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Sample records for a2 receptor expression

  1. TR4 nuclear receptor suppresses HCC cell invasion via downregulating the EphA2 expression.

    PubMed

    Jin, Ren'an; Lin, Hui; Li, Gonghui; Xu, Junjie; Shi, Liang; Chang, Chawnshang; Cai, Xiujun

    2018-02-15

    Early studies indicated that testicular nuclear receptor 4 (TR 4 ) could function as a suppressor in the transcriptional regulation of the HBV core gene expression, which might then influence the development of hepatocellular carcinoma (HCC). The direct linkage between TR 4 and HCC progression, however, remained unclear. Here, via a human clinical sample survey, we found that 13 of the 18 HCC patients studied had lower TR 4 expression in metastatic lesions than in matched primary HCC lesions, suggesting that TR 4 may play a negative role in HCC metastasis. Results from in vitro cell migration/invasion studied confirmed that TR 4 could suppress HCC cell migration/invasion. Mechanism dissection revealed that TR 4 might function through downregulating ephrin type-A receptor 2 (EphA2) expression at the transcriptional level via direct binding to the TR 4 REs located on the 5' promoter of EphA2 to suppress HCC cell migration/invasion. Targeting the EphA2 via EphA2-siRNA partially reversed the enhanced HCC cell migration/invasion with confirmed TR 4 knockdown. Notably, results from preclinical studies using in vivo mouse model with orthotopic xenograft of HCC LM3 cells also confirmed the in vitro findings. Taking these findings together, preclinical studies using multiple in vitro HCC cell lines and an in vivo mouse model all led to the conclusion that TR 4 may function as a suppressor of HCC metastasis and that targeting this newly identified TR 4 -EphA2 signaling may improve our ability to suppress HCC metastasis.

  2. Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

    PubMed

    Menschikowski, Mario; Hagelgans, Albert; Nacke, Brit; Jandeck, Carsten; Sukocheva, Olga; Siegert, Gabriele

    2015-12-16

    It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines. Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system. Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, -181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, -154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression. Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter

  3. High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant.

    PubMed

    Xu, Bing; Chakraborty, Raja; Eilers, Markus; Dakshinamurti, Shyamala; O'Neil, Joe D; Smith, Steven O; Bhullar, Rajinder P; Chelikani, Prashen

    2013-01-01

    G protein-coupled receptors (GPCRs) exhibit some level of basal signaling even in the absence of a bound agonist. This basal or constitutive signaling can have important pathophysiological roles. In the past few years, a number of high resolution crystal structures of GPCRs have been reported, including two crystal structures of constitutively active mutants (CAM) of the dim-light receptor, rhodopsin. The structural characterizations of CAMs are impeded by the lack of proper expression systems. The thromboxane A2 receptor (TP) is a GPCR that mediates vasoconstriction and promotes thrombosis in response to the binding of thromboxane. Here, we report on the expression and purification of a genetic variant and CAM in TP, namely A160T, using tetracycline-inducible HEK293S-TetR and HEK293S (GnTI¯)-TetR cell lines. Expression of the TP and the A160T genes in these mammalian cell lines resulted in a 4-fold increase in expression to a level of 15.8 ±0.3 pmol of receptor/mg of membrane protein. The receptors expressed in the HEK293S (GnTI(-))-TetR cell line showed homogeneous glycosylation. The functional yield of the receptors using a single step affinity purification was 45 µg/10⁶ cells. Temperature- dependent secondary structure changes of the purified TP and A160T receptors were characterized using circular dichroism (CD) spectropolarimetry. The CD spectra shows that the loss of activity or thermal sensitivity that was previously observed for the A160T mutant, is not owing to large unfolding of the protein but rather to a more subtle effect. This is the first study to report on the successful high-level expression, purification, and biophysical characterization of a naturally occurring, diffusible ligand activated GPCR CAM.

  4. The ephrin-A1 ligand and its receptor, EphA2, are expressed during tumor neovascularization.

    PubMed

    Ogawa, K; Pasqualini, R; Lindberg, R A; Kain, R; Freeman, A L; Pasquale, E B

    2000-12-07

    Eph receptor tyrosine kinases and their ephrin ligands have been implicated in embryonic vascular development and in in vivo models of angiogenesis. Eph proteins may also regulate tumor neovascularization, but this role has not been previously investigated. To screen for Eph proteins expressed in tumor blood vessels, we used tumor xenografts grown in nude mice from MDA-MB-435 human breast cancer cells or KS1767 human Kaposi's sarcoma cells. By immunohistochemistry, the ephrin-A1 ligand and one of its receptors, EphA2, were detected throughout tumor vasculature. Double-labeling with anti-CD34 antibodies demonstrated that both ephrin-A1 and EphA2 were expressed in xenograft endothelial cells and also tumor cells. Furthermore, EphA2 was tyrosine-phosphorylated in the xenograft tumors, indicating that it was activated, presumably by interacting with ephrin-A1. Ephrin-A1 and EphA2 were also detected in both the vasculature and tumor cells of surgically removed human cancers. In an in vitro angiogenesis model, a dominant negative form of EphA2 inhibited capillary tube-like formation by human umbilical vein endothelial cells (HUVECs), demonstrating a requirement for EphA receptor signaling. These data suggest that ephrin-A1 and EphA2 play a role in human cancers, at least in part by influencing tumor neovascularization. Eph proteins may represent promising new targets for antiangiogenic cancer treatments.

  5. 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.

  6. Endogenous expression of adenosine A1, A2 and A3 receptors in rat C6 glioma cells.

    PubMed

    Castillo, Carlos Alberto; Albasanz, José Luís; Fernández, Mercedes; Martín, Mairena

    2007-06-01

    Inhibitory and stimulatory adenosine receptors have been identified and characterized in both membranes and intact rat C6 glioma cells. In membranes, saturation experiment performed with [(3)H]DPCPX, selective A(1)R antagonist, revealed a single binding site with a K (D) = 9.4 +/- 1.4 nM and B (max) = 62.7 +/- 8.6 fmol/mg protein. Binding of [(3)H]DPCPX in intact cell revealed a K (D) = 17.7 +/- 1.3 nM and B (max )= 567.1 +/- 26.5 fmol/mg protein. On the other hand, [(3)H]ZM241385 binding experiments revealed a single binding site population of receptors with K (D) = 16.5 +/- 1.3 nM and B (max) = 358.9 +/- 52.4 fmol/mg protein in intact cells, and K (D) = 4.7 +/- 0.6 nM and B (max) = 74.3 +/- 7.9 fmol/mg protein in plasma membranes, suggesting the presence of A(2A) receptor in C6 cells. A(1), A(2A), A(2B) and A(3 )adenosine receptors were detected by Western-blotting and immunocytochemistry, and their mRNAs quantified by real time PCR assays. Gialpha and Gsalpha proteins were also detected by Western-blotting and RT-PCR assays. Furthermore, selective A(1)R agonists inhibited forskolin- and GTP-stimulated adenylyl cyclase activity and CGS 21680 and NECA stimulated this enzymatic activity in C6 cells. These results suggest that C6 glioma cells endogenously express A(1) and A(2) receptors functionally coupled to adenylyl cyclase inhibition and stimulation, respectively, and suggest these cells as a model to study the role of adenosine receptors in tumoral cells.

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

    SciTech Connect

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

    2014-04-04

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

  8. 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.

  9. Decreases in yeast expression yields of the human adenosine A2a receptor are a result of translational or post-translational events.

