type calcium channel blocker, was shown to be an antagonist at recombinant rat P2X(2) (IC(50)=25 microM) and P2X(4) (IC(50) approximately 220 microM) receptors expressed in Xenopus oocytes. Thus, this class of compounds represents a suitable lead for enhancement of affinity through chemical synthesis. In an attempt to modify the 1,4-dihydropyridine structure with a predicted P2 receptor recognition moiety, we have replaced one of the ester groups with a negatively charged phosphonate group. Several 4-phenyl-5-phosphonato-1,4-dihydropyridine derivatives, MRS 2154 (2, 6-dimethyl), MRS 2155 (6-methyl-2-phenyl), and MRS 2156 (2-methyl-6-phenyl), were synthesized through three component condensation reactions. These derivatives were not pure antagonists of the effects of ATP at P2X(2) receptors, rather were either inactive (MRS 2156) or potentiated the effects of ATP in a concentration-dependent manner (MRS 2154 in the 0.3-10 microM range and MRS 2155 at >1 microM). Antagonism of the effects of ATP at P2X(2) receptor superimposed on the potentiation was also observed at >10 microM (MRS 2154) or 0.3-1 microM (MRS 2155). Thus, while a conventional dihydropyridine, nicardipine, was found to antagonize rat P2X(2) receptors ninefold more potently than P2X(4) receptors, the effects of novel, anionic 5-phosphonate analogues at the receptor were more complex.

  3. 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

  4. 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.

  5. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    PubMed Central

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  6. 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.

  7. Selective modulation of the androgen receptor AF2 domain rescues degeneration in spinal bulbar muscular atrophy.

    PubMed

    Badders, Nisha M; Korff, Ane; Miranda, Helen C; Vuppala, Pradeep K; Smith, Rebecca B; Winborn, Brett J; Quemin, Emmanuelle R; Sopher, Bryce L; Dearman, Jennifer; Messing, James; Kim, Nam Chul; Moore, Jennifer; Freibaum, Brian D; Kanagaraj, Anderson P; Fan, Baochang; Tillman, Heather; Chen, Ping-Chung; Wang, Yingzhe; Freeman, Burgess B; Li, Yimei; Kim, Hong Joo; La Spada, Albert R; Taylor, J Paul

    2018-05-01

    Spinal bulbar muscular atrophy (SBMA) is a motor neuron disease caused by toxic gain of function of the androgen receptor (AR). Previously, we found that co-regulator binding through the activation function-2 (AF2) domain of AR is essential for pathogenesis, suggesting that AF2 may be a potential drug target for selective modulation of toxic AR activity. We screened previously identified AF2 modulators for their ability to rescue toxicity in a Drosophila model of SBMA. We identified two compounds, tolfenamic acid (TA) and 1-[2-(4-methylphenoxy)ethyl]-2-[(2-phenoxyethyl)sulfanyl]-1H-benzimidazole (MEPB), as top candidates for rescuing lethality, locomotor function and neuromuscular junction defects in SBMA flies. Pharmacokinetic analyses in mice revealed a more favorable bioavailability and tissue retention of MEPB compared with TA in muscle, brain and spinal cord. In a preclinical trial in a new mouse model of SBMA, MEPB treatment yielded a dose-dependent rescue from loss of body weight, rotarod activity and grip strength. In addition, MEPB ameliorated neuronal loss, neurogenic atrophy and testicular atrophy, validating AF2 modulation as a potent androgen-sparing strategy for SBMA therapy.

  8. Expression of AR, 5αR1 and 5αR2 in bladder urothelial carcinoma and relationship to clinicopathological factors.

    PubMed

    Hata, Shuko; Ise, Kazue; Azmahani, Abdullah; Konosu-Fukaya, Sachiko; McNamara, Keely May; Fujishima, Fumiyoshi; Shimada, Keiji; Mitsuzuka, Koji; Arai, Yoichi; Sasano, Hironobu; Nakamura, Yasuhiro

    2017-12-01

    Bladder urothelial carcinoma is increasing in incidence with age and its prognosis could become worse when accompanied with metastasis. Effective treatment of these advanced patients is required and it becomes important to understand its underlying biology of this neoplasm, especially with regard to its biological pathways. A potential proposed pathway is androgen receptor (AR)-mediated intracellular signaling but the details have remained relatively unexplored. The expression of AR, 5α-reductase type1 (5αR1) and 5α-reductase type2 (5αR2) were examined in the bladder cancer cell line T24 and surgical pathology specimens. We also evaluated the status of androgen related cell proliferation and migration using the potent, non-aromatizable androgen agonist 5α-dihydrotestosterone (DHT). DHT treatment significantly increased AR mRNA expression level, but not those of 5αR1 and 5αR2 in T24 cells. DHT also suppressed cellular migration with weaker and opposite effects on cell proliferation. A significant inverse correlation was detected between pT stage and AR, 5αR1 and 5αR2 immunoreactivity. Inverse correlations detected between tumor grade and AR/androgen metabolizing enzyme also suggested that the loss of AR and androgen-producing enzymes could be associated with tumor progression. Effects of DHT on cells also suggest that androgens may regulate cellular behavior. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Selective and non-selective OT receptor agonists induce different locomotor behaviors in male rats via central OT receptors and peripheral V1a receptors.