    PubMed

    Niebauer, Ronald T; Wedekind, Alison; Robinson, Anne Skaja

    2004-09-01

    The human adenosine receptor (A2a), a G-protein-coupled receptor (GPCR), was C-terminally tagged with the green fluorescent protein (GFP) and expressed in the yeast Saccharomyces cerevisiae to gain an understanding of the expression limitations of this medically relevant class of membrane proteins. The A2a-GFP protein was able to bind adenosine analogs indicating that the GFP tag did not alter the ligand binding activity of the receptor. A screen based on whole cell fluorescence was developed and a library of clones with various gene copy numbers was screened via flow cytometry to isolate clones with the highest protein expression levels. All clones studied exhibited a decrease in the net A2a-GFP protein production rate over time as determined by whole cell fluorescence, Western blotting, confocal microscopy, and ligand binding. Quantitative PCR showed that A2a-GFP mRNA levels remained relatively high even as the protein production rate decreased. A cycloheximide chase experiment showed that the mature protein was stable over time and was not significantly degraded. Taken together, these results suggest that heterologous expression of GPCRs is limited by a translational or post-translational bottleneck that is unique from expression limitations seen for soluble proteins.

  10. A novel, potent, and specific ephrinA1-based cytotoxin against EphA2 receptor expressing tumor cells.

    PubMed

    Wykosky, Jill; Gibo, Denise M; Debinski, Waldemar

    2007-12-01

    We have previously shown that the EphA2 receptor tyrosine kinase is overexpressed in glioblastoma multiforme (GBM) and represents a novel, attractive therapeutic target for the treatment of brain tumors. Here, we have developed an EphA2-targeted agent, ephrinA1-PE38QQR, a novel cytotoxin composed of ephrinA1, a ligand for EphA2, and PE38QQR, a mutated form of Pseudomonas aeruginosa exotoxin A. EphrinA1-PE38QQR showed potent and dose-dependent killing of GBM cells overexpressing the EphA2 receptor in cell viability and clonogenic survival assays, with an average IC(50) of approximately 10(-11) mol/L. The conjugate was also highly effective in killing breast and prostate cancer cells overexpressing EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, as it was neutralized by an excess of EphA2 ligands. Moreover, normal human endothelial cells and breast cancer cells that do not overexpress EphA2, as well as GBM cells that have down-regulated EphA2, were not susceptible to the cytotoxin. EphrinA1-PE38QQR-mediated cytotoxicity induced caspase-dependent apoptosis, which was, however, not responsible for cell death in response to the conjugate. In addition, the conjugate elicited no changes in the activity of survival pathways such as phosphoinositide 3-kinase, measured by AKT phosphorylation. This is the first attempt to create a cytotoxic therapy using any of the ephrin ligands of either class (A or B) conjugated to a bacterial toxin. EphrinA1-PE38QQR is very potent and specific, produces cell death that is caspase independent, and forms the basis for the further development of clinically applicable EphA2-targeted cytotoxins.

  11. Prophylactic effects of the histamine H1 receptor antagonist epinastine and the dual thromboxane A2 receptor and chemoattractant receptor-homologous molecule expressed on Th2 cells antagonist ramatroban on allergic rhinitis model in mice.

    PubMed

    Suzuki, Yuh; Inoue, Toshio; Yamamoto, Atsuki; Sugimoto, Yukio

    2011-01-01

    The prophylactic use of anti-allergic drugs has been proposed to be effective in the treatment of seasonal allergic rhinitis in humans. However, there is little information regarding the prophylactic effect of thromboxane A(2) (TXA(2)) receptor antagonist on allergic rhinitis. Recent studies revealed that a TXA(2) receptor antagonist ramatroban could block the prostaglandin D(2) (PGD(2)) receptor and chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). In the present study, we investigated the prophylactic effects of the histamine H(1) receptor antagonist epinastine and the TXA(2) receptor antagonist ramatroban and seratrodast on mouse models of allergic rhinitis. Female BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin and alum on days 0, 5, 14 and 21. Seven days later, mice were sensitized by intranasal application of ovalbumin thrice a week. Drugs were administered once a day from day 22. The severity of allergic rhinitis was assessed by determining the extent of 2 nasal allergic symptoms (sneezing and nasal rubbing). Histamine sensitivity and eosinophil infiltration into the nasal mucosa were also determined. Epinastine and ramatroban significantly reduced nasal symptoms and the number of eosinophils in the nasal mucosa. Seratrodast showed no effect on nasal symptoms and eosinophil infiltration into the nasal mucosa. In addition, histamine sensitivity was reduced by epinastine and ramatroban. These results indicate that epinastine and ramatroban induce the prophylactic effect on allergic rhinitis.

  12. Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

    PubMed

    Hinz, Sonja; Navarro, Gemma; Borroto-Escuela, Dasiel; Seibt, Benjamin F; Ammon, York-Christoph; de Filippo, Elisabetta; Danish, Azeem; Lacher, Svenja K; Červinková, Barbora; Rafehi, Muhammad; Fuxe, Kjell; Schiedel, Anke C; Franco, Rafael; Müller, Christa E

    2018-03-02

    The adenosine receptor (AR) subtypes A 2A and A 2B are rhodopsin-like G s protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A 2A -A 2B AR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A 2A AR when co-expressed with the A 2B AR (A 2B ≥ A 2A ) in recombinant as well as in native cells. In the presence of A 2B ARs, A 2A -selective ligands lost high affinity binding to A 2A ARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A 2A AR ligands as drugs as they will fail to modulate the receptor in an A 2A -A 2B heteromer context. Accordingly, A 2A -A 2B AR heteromers represent novel pharmacological targets.

  13. Transcriptomic Analysis Shows Decreased Cortical Expression of NR4A1, NR4A2 and RXRB in Schizophrenia and Provides Evidence for Nuclear Receptor Dysregulation.

    PubMed

    Corley, Susan M; Tsai, Shan-Yuan; Wilkins, Marc R; Shannon Weickert, Cynthia

    2016-01-01

    Many genes are differentially expressed in the cortex of people with schizophrenia, implicating factors that control transcription more generally. Hormone nuclear receptors dimerize to coordinate context-dependent changes in gene expression. We hypothesized that members of two families of nuclear receptors (NR4As), and retinoid receptors (RARs and RXRs), are altered in the dorsal lateral prefrontal cortex (DLPFC) of people with schizophrenia. We used next generation sequencing and then qPCR analysis to test for changes in mRNA levels for transcripts encoding nuclear receptors: orphan nuclear receptors (3 in the NR4A, 3 in the RAR, 3 in the RXR families and KLF4) in total RNA extracted from the DLPFC from people with schizophrenia compared to controls (n = 74). We also correlated mRNA levels with demographic factors and with estimates of antipsychotic drug exposure (schizophrenia group only). We tested for correlations between levels of transcription factor family members and levels of genes putatively regulated by these transcription factors. We found significantly down regulated expression of NR4A1 (Nurr 77) and KLF4 mRNAs in people with schizophrenia compared to controls, by both NGS and qPCR (p = or <0.01). We also detected decreases in NR4A2 (Nurr1) and RXRB mRNAs by using qPCR in the larger cohort (p<0.05 and p<0.01, respectively). We detected decreased expression of RARG and NR4A2 mRNAs in females with schizophrenia (p<0.05). The mRNA levels of NR4A1, NR4A2 and NR4A3 were all negative correlated with lifetime estimates of antipsychotic exposure. These novel findings, which may be influenced by antipsychotic drug exposure, implicate the orphan and retinoid nuclear receptors in the cortical pathology found in schizophrenia. Genes down stream of these receptors can be dysregulated as well, but the direction of change is not immediately predictable based on the putative transcription factor changes.

  14. Ephrin receptor (Eph) -A1, -A2, -A4 and -A7 expression in mobile tongue squamous cell carcinoma: associations with clinicopathological parameters and patients survival.

    PubMed

    Theocharis, Stamatios; Klijanienko, Jerzy; Giaginis, Constantinos; Alexandrou, Paraskevi; Patsouris, Efstratios; Sastre-Garau, Xavier

    2014-04-01

    Ephrin receptors (Ephs) are frequently overexpressed in a wide variety of human malignant tumors, being associated with tumor growth, invasion, metastasis and angiogenesis. The present study aimed to evaluate the clinical significance of Eph-A1, -A2, -A4 and -A7 protein expression in mobile tongue squamous cell carcinoma (SCC). Eph-A1, -A2, -A4 and -A7 protein expression was assessed immunohistochemically on 37 mobile tongue SCC tissue samples and was analyzed in relation with clinicopathological characteristics, overall and disease-free patients' survival. All the examined mobile tongue SCC cases were found positive for Eph-A1, -A2, -A4 and -A7. Significant associations were noted between high Eph-A1, -A4 and -A7 expression and absence of lymph node metastases (p = 0.0263, p = 0.0461 and p = 0.0461, respectively). High Eph-A1, -A2 and -A7 expression was significantly more frequently observed in patients presenting absence of vascular invasion (p = 0.0444), dense stromal inflammatory reaction (p = 0.0063) and female gender (p = 0.0327), respectively. Mobile tongue SCC patients with high Eph-A7 expression presented longer overall and disease-free survival compared to those with low Eph-A7 expression (log-rank test, p = 0.0093 and p = 0.0164, respectively). In multivariate analysis, Eph-A7 expression was identified as independent prognostic factor of overall survival (Cox-regression analysis, p = 0.0426). The present study supported evidence that Ephs may participate in the malignant transformation of mobile tongue SCC, reinforcing their utility as clinical markers for patients' management and prognosis, as also as targets for potential therapeutic intervention in tongue chemoprevention.

  15. Peroxisome Proliferator-activated Receptor-α-mediated Transcription of miR-199a2 Attenuates Endothelin-1 Expression via Hypoxia-inducible Factor-1α*

    PubMed Central

    Li, Chen; Mpollo, Marthe-Sandrine Eiymo Mwa; Gonsalves, Caryn S.; Tahara, Stanley M.; Malik, Punam; Kalra, Vijay K.

    2014-01-01

    Endothelin-1, a potent vasoconstrictor, plays an important role in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show that higher levels of placenta growth factor (PlGF), secreted by erythroid precursor cells, correlate with increased plasma levels of endothelin-1 (ET-1) and other functional markers of PH in SCD. PlGF-mediated ET-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively less is understood regarding how PlGF-mediated expression of HIF-1α and its downstream effector ET-1 are post-transcriptionally regulated. Herein, we show that PlGF treatment of endothelial cells resulted in reduced levels of miR-199a2, which targeted the 3′-UTR of HIF-1α mRNA and concomitantly led to augmented ET-1 expression. Plasma levels of miR-199a2 in SCD subjects were significantly lower with reciprocally high levels of plasma ET-1, unlike unaffected controls. This observation provided a molecular link between miR-199a2 and high levels of ET-1 in SCD. Furthermore, we show that miR-199a2 located in the DNM3os transcription unit was co-transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Binding of the latter to PPARα cis-elements in the promoter of DNM3os was demonstrated by promoter mutational analysis and ChIP. Additionally, we show that fenofibrate, a PPARα agonist, increased the expression of miR-199a2 and DNM3os; the former was responsible for reduced expression of HIF-1α and ET-1. In vivo studies of fenofibrate-fed Berkeley sickle mice resulted in increased levels of miR-199a2 and reduced levels of ET-1 in lung tissues. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-199a2 expression can ameliorate PH by reduction of ET-1 levels. PMID:25389292

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

    NASA Astrophysics Data System (ADS)

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

    1982-08-01

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

  17. Caffeine Reduces 11β-Hydroxysteroid Dehydrogenase Type 2 Expression in Human Trophoblast Cells through the Adenosine A2B Receptor

    PubMed Central

    Sharmin, Saina; Guan, Haiyan; Williams, Andrew Scott; Yang, Kaiping

    2012-01-01

    Maternal caffeine consumption is associated with reduced fetal growth, but the underlying molecular mechanisms are unknown. Since there is evidence that decreased placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is linked to fetal growth restriction, we hypothesized that caffeine may inhibit fetal growth partly through down regulating placental 11β-HSD2. As a first step in examining this hypothesis, we studied the effects of caffeine on placental 11β-HSD2 activity and expression using our established primary human trophoblast cells as an in vitro model system. Given that maternal serum concentrations of paraxanthine (the primary metabolite of caffeine) were greater in women who gave birth to small-for-gestational age infants than to appropriately grown infants, we also studied the effects of paraxanthine. Our main findings were: (1) both caffeine and paraxanthine decreased placental 11β-HSD2 activity, protein and mRNA in a concentration-dependent manner; (2) this inhibitory effect was mediated by the adenosine A2B receptor, since siRNA-mediated knockdown of this receptor prevented caffeine- and paraxanthine-induced inhibition of placental 11β-HSD2; and (3) forskolin (an activator of adenyl cyclase and a known stimulator of 11β-HSD2) abrogated the inhibitory effects of both caffeine and paraxanthine, which provides evidence for a functional link between exposure to caffeine and paraxanthine, decreased intracellular levels of cAMP and reduced placental 11β-HSD2. Taken together, these findings reveal that placental 11β-HSD2 is a novel molecular target through which caffeine may adversely affect fetal growth. They also uncover a previously unappreciated role for the adenosine A2B receptor signaling in regulating placental 11β-HSD2, and consequently fetal development. PMID:22701600

  18. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  19. Transcriptional profiling of striatal neurons in response to single or concurrent activation of dopamine D2, adenosine A(2A) and metabotropic glutamate type 5 receptors: focus on beta-synuclein expression.

    PubMed

    Canela, Laia; Selga, Elisabet; García-Martínez, Juan Manuel; Amaral, Olavo B; Fernández-Dueñas, Víctor; Alberch, Jordi; Canela, Enric I; Franco, Rafael; Noé, Véronique; Lluís, Carme; Ciudad, Carlos J; Ciruela, Francisco

    2012-10-25

    G protein-coupled receptor oligomerization is a concept which is changing the understanding of classical pharmacology. Both, oligomerization and functional interaction between adenosine A(2A,) dopamine D(2) and metabotropic glutamate type 5 receptors have been demonstrated in the striatum. However, the transcriptional consequences of receptors co-activation are still unexplored. We aim here to determine the changes in gene expression of striatal primary cultured neurons upon isolated or simultaneous receptor activation. Interestingly, we found that 95 genes of the total analyzed (15,866 transcripts and variants) changed their expression in response to simultaneous stimulation of all three receptors. Among these genes, we focused on the β-synuclein (β-Syn) gene (SCNB). Quantitative PCR verified the magnitude and direction of change in expression of SCNB. Since β-Syn belongs to the homologous synuclein family and may be considered a natural regulator of α-synuclein (α-Syn), it has been proposed that β-Syn might act protectively against α-Syn neuropathology. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Selective early expression of the orphan nuclear receptor Nr4a2 identifies the claustrum homolog in the avian mesopallium: Impact on sauropsidian/mammalian pallium comparisons.