    PubMed

    Wolfe, Monica; Wisniewska, Halina; Tariga, Hiroe; Ibanez, Gerardo; Collins, James C; Wisniewski, Kazimierz; Qi, Steve; Srinivasan, Karthik; Hargrove, Diane; Lindstrom, Beatriz Fioravanti

    2018-05-21

    Oxytocin (OT) continues to inspire much research due to its diverse physiological effects. While the best-understood actions of OT are uterine contraction and milk ejection, OT is also implicated in maternal and bonding behaviors, and potentially in CNS disorders such as autism, schizophrenia, and pain. The dissection of the mechanism of action of OT is complicated by the fact that this peptide activates not only its cognate receptor but also vasopressin type 1a (V1a) receptors. In this study, we evaluated OT and a selective OT receptor (OTR) agonist, FE 204409, in an automated assay that measures rat locomotor activity. The results showed: 1) Subcutaneous (sc) administration of OT decreased locomotor behavior (distance traveled, stereotypy, and rearing). This effect was reversed by a V1a receptor (V1aR) antagonist ([Pmp1,Tyr(ME)2]AVP, sc), suggesting that OT acts through peripheral V1aR to inhibit locomotor activity. 2) A selective OTR agonist (FE 204409, sc) increased stereotypy. This effect was reversed by an OTR antagonist dosed icv, suggesting a central OTR site of action. Our findings identify distinct behavioral effects for OT and the selective agonist FE 204409, adding to the growing body of evidence that the V1aR mediates many effects attributed to OT and that peptides administered systemically at supra-physiological doses may activate receptors in the brain. Our studies further emphasize the importance of utilizing selective agonists and antagonists to assess therapeutic indications. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Europium-Labeled Synthetic C3a Protein as a Novel Fluorescent Probe for Human Complement C3a Receptor.

    PubMed

    Dantas de Araujo, Aline; Wu, Chongyang; Wu, Kai-Chen; Reid, Robert C; Durek, Thomas; Lim, Junxian; Fairlie, David P

    2017-06-21

    Measuring ligand affinity for a G protein-coupled receptor is often a crucial step in drug discovery. It has been traditionally determined by binding putative new ligands in competition with native ligand labeled with a radioisotope of finite lifetime. Competing instead with a lanthanide-based fluorescent ligand is more attractive due to greater longevity, stability, and safety. Here, we have chemically synthesized the 77 residue human C3a protein and conjugated its N-terminus to europium diethylenetriaminepentaacetate to produce a novel fluorescent protein (Eu-DTPA-hC3a). Time-resolved fluorescence analysis has demonstrated that Eu-DTPA-hC3a binds selectively to its cognate G protein-coupled receptor C3aR with full agonist activity and similar potency and selectivity as native C3a in inducing calcium mobilization and phosphorylation of extracellular signal-regulated kinases in HEK293 cells that stably expressed C3aR. Time-resolved fluorescence analysis for saturation and competitive binding gave a dissociation constant (K d ) of 8.7 ± 1.4 nM for Eu-DTPA-hC3a and binding affinities for hC3a (pK i of 8.6 ± 0.2 and K i of 2.5 nM) and C3aR ligands TR16 (pK i of 6.8 ± 0.1 and K i of 138 nM), BR103 (pK i of 6.7 ± 0.1 and K i of 185 nM), BR111 (pK i of 6.3 ± 0.2 and K i of 544 nM) and SB290157 (pK i of 6.3 ± 0.1 and K i of 517 nM) via displacement of Eu-DTPA-hC3a from hC3aR. The macromolecular conjugate Eu-DTPA-hC3a is a novel nonradioactive probe suitable for studying ligand-C3aR interactions with potential value in accelerating drug development for human C3aR in physiology and disease.

  11. Computational Study on the Different Ligands Induced Conformation Change of β2 Adrenergic Receptor-Gs Protein Complex

    PubMed Central

    Bai, Qifeng; Zhang, Yang; Ban, Yihe; Liu, Huanxiang; Yao, Xiaojun

    2013-01-01

    β2 adrenergic receptor2AR) regulated many key physiological processes by activation of a heterotrimeric GTP binding protein (Gs protein). This process could be modulated by different types of ligands. But the details about this modulation process were still not depicted. Here, we performed molecular dynamics (MD) simulations on the structures of β2AR-Gs protein in complex with different types of ligands. The simulation results demonstrated that the agonist BI-167107 could form hydrogen bonds with Ser2035.42, Ser2075.46 and Asn2936.55 more than the inverse agonist ICI 118,551. The different binding modes of ligands further affected the conformation of β2AR. The energy landscape profiled the energy contour map of the stable and dissociated conformation of Gαs and Gβγ when different types of ligands bound to β2AR. It also showed the minimum energy pathway about the conformational change of Gαs and Gβγ along the reaction coordinates. By using interactive essential dynamics analysis, we found that Gαs and Gβγ domain of Gs protein had the tendency to separate when the inverse agonist ICI 118,551 bound to β2AR. The α5-helix had a relatively quick movement with respect to transmembrane segments of β2AR when the inverse agonist ICI 118,551 bound to β2AR. Besides, the analysis of the centroid distance of Gαs and Gβγ showed that the Gαs was separated from Gβγ during the MD simulations. Our results not only could provide details about the different types of ligands that induced conformational change of β2AR and Gs protein, but also supplied more information for different efficacies of drug design of β2AR. PMID:23922653

  12. 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.

  13. 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors.

    PubMed

    Newell, Elizabeth A; Exo, Jennifer L; Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Kochanek, Patrick M; Jackson, Edwin K

    2015-01-12

    Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 μM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 μM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 μM; agonist for all adenosine receptor subtypes) and CGS21680 (10 μM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Discovery of a Novel Series of CRTH2 (DP2) Receptor Antagonists Devoid of Carboxylic Acids

    PubMed Central

    2011-01-01

    Antagonism of the CRTH2 receptor represents a very attractive target for a variety of allergic diseases. Most CRTH2 antagonists known to date possess a carboxylic acid moiety, which is essential for binding. However, potential acid metabolites O-acyl glucuronides might be linked to idiosynchratic toxicity in humans. In this communication, we describe a new series of compounds that lack the carboxylic acid moiety. Compounds with high affinity (Ki < 10 nM) for the receptor have been identified. Subsequent optimization succeeded in reducing the high metabolic clearance of the first compounds in human and rat liver microsomes. At the same time, inhibition of the CYP isoforms was optimized, giving rise to stable compounds with an acceptable CYP inhibition profile (IC50 CYP2C9 and 2C19 > 1 μM). Taken together, these data show that compounds devoid of carboxylic acid groups could represent an interesting alternative to current CRTH2 antagonists in development. PMID:24900284

  15. The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation.

    PubMed

    Nakata, Daisuke; Nakao, Shoichi; Nakayama, Kazuhide; Araki, Shinsuke; Nakayama, Yusuke; Aparicio, Samuel; Hara, Takahito; Nakanishi, Atsushi

    2017-01-29

    Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. The Hinge Region as a Key Regulatory Element of Androgen Receptor Dimerization, DNA Binding and Transactivation

    DTIC Science & Technology

    2006-05-01

    Mutations in the human androgen receptor gene as a learning tool for molecular endocrinology’ III. Poster presentations at international meetings...nonconsensus half-site, the cognate half-complex buries slightly more surface area from solvent (1,230 Å2) than the noncognate one (960 Å2). AR Mutations ...energetic penalty in- Fig. 4. (A) The AR DBD dimer interface. The molecular surfaces of the AR subunits are shown in red and blue. Dashed black lines

  17. Simultaneous Activation of Induced Heterodimerization between CXCR4 Chemokine Receptor and Cannabinoid Receptor 2 (CB2) Reveals a Mechanism for Regulation of Tumor Progression.

    PubMed

    Coke, Christopher J; Scarlett, Kisha A; Chetram, Mahandranauth A; Jones, Kia J; Sandifer, Brittney J; Davis, Ahriea S; Marcus, Adam I; Hinton, Cimona V

    2016-05-06

    The G-protein-coupled chemokine receptor CXCR4 generates signals that lead to cell migration, cell proliferation, and other survival mechanisms that result in the metastatic spread of primary tumor cells to distal organs. Numerous studies have demonstrated that CXCR4 can form homodimers or can heterodimerize with other G-protein-coupled receptors to form receptor complexes that can amplify or decrease the signaling capacity of each individual receptor. Using biophysical and biochemical approaches, we found that CXCR4 can form an induced heterodimer with cannabinoid receptor 2 (CB2) in human breast and prostate cancer cells. Simultaneous, agonist-dependent activation of CXCR4 and CB2 resulted in reduced CXCR4-mediated expression of phosphorylated ERK1/2 and ultimately reduced cancer cell functions such as calcium mobilization and cellular chemotaxis. Given that treatment with cannabinoids has been shown to reduce invasiveness of cancer cells as well as CXCR4-mediated migration of immune cells, it is plausible that CXCR4 signaling can be silenced through a physical heterodimeric association with CB2, thereby inhibiting subsequent functions of CXCR4. Taken together, the data illustrate a mechanism by which the cannabinoid system can negatively modulate CXCR4 receptor function and perhaps tumor progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. 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

  19. MEIS1 functions as a potential AR negative regulator

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cui, Liang; Department of Urology, Civil Aviation General Hospital/Civil Aviation Medical College of Peking University, Beijing 100123; Li, Mingyang

    2014-10-15

    The androgen receptor (AR) plays critical roles in human prostate carcinoma progression and transformation. However, the activation of AR is regulated by co-regulators. MEIS1 protein, the homeodomain transcription factor, exhibited a decreased level in poor-prognosis prostate tumors. In this study, we investigated a potential interaction between MEIS1 and AR. We found that overexpression of MEIS1 inhibited the AR transcriptional activity and reduced the expression of AR target gene. A potential protein–protein interaction between AR and MEIS1 was identified by the immunoprecipitation and GST pull-down assays. Furthermore, MEIS1 modulated AR cytoplasm/nucleus translocation and the recruitment to androgen response element in prostatemore » specific antigen (PSA) gene promoter sequences. In addition, MEIS1 promoted the recruitment of NCoR and SMRT in the presence of R1881. Finally, MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells. Taken together, our data suggests that MEIS1 functions as a novel AR co-repressor. - Highlights: • A potential interaction was identified between MEIS1 and AR signaling. • Overexpression of MEIS1 reduced the expression of AR target gene. • MEIS1 modulated AR cytoplasm/nucleus translocation. • MEIS1 inhibited the proliferation and anchor-independent growth of LNCaP cells.« less

  20. 5-HT2A receptor gene polymorphisms in Croatian subjects with autistic disorder.