    PubMed

    Puelles, L; Ayad, A; Alonso, A; Sandoval, J E; MartÍnez-de-la-Torre, M; Medina, L; Ferran, J L

    2016-02-15

    The transcription factor Nr4a2 was recently revealed as a very early developmental marker of the claustrum (CL) proper in the mouse. The earliest claustral primordium was identified superficially, dorsal to the olfactory cortex, and was subsequently covered by the Nr4a2-negative cells of the insular cortex. Some tangentially migrating claustral derivatives (subplate cells and some endopiriform elements) also expressed this marker. The present study employs the same genetic marker to explore the presence of a comparable pallial division in chicken in which, in principle, the same pallial sectors exist as in mammals. We were indeed able to delineate an early-developing Nr4a2-positive mantle domain at the expected topologic position within the developing chicken lateral pallium. In the chicken as well as in the turtle (from data in the literature), the earliest postmitotic lateropallial cells likewise express Nr4a2 and occupy a corticoid superficial stratum of the mesopallium, which is clearly comparable in spatial and chronological profile to the mouse CL. Other cells produced in this pallial sector include various tangentially migrating Nr4a2-labeled derivatives as well as Nr4a2-negative and Nr4a2-positive local deeper subpopulations that partially interdigitate, forming mesopallial core and shell populations. We hold that the deep avian and reptilian mesopallial formation developing under the superficial corticoid CL homolog represents a field homolog of the insula, although additional studies are required to underpin this hypothesis. © 2015 Wiley Periodicals, Inc.

  1. 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

  2. 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

  3. A2A receptor ligands: past, present and future trends.

    PubMed

    Clementina, Manera; Giuseppe, Saccomanni

    2010-01-01

    The adenosine A(2A) receptor is a member of the G protein-coupled receptor family and mediates multiple physiological effects of adenosine, both at the central nervous system and at peripheral tissues. Increasing evidence relates the A(2A) receptor with several pathological conditions such as neurodegenerative disorders, inflammation, pharmacological stress, and wound healing renewing the interest in A(2A) receptor agonists and antagonists as promising leads for drugs. However some of them initially tested in clinical trials presented several side effects, short half-life, lower solubility, and in some cases a lack of effects, perhaps attributable to receptor desensitization or to low receptor density in the targeted tissue. For these reasons it is evident that additional rational chemical modifications of the existing A(2A) receptor ligands to improve their affinity/selectivity and bioavailability as well as further studies to get new template for A(2A)AR ligands are necessary. The purpose of this review is to analyze and summarize the past and the present aspects related to the medicinal chemistry of A(2A) receptor ligands. Moreover their current and possible therapeutic applications have been also highlighted.

  4. 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.

  5. 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.

  6. Silencing receptor EphA2 induces apoptosis and attenuates tumor growth in malignant mesothelioma

    PubMed Central

    Mohammed, Kamal A; Wang, Xiaohong; Goldberg, Eugene P; Antony, Veena B; Nasreen, Najmunnisa

    2011-01-01

    Receptor EphA2 over-expression is associated with the aggressive nature of growth in malignant mesothelioma (MM) and silencing EphA2 with interference RNA suppressed MM proliferation. The mechanisms associated with targeting the EphA2 gene in MM were not clear. We sought to determine whether silencing EphA2 induces apoptosis in MM cells by either extrinsic or intrinsic pathways. The receptor EphA2 signaling pathway may provide attractive therapeutic strategy for MM. Apoptosis was determined by Cell Death ELISA in MM Cells transfected with siRNA-EphA2 and control siRNA. The gene expression profile of apoptosis pathways were analyzed by GEArray. Selected genes were further studied by quantitative PCR, Western analysis, and immunofluorescence. Caspases activities were measured by fluorescence spectrometer. Silencing EphA2 expression induced apoptosis in MMC. Apoptosis was characterized by FADD expression, activated caspase-8, caspase-3 and induction of Bax, Bak, and Bid as revealed by GEArray and protein fractionation assays. The expression of FADD, Bid, caspase-8, cytochrome-c and apaf-1 were significantly higher in the cytosolic fractions of EphA2-siRNA transfected cells. Furthermore, blocking the expression of caspase-8 by an inhibitor blunted FADD expression, indicating that caspase-8 is implicated in EphA2-siRNA induced apoptosis in MMC. Our data indicates that targeting the EphA2 gene by siRNA induced both extrinsic and intrinsic apoptotic pathways in MM Cells. Receptor EphA2 inhibition may be an effective approach for inhibiting MM growth and a promising direction for MM therapy. PMID:21968554

  7. The Ephrin Receptor Tyrosine Kinase A2 is a Cellular Receptor for Kaposi’s Sarcoma-Associated Herpesvirus

    PubMed Central

    Hahn, Alexander; Kaufmann, Johanna; Wies, Effi; Naschberger, Elisabeth; Panteleev-Ivlev, Julia; Schmidt, Katharina; Holzer, Angela; Schmidt, Martin; Chen, Jin; König, Simone; Ensser, Armin; Myoung, Jinjong; Brockmeyer, Norbert H.; Stürzl, Michael; Fleckenstein, Bernhard; Neipel, Frank

    2013-01-01

    Kaposi’s sarcoma associated herpesvirus (KSHV) is the human oncovirus which causes Kaposi’s sarcoma (KS), a highly vascularised tumour originating from lymphatic endothelial cells. Amongst others, the dimeric complex formed by the KSHV virion envelope glycoproteins H and L (gH/gL) is required for entry of herpesviruses into the host cell. We show that the Ephrin receptor tyrosine kinase A2 (EphA2) is a cellular receptor for KSHV gH/gL. EphA2 co-precipitated with both gH/gL and KSHV virions. KSHV infection rates were increased upon over-expression of EphA2. In contrast, antibodies against EphA2 and siRNAs directed against EphA2 inhibited KSHV infection of lymphatic endothelial cells. Pretreatment of KSHV virions with soluble EphA2 resulted in a dose-dependent inhibition of KSHV infection by up to 90%. Similarly, pretreating cells with the soluble EphA2 ligand EphrinA4 but not with EphA2 itself impaired KSHV infection. Notably, deletion of the EphA2 gene essentially abolished KSHV infection of murine vascular endothelial cells. Binding of gH/gL to EphA2 triggered EphA2 phosphorylation and endocytosis, a major pathway of KSHV entry. Quantitative RT-PCR and situ histochemistry revealed a close correlation between KSHV infection and EphA2 expression both in cultured cells derived from KS or lymphatic endothelium and in KS specimens, respectively. Taken together, these results identify EphA2, a tyrosine kinase with known functions in neo-vascularisation and oncogenesis, as receptor for KSHV gH/gL and implicate an important role for EphA2 in KSHV infection especially of endothelial cells and in KS. PMID:22635007

  8. Adenosine A2A Receptors Modulate Acute Injury and Neuroinflammation in Brain Ischemia

    PubMed Central

    Pedata, Felicita; Pugliese, Anna Maria; Coppi, Elisabetta; Dettori, Ilaria; Maraula, Giovanna; Cellai, Lucrezia; Melani, Alessia

    2014-01-01

    The extracellular concentration of adenosine in the brain increases dramatically during ischemia. Adenosine A2A receptor is expressed in neurons and glial cells and in inflammatory cells (lymphocytes and granulocytes). Recently, adenosine A2A receptor emerged as a potential therapeutic attractive target in ischemia. Ischemia is a multifactorial pathology characterized by different events evolving in the time. After ischemia the early massive increase of extracellular glutamate is followed by activation of resident immune cells, that is, microglia, and production or activation of inflammation mediators. Proinflammatory cytokines, which upregulate cell adhesion molecules, exert an important role in promoting recruitment of leukocytes that in turn promote expansion of the inflammatory response in ischemic tissue. Protracted neuroinflammation is now recognized as the predominant mechanism of secondary brain injury progression. A2A receptors present on central cells and on blood cells account for important effects depending on the time-related evolution of the pathological condition. Evidence suggests that A2A receptor antagonists provide early protection via centrally mediated control of excessive excitotoxicity, while A2A receptor agonists provide protracted protection by controlling massive blood cell infiltration in the hours and days after ischemia. Focus on inflammatory responses provides for adenosine A2A receptor agonists a wide therapeutic time-window of hours and even days after stroke. PMID:25165414