    PubMed

    Hranilovic, Dubravka; Blazevic, Sofia; Babic, Marina; Smurinic, Maja; Bujas-Petkovic, Zorana; Jernej, Branimir

    2010-08-15

    Disturbances in the expression/function of the 5-HT2A receptor are implicated in autism. The association of the 5-HT2A receptor gene with autism was studied in the Croatian population. Distribution frequencies for alleles, genotypes and haplotypes of -1438 A/G and His452Tyr polymorphisms were compared in samples of 103 autistic and 214 control subjects. Significant overrepresentation of the G allele and the GG genotype of the -1438 A/G polymorphism was observed in group of autistic subjects, supporting the possible involvement of the 5-HT2A receptor in the development of autism. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  1. Accretion disks around neutron and strange stars in R + aR {sup 2} gravity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Staykov, Kalin V.; Yazadjiev, Stoytcho S.; Doneva, Daniela D., E-mail: kstaykov@phys.uni-sofia.bg, E-mail: daniela.doneva@uni-tuebingen.de, E-mail: yazad@phys.uni-sofia.bg

    2016-08-01

    We study the electromagnetic spectrum of accretion disks around neutron and strange stars in R + aR {sup 2} gravity. Both static and rapidly rotating models are investigated. The results are compared with the General Relativistic results. We found difference between the results in both theories of about 50% for the electromagnetic flux and about 20% in the luminosity for models with equal mass and angular velocity in both theories. The observed differences are much lower for models rotating with Keplerian velocity and with equal masses.

  2. 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.

  3. Predictions Suggesting a Participation of β-Sheet Configuration in the M2 Domain of the P2X7 Receptor: A Novel Conformation?

    PubMed Central

    Teixeira, Pedro Celso Nogueira; de Souza, Cristina Alves Magalhães; de Freitas, Mônica Santos; Foguel, Débora; Caffarena, Ernesto Raul; Alves, Luiz Anastacio

    2009-01-01

    Scanning experiments have shown that the putative TM2 domain of the P2X7 receptor (P2X7R) lines the ionic pore. However, none has identified an α-helix structure, the paradigmatic secondary structure of ion channels in mammalian cells. In addition, some researchers have suggested a β-sheet conformation in the TM2 domain of P2X2. These data led us to investigate a new architecture within the P2X receptor family. P2X7R is considered an intriguing receptor because its activation induces nonselective large pore formation, in contrast to the majority of other ionic channel proteins in mammals. This receptor has two states: a low-conductance channel (∼10 pS) and a large pore (>400 pS). To our knowledge, one fundamental question remains unanswered: Are the P2X7R channel and the pore itself the same entity or are they different structures? There are no structural data to help solve this question. Thus, we investigated the hydrophobic M2 domain with the aim of predicting the fitted position and the secondary structure of the TM2 segment from human P2X7R (hP2X7R). We provide evidence for a β-sheet conformation, using bioinformatics algorithms and molecular-dynamics simulation in conjunction with circular dichroism in different environments and Fourier transform infrared spectroscopy. In summary, our study suggests the possibility that a segment composed of residues from part of the M2 domain and part of the putative TM2 segment of P2X7R is partially folded in a β-sheet conformation, and may play an important role in channel/pore formation associated with P2X7R activation. It is important to note that most nonselective large pores have a transmembrane β-sheet conformation. Thus, this study may lead to a paradigmatic change in the P2X7R field and/or raise new questions about this issue. PMID:19186133

  4. 2.7 MeV Ar11+ ion irradiation induced structural evolution in Lu2(Ti2-xLux)O7-x/2 pyrochlores

    NASA Astrophysics Data System (ADS)

    Yang, D. Y.; Liu, C. G.; Zhang, K. Q.; Xia, Y.; Chen, L. J.; Liu, H.; Li, Y. H.

    2015-11-01

    This paper aims to study the radiation effects of nonstoichiometric pyrochlore series Lu2(Ti2-xLux)O7-x/2 (x = 0-0.667). Polycrystalline Lu2(Ti2-xLux)O7-x/2 samples were irradiated with 2.7 MeV Ar11+ ions up to a fluence of 8 × 1014 ions/cm2. The irradiated samples were characterized using grazing incidence X-ray diffraction technique. The results reveal that Lu2(Ti2-xLux)O7-x/2 samples undergo significant amorphization and lattice swelling upon irradiation. Specifically, the amorphization process is predominantly driven by ballistic nuclear energy deposition of Ar11+ ions at this energy regime, which can be well described by a direct-impact/defect-stimulated model. Both the amorphization fraction and the relative variation of lattice parameter decrease with increasing x, showing a strong dependence on the chemical composition. The results are then discussed in the framework of the structural disorder and recovery ability from damage, applying an atomic layer model.

  5. Psychotropic and nonpsychotropic cannabis derivatives inhibit human 5-HT(3A) receptors through a receptor desensitization-dependent mechanism.

    PubMed

    Xiong, W; Koo, B-N; Morton, R; Zhang, L

    2011-06-16

    Δ⁹ tetrahydrocannabinol (THC) and cannabidiol (CBD) are the principal psychoactive and nonpsychoactive components of cannabis. While most THC-induced behavioral effects are thought to depend on endogenous cannabinoid 1 (CB1) receptors, the molecular targets for CBD remain unclear. Here, we report that CBD and THC inhibited the function of human 5-HT(3A) receptors (h5-HT(3A)Rs) expressed in HEK 293 cells. The magnitude of THC and CBD inhibition was maximal 5 min after a continuous incubation with cannabinoids. The EC₅₀ values for CBD and THC-induced inhibition were 110 nM and 322 nM, respectively in HEK 293 cells expressing h5-HT(3A)Rs. In these cells, CBD and THC did not stimulate specific [³⁵S]-GTP-γs binding in membranes, suggesting that the inhibition by cannabinoids is unlikely mediated by a G-protein dependent mechanism. On the other hand, both CBD and THC accelerated receptor desensitization kinetics without significantly changing activation time. The extent of cannabinoid inhibition appeared to depend on receptor desensitization. Reducing receptor desensitization by nocodazole, 5-hydroxyindole and a point-mutation in the large cytoplasmic domain of the receptor significantly decreased CBD-induced inhibition. Similarly, the magnitude of THC and CBD-induced inhibition varied with the apparent desensitization rate of h5-HT(3A)Rs expressed in Xenopus oocytes. For instance, with increasing amount of h5-HT(3A)R cRNA injected into the oocytes, the receptor desensitization rate at steady state decreased. THC and CBD-induced inhibition was correlated with the change in the receptor desensitization rate. Thus, CBD and THC inhibit h5-HT(3A) receptors through a mechanism that is dependent on receptor desensitization. Published by Elsevier Ltd.

  6. The Activating Human NK Cell Receptor KIR2DS2 Recognizes a β2-Microglobulin-Independent Ligand on Cancer Cells.

    PubMed

    Thiruchelvam-Kyle, Lavanya; Hoelsbrekken, Sigurd E; Saether, Per C; Bjørnsen, Elisabeth Gyllensten; Pende, Daniela; Fossum, Sigbjørn; Daws, Michael R; Dissen, Erik

    2017-04-01

    The functions of activating members of the killer cell Ig-like receptor (KIR) family are not fully understood, as the ligands for these receptors are largely unidentified. In this study, we report that KIR2DS2 reporter cells recognize a ligand expressed by cancer cell lines. All cancer targets recognized by KIR2DS2 were also recognized by KIR2DL2 and KIR2DL3 reporters. Trogocytosis of membrane proteins from the cancer targets was observed with responding reporter cells, indicating the formation of KIR2DS2 ligand-specific immunological synapses. HLA-C typing of target cells showed that KIR2DS2 recognition was independent of the HLA C1 or C2 group, whereas targets cells that were only recognized by KIR2DL3 expressed C1 group alleles. Anti-HLA class I Abs blocked KIR2DL3 responses toward C1-expressing targets, but they did not block KIR2DS2 recognition of cancer cells. Small interfering RNA knockdown of β 2 -microglobulin reduced the expression of class I H chain on the cancer targets by >97%, but it did not reduce the KIR2DS2 reporter responses, indicating a β 2 -microglobulin-independent ligand for KIR2DS2. Importantly, KIR2DL3 responses toward some KIR2DS2 ligand-expressing cells were also undiminished after β 2 -microglobulin knockdown, and they were not blocked by anti-HLA class I Abs, suggesting that KIR2DL3, in addition to the traditional HLA-C ligands, can bind to the same β 2 -microglobulin-independent ligand as KIR2DS2. These observations indicate the existence of a novel, presently uncharacterized ligand for the activating NK cell receptor KIR2DS2. Molecular identification of this ligand may lead to improved KIR-HLA mismatching in hematopoietic stem cell transplantation therapy for leukemia and new, more specific NK cell-based cancer therapies. Copyright © 2017 by The American Association of Immunologists, Inc.

  7. Old dance with a new partner: EGF receptor as the phenobarbital receptor mediating Cyp2B expression.

    PubMed

    Meyer, Sharon A; Jirtle, Randy L

    2013-05-07

    The decades-long quest for the phenobarbital (PhB) receptor that mediates activation of Cyp2B would appear fulfilled with the discovery by Mutoh et al., who found that PhB binds with pharmacological affinity to the epidermal growth factor receptor (EGFR). This finding provides a molecular basis for the suppression of hepatocyte EGFR signaling observed with PhB treatment, as previously noted in the context of tumor promotion. Although the PhB-mediated induction of Cyp2B expression through the association of a canonical nuclear receptor with the 5'-enhancer PBREM of Cyp2B is well known, direct binding of PhB to constitutive active androstane receptor (CAR, also known as NR1I3) typical of other xenobiotic-activated nuclear receptors has eluded detection. One EGF-activated pathway affected by the PhB-EGFR interaction is the loss of tyrosine phosphorylation of the scaffold protein RACK1. Dephosphorylated RACK1 provides the mechanistic link between the binding of PhB to EGFR and its effects on CAR by facilitating the interaction of serine/threonine phosphatase PP2A with inactive phosphorylated CAR. The dephosphorylation of CAR enables its translocation to the nucleus and activation of Cyp2B expression. Because EGFR and transducers RACK1, PP2A, and other partners are highly networked in numerous cellular pathways, this newly discovered partnership will surely reveal new fundamental roles for PhB beyond the regulation of drug metabolism.