  9. Adenosine A2A receptor antagonists are potential antidepressants: evidence based on pharmacology and A2A receptor knockout mice

    PubMed Central

    Yacoubi, Malika El; Ledent, Catherine; Parmentier, Marc; Bertorelli, Rosalia; Ongini, Ennio; Costentin, Jean; Vaugeois, Jean-Marie

    2001-01-01

    Adenosine, an ubiquitous neuromodulator, and its analogues have been shown to produce ‘depressant' effects in animal models believed to be relevant to depressive disorders, while adenosine receptor antagonists have been found to reverse adenosine-mediated ‘depressant' effect. We have designed studies to assess whether adenosine A2A receptor antagonists, or genetic inactivation of the receptor would be effective in established screening procedures, such as tail suspension and forced swim tests, which are predictive of clinical antidepressant activity. Adenosine A2A receptor knockout mice were found to be less sensitive to ‘depressant' challenges than their wildtype littermates. Consistently, the adenosine A2A receptor blockers SCH 58261 (1 – 10 mg kg−1, i.p.) and KW 6002 (0.1 – 10 mg kg−1, p.o.) reduced the total immobility time in the tail suspension test. The efficacy of adenosine A2A receptor antagonists in reducing immobility time in the tail suspension test was confirmed and extended in two groups of mice. Specifically, SCH 58261 (1 – 10 mg kg−1) and ZM 241385 (15 – 60 mg kg−1) were effective in mice previously screened for having high immobility time, while SCH 58261 at 10 mg kg−1 reduced immobility of mice that were selectively bred for their spontaneous ‘helplessness' in this assay. Additional experiments were carried out using the forced swim test. SCH 58261 at 10 mg kg−1 reduced the immobility time by 61%, while KW 6002 decreased the total immobility time at the doses of 1 and 10 mg kg−1 by 75 and 79%, respectively. Administration of the dopamine D2 receptor antagonist haloperidol (50 – 200 μg kg−1 i.p.) prevented the antidepressant-like effects elicited by SCH 58261 (10 mg kg−1 i.p.) in forced swim test whereas it left unaltered its stimulant motor effects. In conclusion, these data support the hypothesis that A2A receptor antagonists prolong escape

  10. Interferon receptor expression in multiple sclerosis patients.

    PubMed

    Oliver, Begoña; Mayorga, Cristobalina; Fernández, Victoria; Leyva, Laura; León, Antonio; Luque, Gloria; López, Juan C; Tamayo, Jose A; Pinto-Medel, Maria J; de Ramon, Enrique; Blanco, Eva; Alonso, Ana; Fernández, Oscar

    2007-02-01

    To determine the gene expression of IFNAR1, IFNAR2 and MxA protein and the association with IFNbeta treatment response in MS patients. MS patients treated with IFNbeta had a significant decrease in IFNAR1 and IFNAR2 expression, and a significant increase in MxA compared to non-treated patients and healthy controls. Also, those patients who had a good response to treatment had a significant decrease in IFNAR1 and IFNAR2 expression compared to non-responders, non-treated patients and healthy controls. IFNbeta influences the expression of its receptors, and is greater in patients who respond to IFNbeta treatment. This down-regulation could be indicative of the response to IFNbeta.

  11. Downregulation of GluA2 AMPA Receptor Subunits Reduces the Dendritic Arborization of Developing Spinal Motoneurons

    PubMed Central

    Yoon, Yone J.; White, Sheryl L.; Ni, Xianglian; Gokin, Alexander P.; Martin-Caraballo, Miguel

    2012-01-01

    AMPA receptors lacking the GluA2 subunit allow a significant influx of Ca2+ ions. Although Ca2+-permeable AMPA receptors are a familiar feature at early stages of development, the functional significance of these receptors during the maturation of the nervous system remains to be established. Chicken lumbar motoneurons express Ca2+-permeable AMPA receptors at E6 but the Ca2+ permeability of AMPA receptors decreases ∼3-fold by E11. Considering that activity-dependent changes in intracellular Ca2+ regulates dendritic outgrowth, in this study we investigated whether downregulation of GluA2 expression during a critical period of development alters the dendritic arborization of spinal motoneurons in ovo. We use an avian replication-competent retroviral vector RCASBP (B) carrying the marker red fluorescent protein (RFP) and a GluA2 RNAi construct to downregulate GluA2 expression. Chicken embryos were infected at E2 with one of the following constructs: RCASBP(B)-RFP, RCASBP(B)-RFP-scrambled RNAi, or RCASBP(B)-RFP-GluA2 RNAi. Infection of chicken embryos at E2 resulted in widespread expression of RFP throughout the spinal cord with ≥60% of Islet1/2-positive motoneurons infected, resulting in a significant reduction in GluA2 protein expression. Downregulation of GluA2 expression had no effect on the dendritic arborization of E6 motoneurons. However, downregulation of GluA2 expression caused a significant reduction in the dendritic arborization of E11 motoneurons. Neither motoneuron survival nor maturation of network activity was affected by changes in GluA2 expression. These findings demonstrate that increased GluA2 expression and changes in the Ca2+ permeability of AMPA receptors regulate the dendritic arborization of spinal cord motoneurons during a critical period of development. PMID:23226228

  12. Vitamin D Receptor Expression in Dogs

    PubMed Central

    Gow, A.G.; Milne, E.; Drummond, D.; Smith, S.; Handel, I.; Mellanby, R.J.

    2018-01-01

    Background There is growing evidence linking low blood vitamin D concentration to numerous diseases in people and in dogs. Vitamin D influences cellular function by signaling through the vitamin D receptor (VDR). Little is known about which non‐skeletal tissues express the VDR or how inflammation influences its expression in the dog. Objectives To define which non‐skeletal canine tissues express the VDR and to investigate expression in inflamed small intestine. Animals Thirteen non‐skeletal tissues were collected prospectively from 6 control dogs. Thirty‐five dogs diagnosed with a chronic enteropathy (CE) and 24 control dogs were prospectively enrolled and duodenal biopsies were evaluated for VDR expression. Methods Prospective; blinded assessment of canine intestinal VDR. Dogs with CE were included once other identifiable causes of intestinal disease were excluded. Age matched controls were included with no intestinal clinical signs. VDR expression was assessed immunohistochemically in all samples, using a Rat IgG VDR monoclonal antibody. Quantitative real‐time polymerase chain reaction (qPCR) was also used for duodenal biopsies. Results VDR expression as assessed by immunohistochemistry (IHC) was highest in the kidney, duodenum, skin, ileum and spleen, and weak in the colon, heart, lymph node, liver, lung, and ovary. Gastric and testicular tissue did not express the VDR. There was no statistical difference in duodenal VDR expression between the 24 healthy dogs and 34 dogs with CE when quantified by either qPCR (P = 0.87) or IHC (P = 0.099). Conclusions and Clinical Importance The lack of down regulation of VDR expression in inflamed intestine contrasts with previous studies in humans. Our findings support future studies to investigate whether vitamin D and its analogues can be used to modulate intestinal inflammation in the dog. PMID:29469965

  13. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons.

    PubMed

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L; Anggono, Victor; Gether, Ulrik; Huganir, Richard L; Madsen, Kenneth L

    2013-08-27

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na(+)/H(+)-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity.