  8. α7 nicotinic ACh receptors as a ligand-gated source of Ca(2+) ions: the search for a Ca(2+) optimum.

    PubMed

    Uteshev, Victor V

    2012-01-01

    The spatiotemporal distribution of cytosolic Ca(2+) ions is a key determinant of neuronal behavior and survival. Distinct sources of Ca(2+) ions including ligand- and voltage-gated Ca(2+) channels contribute to intracellular Ca(2+) homeostasis. Many normal physiological and therapeutic neuronal functions are Ca(2+)-dependent, however an excess of cytosolic Ca(2+) or a lack of the appropriate balance between Ca(2+) entry and clearance may destroy cellular integrity and cause cellular death. Therefore, the existence of optimal spatiotemporal patterns of cytosolic Ca(2+) elevations and thus, optimal activation of ligand- and voltage-gated Ca(2+) ion channels are postulated to benefit neuronal function and survival. Alpha7 nicotinic -acetylcholine receptors (nAChRs) are highly permeable to Ca(2+) ions and play an important role in modulation of neurotransmitter release, gene expression and neuroprotection in a variety of neuronal and non-neuronal cells. In this review, the focus is placed on α7 nAChR-mediated currents and Ca(2+) influx and how this source of Ca(2+) entry compares to NMDA receptors in supporting cytosolic Ca(2+) homeostasis, neuronal function and survival.

  9. 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.

  10. A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

    PubMed

    Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

    2014-05-01

    The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

  11. αConotoxin ArIB[V11L,V16D] is a potent and selective antagonist at rat and human native α7 nicotinic acetylcholine receptors

    PubMed Central

    Innocent, Neal; Livingstone, Phil D.; Hone, Arik; Kimura, Atsuko; Young, Tracey; Whiteaker, Paul; McIntosh, J. Michael; Wonnacott, Susan

    2008-01-01

    A recently developed α-conotoxin, α-CtxArIB[V11L,V16D] is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues where they are implicated in a variety of functions. Here we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 in rat PC12 cells and human SHSY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli (5-iodo-A-85380, nicotine or KCl) that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]: ACh-evoked currents in X. laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the timecourse of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces: α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions. PMID:18664588

  12. Freud-2/CC2D1B mediates dual repression of the serotonin-1A receptor gene.

    PubMed

    Hadjighassem, Mahmoud R; Galaraga, Kimberly; Albert, Paul R

    2011-01-01

    The serotonin-1A (5-HT1A) receptor functions as a pre-synaptic autoreceptor in serotonin neurons that regulates their activity, and is also widely expressed on non-serotonergic neurons as a post-synaptic heteroreceptor to mediate serotonin action. The 5-HT1A receptor gene is strongly repressed by a dual repressor element (DRE), which is recognized by two proteins: Freud-1/CC2D1A and another unknown protein. Here we identify mouse Freud-2/CC2D1B as the second repressor of the 5-HT1A-DRE. Freud-2 shares 50% amino acid identity with Freud-1, and contains conserved structural domains. Mouse Freud-2 bound specifically to the rat 5-HT1A-DRE adjacent to, and partially overlapping, the Freud-1 binding site. By supershift assay using nuclear extracts from L6 myoblasts, Freud-2-DRE complexes were distinguished from Freud-1-DRE complexes. Freud-2 mRNA and protein were detected throughout mouse brain and peripheral tissues. Freud-2 repressed 5-HT1A promoter-reporter constructs in a DRE-dependent manner in non-neuronal (L6) or 5-HT1A-expressing neuronal (NG108-15, RN46A) cell models. In NG108-15 cells, knockdown of Freud-2 using a specific short-interfering RNA reduced endogenous Freud-2 protein levels and decreased Freud-2 bound to the 5-HT1A-DRE as detected by chromatin immunoprecipitation assay, but increased 5-HT1A promoter activity and 5-HT1A protein levels. Taken together, these data show that Freud-2 is the second component that, with Freud-1, mediates dual repression of the 5-HT1A receptor gene at the DRE. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  13. [(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

    PubMed

    Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

    2002-02-11

    This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

  14. Classics in Neuroimaging: The Serotonergic 2A Receptor System-from Discovery to Modern Molecular Imaging.

    PubMed

    T L'Estrade, Elina; Hansen, Hanne D; Erlandsson, Maria; Ohlsson, Tomas G; Knudsen, Gitte M; Herth, Matthias M

    2018-06-20

    Already in 1953, Woolley and Shaw speculated that serotonin could be involved in a range of central nervous system (CNS) disorders. Lysergic acid diethylamide (LSD) displayed an important role in this respect. It was used not only to antagonize biological effects of serotonin and to study the system itself, but also to identify serotonergic subtype receptors. The 5-HT 2A receptor was discovered in the 1970s and identified as the responsible receptor mediating psychedelic effects of LSD. The development of positron emission tomography (PET) allowed to study this receptor system in vivo. Parameters such as abundance of 5-HT 2A neuroreceptors or receptor occupancy can be determined using PET. As such, the development of 5-HT 2A receptor tracers started immediately after the introduction of PET in the mid-1970s. In this Viewpoint, we provide a historical overview from the discovery of serotonin to the identification of the 5-HT 2A receptor subtype and the subsequent development of 5-HT 2A receptor subtype specific PET tracers over the last four decades. We emphasize the interplay between pharmacology, medicinal chemistry, radiochemistry, and nuclear medicine that is important while developing a PET tracer. Moreover, we highlight selected examples applying 5-HT 2A receptor PET tracers within neurological diseases and drug occupancy studies.

  15. GSNO Reductase and β2 Adrenergic Receptor Gene-gene Interaction: Bronchodilator Responsiveness to Albuterol

    PubMed Central

    Choudhry, Shweta; Que, Loretta G.; Yang, Zhonghui; Liu, Limin; Eng, Celeste; Kim, Sung O.; Kumar, Gunjan; Thyne, Shannon; Chapela, Rocio; Rodriguez-Santana, Jose R.; Rodriguez-Cintron, William; Avila, Pedro C.; Stamler, Jonathan S.; Burchard, Esteban G.

    2010-01-01

    Background Short-acting inhaled β2-agonists such as albuterol are used for bronchodilation and are the mainstay of asthma treatment worldwide. There is significant variation in bronchodilator responsiveness to albuterol not only between individuals but also across racial/ethnic groups. The β2-adrenergic receptor2AR) is the target for β2-agonist drugs. The enzyme S-nitrosoglutathione reductase (GSNOR), which regulates levels of the endogenous bronchodilator S-nitrosoglutathione, has been shown to modulate the response to β2-agonists. Objective We hypothesized that there are pharmacogenetic interactions between GSNOR and β2AR gene variants which are associated with variable response to albuterol. Methods We performed family-based analyses to test for association between GSNOR gene variants and asthma and related phenotypes in 609 Puerto Rican and Mexican families with asthma. In addition, we tested these subjects for pharmacogenetic interaction between GSNOR and β2AR gene variants and responsiveness to albuterol using linear regression. Cell transfection experiments were performed to test the potential effect of the GSNOR gene variants. Results Among Puerto Ricans, several GSNOR SNPs and a haplotype in the 3′UTR were significantly associated with increased risk for asthma and lower bronchodilator responsiveness (p = 0.04 to 0.007). The GSNOR risk haplotype affects expression of GSNOR mRNA and protein, suggesting a gain of function. Furthermore, gene-gene interaction analysis provided evidence of pharmacogenetic interaction between GSNOR and β2AR gene variants and the response to albuterol in Puerto Rican (p = 0.03), Mexican (p = 0.15) and combined Puerto Rican and Mexican asthmatics (p = 0.003). Specifically, GSNOR+17059*β2AR+46 genotype combinations (TG+GG*AG and TG+GG*GG) were associated with lower bronchodilator response. Conclusion Genotyping of GSNOR and β2AR genes may be a useful in identifying Latino subjects, who might benefit from adjuvant

  16. Novel Confocal Microscopic and Flow Cytometric Based Assays to Visualize and Detect the (Beta)2-Adrenergic Receptor in Human Lymphocyte and Mononuclear Cell Populations

    NASA Technical Reports Server (NTRS)

    Salicru, A. N.; Crucian, B. E.; Nelman, M. A.; Sams, C. F.; Actor, J. K.; Marshall, G. D.

    2006-01-01

    The data show that immunophenotyping of leukocyte populations with (beta)2AR is possible with the commercially available Ab, although the FC assay is limited to the IST as a result of the Ab binding site to the intracellular C-terminus of the 2AR. The FC assay has applications for measuring alterations in total (beta)2AR in human leukocyte populations as changes in fluorescence. In addition, CM confirms that both surface and intracellular compartments stain positively for the (beta)2AR and can be used for qualitative assays that screen for changes in receptor compartmentalization and localization.

  17. 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

  18. Housing Temperature-Induced Stress Is Suppressing Murine Graft-versus-Host Disease through β2-Adrenergic Receptor Signaling.

    PubMed

    Leigh, Nicholas D; Kokolus, Kathleen M; O'Neill, Rachel E; Du, Wei; Eng, Jason W-L; Qiu, Jingxin; Chen, George L; McCarthy, Philip L; Farrar, J David; Cao, Xuefang; Repasky, Elizabeth A