  14. AMPA receptor pHluorin-GluA2 reports NMDA receptor-induced intracellular acidification in hippocampal neurons

    PubMed Central

    Rathje, Mette; Fang, Huaqiang; Bachman, Julia L.; Anggono, Victor; Gether, Ulrik; Huganir, Richard L.; Madsen, Kenneth L.

    2013-01-01

    NMDA receptor activation promotes endocytosis of AMPA receptors, which is an important mechanism underlying long-term synaptic depression. The pH-sensitive GFP variant pHluorin fused to the N terminus of GluA2 (pH-GluA2) has been used to assay NMDA-mediated AMPA receptor endocytosis and recycling. Here, we demonstrate that in somatic and dendritic regions of hippocampal neurons a large fraction of the fluorescent signal originates from intracellular pH-GluA2, and that the decline in fluorescence in response to NMDA and AMPA primarily describes an intracellular acidification, which quenches the pHluorin signal from intracellular receptor pools. Neurons expressing an endoplasmic reticulum-retained mutant of GluA2 (pH-GluA2 ΔC49) displayed a larger response to NMDA than neurons expressing wild-type pH-GluA2. A similar NMDA-elicited decline in pHluorin signal was observed by expressing cytosolic pHluorin alone without fusion to GluA2 (cyto-pHluorin). Intracellular acidification in response to NMDA was further confirmed by using the ratiometric pH indicator carboxy-SNARF-1. The NMDA-induced decline was followed by rapid recovery of the fluorescent signal from both cyto-pHluorin and pH-GluA2. The recovery was sodium-dependent and sensitive to Na+/H+-exchanger (NHE) inhibitors. Moreover, recovery was more rapid after shRNA-mediated knockdown of the GluA2 binding PDZ domain-containing protein interacting with C kinase 1 (PICK1). Interestingly, the accelerating effect of PICK1 knockdown on the fluorescence recovery was eliminated in the presence of the NHE1 inhibitor zoniporide. Our results indicate that the pH-GluA2 recycling assay is an unreliable assay for studying AMPA receptor trafficking and also suggest a role for PICK1 in regulating intracellular pH via modulation of NHE activity. PMID:23940334

  15. Expression of melanocortin receptors in human endometrium.

    PubMed

    Lantang, Anastasia M; Innes, Barbara A; Gan, Earn H; Pearce, Simon H; Lash, Gendie E

    2015-10-01

    Are melanocortin receptors (MCR1-5) expressed in the endometrium? MCR1-5 are expressed in endometrium to varying degrees, with MC2R, MC3R and MC5R being the most abundant and the majority of expression being observed in glandular epithelium. Women with Addison's disease who were being administered synthetic ACTH reported menstrual complications as a side effect. There is no previous literature on expression of the melanocortin receptors within the endometrium, and therefore whether ACTH may directly affect the endometrial vasculature. Endometrial biopsies were taken from hysterectomy specimens in control women without endometrial pathology (n = 4 for each of proliferative and late-secretory phases). Biopsies were formalin fixed and embedded in paraffin wax. Decidual samples (n = 7) were cultured in a range of concentrations of synthetic ACTH for 3 days before being formalin fixed and embedded in paraffin wax. Endometrial paraffin embedded sections were immunostained for MCR1-5 and assessed using a modified quickscore with luminal epithelium, glandular epithelium, stromal cells, endothelial cells and vascular smooth muscle cells all being assessed separately. Cultured decidual biopsy paraffin embedded sections were immunostained for H-caldesmon and the number of layers of vascular smooth muscle cells surrounding the vessel assessed. All five melanocortin receptors were shown to be immunolocalised to the endometrium, with MC5R, MC2R and MC3R being the most abundant and limited immunostaining being observed for MC1R and MC4R. Treatment of decidual biopsies with synthetic adrenocorticotropin (ACTH) resulted in loss of vascular integrity. This is an observational study and does not definitively demonstrate a link between synthetic ACTH administration and menstrual complications. This is the first study to demonstrate widespread expression of melanocortin receptors within the endometrium. Further study is required to determine the role of this hormone family in

  16. Adenovirus transduction: More complicated than receptor expression.

    PubMed

    Sharma, Priyanka; Martis, Prithy C; Excoffon, Katherine J D A

    2017-02-01

    The abundance and accessibility of a primary virus receptor are critical factors that impact the susceptibility of a host cell to virus infection. The Coxsackievirus and adenovirus receptor (CAR) has two transmembrane isoforms that occur due to alternative splicing and differ in localization and function in polarized epithelia. To determine the relevance of isoform-specific expression across cell types, the abundance and localization of both isoforms were determined in ten common cell lines, and correlated with susceptibility to adenovirus transduction relative to polarized primary human airway epithelia. Data show that the gene and protein expression for each isoform of CAR varies significantly between cell lines and polarization, as indicated by high transepithelial resistance, is inversely related to adenovirus transduction. In summary, the variability of polarity and isoform-specific expression among model cells are critical parameters that must be considered when evaluating the clinical relevance of potential adenovirus-mediated gene therapy and anti-adenovirus strategies. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Structure-activity relationship analysis of peptides targeting the EphA2 receptor.

    PubMed

    Mitra, Sayantan; Duggineni, Srinivas; Koolpe, Mitchell; Zhu, Xuejun; Huang, Ziwei; Pasquale, Elena B

    2010-08-10

    The EphA2 receptor tyrosine kinase has emerged as a promising new therapeutic target in cancer because of its high level of expression in tumors. EphA2-specific antibodies have been used to deliver drugs and toxins to tumor cells, leading to inhibition of tumor growth and metastatic dissemination. We previously identified two related peptides, YSA and SWL, that selectively bind to the ligand-binding domain of EphA2 but not other Eph receptors and could therefore be useful as selective targeting agents. Here we characterize the two peptides and a series of derivatives. On the basis of systematic amino acid replacements, only five YSA residues appear to be critical for high-affinity receptor binding. Furthermore, a peptide comprising only the first five residues of YSA retains selectivity for EphA2. Similar to ephrin-A1, the physiological ligand for EphA2, both YSA and SWL activate EphA2 and inhibit downstream oncogenic signaling pathways in PC3 cancer cells. The two peptides and derivatives are quite stable in conditioned cell culture medium and show promise for delivering drugs and imaging agents to EphA2-expressing tumors.

  18. 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.

  19. Androgen receptor in estrogen receptor positive breast cancer: Beyond expression.

    PubMed

    Basile, Debora; Cinausero, Marika; Iacono, Donatella; Pelizzari, Giacomo; Bonotto, Marta; Vitale, Maria Grazia; Gerratana, Lorenzo; Puglisi, Fabio

    2017-12-01

    In recent years, new therapeutic approaches have reshaped the overall strategy of breast cancer (BC) treatment and have markedly improved patient survival. This is, in part, due to novel therapies for estrogen receptor (ER)-positive BC. Unfortunately, many patients present de novo resistance to these therapies or develop an acquired resistance over time. Therefore, research is now focused on discovering new molecular targets to overcome these resistances. Interestingly, preclinical and clinical studies have shown a critical role for the cross-talk between androgen receptor (AR) and ER in luminal-like BC. AR is expressed in >60% of BC and in up to 90% of ERα-positive tumors. Multiple studies suggest that AR is associated with a favorable prognosis. However, AR overexpression and, in particular, the high AR:ER ratio, seem to be involved in resistance to hormonal treatment. In this setting, a group of BCs could benefit from AR-inhibitors; nevertheless, some ER-positive BC patients do not seem to benefit from this strategy. Therefore, it is crucial to identify biomarkers that would enable the selection of patients who might benefit from combination treatment with ER and AR inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Cell adhesion and EGFR activation regulate EphA2 expression in cancer.

    PubMed

    Larsen, Alice Bjerregaard; Stockhausen, Marie-Thérése; Poulsen, Hans Skovgaard

    2010-04-01

    EphA2 is frequently overexpressed in cancer, and increasing amounts of evidence show that EphA2 contributes to multiple aspects of the malignant character including angiogenesis and metastasis. Several aspects of the regulation and functional significance of EphA2 expression in cancer are still largely unknown. Here we show that the expression of EphA2 in in vitro cultured cells, is restricted to cells growing adherently and that adhesion-induced EphA2 expression is dependent upon activation of the epidermal growth factor receptor (EGFR), mitogen activated protein kinase kinase (MEK) and Src family kinases (SRC). Moreover, the results show that adhesion-induced EGFR activation and EphA2 expression is affected by interactions with extracellular matrix (ECM) proteins working as integrin ligands. Stimulation with the EphA2 ligand, ephrinA1 inhibited ERK phosphorylation and cancer cell viability. These effects were however abolished by activation of the EGF-receptor ligand system favoring Ras/MAPK signaling and cell proliferation. Based on our results, we propose a regulatory mechanism where cell adhesion induces EGFR kinase activation and EphA2 expression; and where the effect of ephrinA1 mediated reduction in cell viability by inhibiting EphA2 expression is overruled by activated EGFR in human cancer cells. Copyright 2009 Elsevier Inc. All rights reserved.

  1. Adenosine A2A receptor hyperexpression in patients with severe SIRS after cardiopulmonary bypass.

    PubMed

    Kerbaul, François; Bénard, Frédéric; Giorgi, Roch; Youlet, By; Carrega, Louis; Zouher, Ibrahim; Mercier, Laurence; Gérolami, Victoria; Bénas, Vincent; Blayac, Dorothée; Gariboldi, Vlad; Collart, Frédéric; Guieu, Régis

    2008-08-01

    Adenosine (ADO) is an endogenous nucleoside, which has been involved in blood pressure failure during severe systemic inflammatory response syndrome (severe SIRS) after cardiac surgery with cardiopulmonary bypass (CPB). Adenosine acts via its receptor subtypes, namely A1, A2A, A2B, or A3. Because A2A receptors are implicated in vascular tone, their expression might contribute to severe SIRS. We compared adenosine plasma levels (APLs) and A2A ADO receptor expression (ie, B, K, and mRNA amount) in patients with or without postoperative SIRS. : This was a prospective comparative observational study. Forty-four patients who underwent cardiac surgery involving CPB. Ten healthy subjects served as controls. Among the patients, 11 presented operative vasoplegia and postoperative SIRS (named complicated patients) and 33 were without vasoplegia or SIRS (named uncomplicated patients). Adenosine plasma levels, K, B, and mRNA amount (mean +/- SD) were measured on peripheral blood mononuclear cells. Adenosine plasma levels, B, and K were significantly higher in complicated patients than in uncomplicated patients (APLs: 2.7 +/- 1.0 vs 1.0 +/- 0.5 micromol l, P < 0.05; B: 210 +/- 43 vs 65 +/- 26 fmol/mg, P < 0.05; K: 35 +/- 10 vs 2 +/- 1 nM, P < 0.05). In uncomplicated patients, APLs remain higher than in controls (1 +/- 0.5 vs 0.6 +/- 0.25 micromol/L; P < 0.05). Mean arterial pressure was inversely correlated to APLs (R = -0.58; P < 0.001) and B (R = -0.64; P < 0.001) leading to an increased requirement of vasoactive drugs during the postoperative period in vasoplegic patients. High expression of A2A ADO receptor and high APLs may be a predictive factor of postoperative severe SIRS after CPB.

  2. Interleukin-7 receptor expression: intelligent design.

    PubMed

    Mazzucchelli, Renata; Durum, Scott K

    2007-02-01

    Interleukin-7 (IL-7) is produced by stromal cells in lymphoid tissues and is required for the development of T cells and for their persistence in the periphery. Unlike many other cytokines that act on lymphocytes, IL-7 production by stromal cells is not substantially affected by extrinsic stimuli. So, the amount of available IL-7 protein is thought to be regulated by the rate that it is scavenged by T cells. As we review here, there is mounting evidence indicating that the amount of IL-7 receptor expressed on a cell not only determines how vigorously the cell responds to IL-7, but it can also determine how efficiently the cell consumes IL-7 and, therefore, affect the supply of this limiting resource in the niche.

  3. Regulation of serotonin2A receptors in heterologous expression systems.

    PubMed

    Grotewiel, M S; Sanders-Bush, E

    1994-10-01

    The serotonin2A and serotonin2C receptors are unique among receptors coupled to guanine nucleotide binding proteins in that chronic treatment in vivo with agonists as well as antagonists decreases receptor density. In an attempt to uncover molecular events involved in down-regulation of the serotonin2A receptor, the ability of agonists and antagonists to alter receptor density was examined in three heterologous expression systems, i.e., transfected NIH 3T3, transfected Madin-Darby canine kidney, and transfected AtT-20 cells. All three transfected cell lines exhibited pharmacological properties consistent with that predicted for cells expressing the serotonin2A receptor. However, the three cell lines displayed different receptor regulation properties in response to drugs acting at the serotonin2A receptor. In transfected NIH 3T3 cells, neither agonist nor antagonist treatment altered receptor density. Treatment with agonist as well as antagonist led to up-regulation of the serotonin2A receptor in transfected Madin-Darby canine kidney cells. In transfected AtT-20 cells, treatment with agonist led to receptor down-regulation, whereas antagonist treatment increased receptor density. Thus, the cellular background in which the serotonin2A receptor is expressed appears to determine the regulation properties of the receptor.

  4. Striatal Pre- and Postsynaptic Profile of Adenosine A2A Receptor Antagonists

    PubMed Central

    Quiroz, César; Beaumont, Vahri; Goldberg, Steven R.; Lluís, Carme; Cortés, Antoni; Franco, Rafael; Casadó, Vicent; Canela, Enric I.; Ferré, Sergi

    2011-01-01

    Striatal adenosine A2A receptors (A2ARs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D2 receptors (D2Rs). A2ARs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A1 receptors (A1Rs). It has been hypothesized that postsynaptic A2AR antagonists should be useful in Parkinson's disease, while presynaptic A2AR antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A2AR antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A2AR-D2R and A1R-A2AR heteromers to determine possible differences in the affinity of these compounds for different A2AR heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A2AR when co-expressed with D2R than with A1R. KW-6002 showed the best relative affinity for A2AR co-expressed with D2R than co-expressed with A1R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential

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

    SciTech Connect

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

    2009-09-09

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

  6. 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

  7. Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors*

    PubMed Central

    Navarro, Gemma; Aymerich, Marisol S.; Marcellino, Daniel; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I.; Agnati, Luigi; Woods, Amina S.; Fuxe, Kjell; Lluís, Carmen; Lanciego, Jose Luis; Ferré, Sergi; Franco, Rafael

    2009-01-01

    The Ca2+-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D2 receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is also involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A2A receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A2A-D2 receptor oligomerization. BRET competition experiments indicated that, in the A2A-D2 receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A2A receptor. Furthermore, Ca2+ was found to induce conformational changes in the CaM-A2A-D2 receptor oligomer and to selectively modulate A2A and D2 receptor-mediated MAPK signaling in the A2A-D2 receptor heteromer. These results may have implications for basal ganglia disorders, since A2A-D2 receptor heteromers are being considered as a target for anti-parkinsonian agents. PMID:19632986