    2015-11-15

    Graft-versus-host disease (GVHD) is the major complication of allogeneic hematopoietic cell transplantation, a potentially curative therapy for hematologic diseases. It has long been thought that murine bone marrow-derived T cells do not mediate severe GVHD because of their quantity and/or phenotype. During the course of experiments testing the impact of housing temperatures on GVHD, we discovered that this apparent resistance is a function of the relatively cool ambient housing temperature. Murine bone marrow-derived T cells have the ability to mediate severe GVHD in mice housed at a thermoneutral temperature. Specifically, mice housed at Institutional Animal Care and Use Committee-mandated, cool standard temperatures (∼ 22°C) are more resistant to developing GVHD than are mice housed at thermoneutral temperatures (∼ 30°C). We learned that the mechanism underlying this housing-dependent immunosuppression is associated with increased norepinephrine production and excessive signaling through β-adrenergic receptor signaling, which is increased when mice are cold stressed. Treatment of mice housed at 22°C with a β2-adrenergic antagonist reverses the norepinephrine-driven suppression of GVHD and yields similar disease to mice housed at 30°C. Conversely, administering a β2-adrenergic agonist decreases GVHD in mice housed at 30°C. In further mechanistic studies using β2-adrenergic receptor-deficient (β2-AR(-/-)) mice, we found that it is host cell β2-AR signaling that is essential for decreasing GVHD. These data reveal how baseline levels of β-adrenergic receptor signaling can influence murine GVHD and point to the feasibility of manipulation of β2-AR signaling to ameliorate GVHD in the clinical setting. Copyright © 2015 by The American Association of Immunologists, Inc.

  19. Anti-nociceptive interactions between opioids and a cannabinoid receptor 2 agonist in inflammatory pain

    PubMed Central

    Hale, David E; Guindon, Josée; Morgan, Daniel J

    2017-01-01

    The cannabinoid 1 receptor and cannabinoid 2 receptor can both be targeted in the treatment of pain; yet, they have some important differences. Cannabinoid 1 receptor is expressed at high levels in the central nervous system, whereas cannabinoid 2 receptor is found predominantly, although not exclusively, outside the central nervous system. The objective of this study was to investigate potential interactions between cannabinoid 2 receptor and the mu-opioid receptor in pathological pain. The low level of adverse side effects and lack of tolerance for cannabinoid 2 receptor agonists are attractive pharmacotherapeutic traits. This study assessed the anti-nociceptive effects of a selective cannabinoid 2 receptor agonist (JWH-133) in pathological pain using mice subjected to inflammatory pain using the formalin test. Furthermore, we examined several ways in which JWH-133 may interact with morphine. JWH-133 produces dose-dependent anti-nociception during both the acute and inflammatory phases of the formalin test. This was observed in both male and female mice. However, a maximally efficacious dose of JWH-133 (1 mg/kg) was not associated with somatic withdrawal symptoms, motor impairment, or hypothermia. After eleven once-daily injections of 1 mg/JWH-133, no tolerance was observed in the formalin test. Cross-tolerance for the anti-nociceptive effects of JWH-133 and morphine were assessed to gain insight into physiologically relevant cannabinoid 2 receptor and mu-opioid receptor interaction. Mice made tolerant to the effects of morphine exhibited a lower JWH-133 response in both phases of the formalin test compared to vehicle-treated morphine-naïve animals. However, repeated daily JWH-133 administration did not cause cross-tolerance for morphine, suggesting opioid and cannabinoid 2 receptor cross-tolerance is unidirectional. However, preliminary data suggest co-administration of JWH-133 with morphine modestly attenuates morphine tolerance. Isobolographic analysis

  20. Crosstalk Between Adrenergic and Toll-Like Receptors in Human Mesenchymal Stem Cells and Keratinocytes: A Recipe for Impaired Wound Healing

    PubMed Central

    Ramirez, Sandra R.; La, Thi Dinh; Gorouhi, Farzam; Nguyen, Chuong; Lin, Benjamin R.; Mashburn, Chelcy; Stewart, Heather; Peavy, Thomas R.; Nolta, Jan A.

    2014-01-01

    Previous studies demonstrate that skin wounds generate epinephrine (EPI) that can activate local adrenergic receptors (ARs), impairing healing. Bacterially derived activators of Toll-like receptors (TLRs) within the wound initiate inflammatory responses and can also impair healing. In this study, we examined the hypothesis that these two pathways crosstalk to one another, using EPI and macrophage-activating lipopeptide-2 (MALP2) to activate ARs and TLR2, respectively, in human bone marrow-derived mesenchymal stem cells (BM-MSCs) and neonatal keratinocytes (NHKs). BM-MSCs exposed to EPI significantly (p < .05) increased TLR2 message (sevenfold BM-MSCs), TLR2 protein (twofold), and myeloid differentiation factor 88 (MyD88) (fourfold). Conversely, activation of TLR2 by MALP2 in these cells increased β2-AR message (twofold in BM-MSCs, 2.7-fold in NHKs), β2-AR protein (2.5-fold), phosphorylation of β-AR-activated kinase (p-BARK, twofold), and induced release of EPI from both cell types (twofold). Treating cells with EPI and MALP2 together, as would be encountered in a wound, increased β2-AR and p-BARK protein expression (sixfold), impaired cell migration (BM-MSCs- 21%↓ and NHKs- 60%↓, p < .002), and resulted in a 10-fold (BM-MSCs) and 51-fold (NHKs) increase in release of IL-6 (p < .001) responses that were remarkably reduced by pretreatment with β2-AR antagonists. In vivo, EPI-stressed animals exhibited impaired healing, with elevated levels of TLR2, MyD88, and IL-6 in the wounds (p < .05) relative to nonstressed controls. Thus, our data describe a recipe for decreasing cell migration and exacerbating inflammation via novel crosstalk between the adrenergic and Toll-like receptor pathways in BM-MSCs and NHKs. PMID:24760207