  8. Estrogen stimulates adenosine receptor expression subtypes in human breast cancer MCF-7 cell line.

    PubMed

    Mohamadi, Azam; Aghaei, Mahmoud; Panjehpour, Mojtaba

    2018-02-01

    Estrogen is a steroid hormone that plays a key role in the development and regulation of reproductive system. It has been shown that estrogen is related to breast cancer development through binding to its receptors. In order to uncover the estrogen effects on adenosine receptor expression, estrogen-positive MCF-7 cells were used to treat with agonist and antagonist of estrogen and then the mRNA expression of adenosine receptor subtypes were evaluated. Estrogen-positive MCF-7 cells were treated with various concentrations of 17β estradiol (E2) as an estrogen agonist, and ICI 182,780 as an estrogen antagonist. The gene expression of adenosine receptor subtypes were detected by real time RT-PCR. The results of MTT assay showed that E2 increased cell viability in a dose dependent manner. The expression pattern of all adenosine receptor subtypes are as follow; A2b > A1 > A2a > A3 in untreated MCF-7 cells. Obtained results showed that E2 incubation at 0.001-0.01 μM led to up-regulation of A1ARs, A2aARs and A3ARs dose dependently. E2 at 0.001 μM also had no significant effect on A2bARs expression but, at higher doses induced a considerable decrease in mRNA A2bARs expression. Treatment with antagonist confirmed that up-regulation of these receptors is mediated by estrogen receptor. Taken together, our results indicate that treatment of MCF-7 cells with E2 led to up-regulation of adenosine receptors. However, these effects were partially restored by treatment with antagonist suggesting that such effects are mediated by estrogen receptors.

  9. Emerging strategies for EphA2 receptor targeting for cancer therapeutics

    PubMed Central

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

    2010-01-01

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

  10. Ephrin receptor A2 is an epithelial cell receptor for Epstein-Barr virus entry.

    PubMed

    Zhang, Hua; Li, Yan; Wang, Hong-Bo; Zhang, Ao; Chen, Mei-Ling; Fang, Zhi-Xin; Dong, Xiao-Dong; Li, Shi-Bing; Du, Yong; Xiong, Dan; He, Jiang-Yi; Li, Man-Zhi; Liu, Yan-Min; Zhou, Ai-Jun; Zhong, Qian; Zeng, Yi-Xin; Kieff, Elliott; Zhang, Zhiqiang; Gewurz, Benjamin E; Zhao, Bo; Zeng, Mu-Sheng

    2018-02-01

    Epstein-Barr virus (EBV) is causally associated with nasopharyngeal carcinoma, 10% of gastric carcinoma and various B cell lymphomas 1 . EBV infects both B cells and epithelial cells 2 . Recently, we reported that epidermal growth factor and Neuropilin 1 markedly enhanced EBV entry into nasopharyngeal epithelial cells 3 . However, knowledge of how EBV infects epithelial cells remains incomplete. To understand the mechanisms through which EBV infects epithelial cells, we integrated microarray and RNA interference screen analyses and found that Ephrin receptor A2 (EphA2) is important for EBV entry into the epithelial cells. EphA2 short interfering RNA knockdown or CRISPR-Cas9 knockout markedly reduced EBV epithelial cell infection, which was mostly restored by EphA2 complementary DNA rescue. EphA2 overexpression increased epithelial cell EBV infection. Soluble EphA2 protein, antibodies against EphA2, soluble EphA2 ligand EphrinA1, or the EphA2 inhibitor 2,5-dimethylpyrrolyl benzoic acid efficiently blocked EBV epithelial cell infection. Mechanistically, EphA2 interacted with EBV entry proteins gH/gL and gB to facilitate EBV internalization and fusion. The EphA2 Ephrin-binding domain and fibronectin type III repeats domain were essential for EphA2-mediated EBV infection, while the intracellular domain was dispensable. This is distinct from Kaposi's sarcoma-associated herpesvirus infection through EphA2 4 . Taken together, our results identify EphA2 as a critical player for EBV epithelial cell entry.

  11. Retinoid receptors in ovarian cancer: expression and prognosis.

    PubMed

    Kaiser, P C; Körner, M; Kappeler, A; Aebi, S

    2005-09-01

    Ovarian cancer is frequently lethal despite aggressive multimodal therapy, and new therapies are therefore needed. Retinoids are potential candidate drugs: they prevent the development of ovarian carcinoma and enhance the efficacy of cytotoxic drugs in ovarian cancer cells. At present, little is known about the retinoid receptor expression in ovarian cancer. The retinoid receptors comprise two classes, retinoic acid receptors (RARs) and retinoid X receptors (RXRs), each with three subclasses, alpha, beta and gamma. We investigated the expression of the subtypes RARalpha, RARgamma, RXRalpha and RXRbeta by immunohistochemistry in ovarian cancers of 80 patients, and assessed their prognostic significance. In addition, we quantified the expression of retinoid receptor mRNA using real-time PCR and correlated the results with clinical characteristics. RARalpha and RXRbeta were highly expressed in a majority of ovarian cancers, particularly in advanced stages. High expression of RARalpha was an independent negative prognostic factor of survival in addition to FIGO stage, age and p53 accumulation. The mRNA expression of retinoid receptors did not correlate with clinical properties of the tumors. Retinoic acid receptors are frequently and strongly expressed in epithelial ovarian cancer and may be indicators of an adverse prognosis. This study provides the molecular basis for the therapeutic use of retinoids in ovarian cancer.

  12. 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

  13. Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

    PubMed Central

    Varani, K; Vincenzi, F; Tosi, A; Targa, M; Masieri, FF; Ongaro, A; De Mattei, M; Massari, L; Borea, PA

    2010-01-01

    Background and purpose: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors (A1, A2A, A2B and A3) and acts as a potent inhibitor of inflammatory processes in several tissues. So far, the functional effects modulated by adenosine receptors on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of adenosine receptors and their pharmacological modulation in human synoviocytes. Experimental approach: mRNA, Western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB activation, tumour necrosis factor α (TNF-α) and interleukin-8 (IL-8) release were assessed in human synoviocytes isolated from patients with osteoarthritis. Key results: mRNA and protein for A1, A2A, A2B and A3 adenosine receptors are expressed in human synoviocytes. Standard adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. Activation of A2A and A3 adenosine receptors inhibited p38 MAPK and NF-κB pathways, an effect abolished by selective adenosine antagonists. A2A and A3 receptor agonists decreased TNF-α and IL-8 production. The phosphoinositide 3-kinase or Gs pathways were involved in the functional responses of A3 or A2A adenosine receptors. Synoviocyte A1 and A2B adenosine receptors were not implicated in the inflammatory process whereas stimulation of A2A and A3 adenosine receptors was closely associated with a down-regulation of the inflammatory status. Conclusions and implications: These results indicate that A2A and A3 adenosine receptors may represent a potential target in therapeutic modulation of joint inflammation. PMID:20331607

  14. A novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells

    PubMed Central

    Wang, Si; Placzek, William J.; Stebbins, John L.; Mitra, Sayantan; Noberini, Roberta; Koolpe, Mitchell; Zhang, Ziming; Dahl, Russell; Pasquale, Elena B.; Pellecchia, Maurizio

    2012-01-01

    The efficacy of anti-cancer drugs is often limited by their systemic toxicities and adverse side effects. We report that the EphA2 receptor is over-expressed preferentially in several human cancer cell lines compared to normal tissues and that an EphA2 targeting peptide (YSAYPDSVPMMS) can be effective in delivering anti-cancer agents to such tumors. Hence, we report on the synthesis and characterizations of a novel EphA2-targeting agent conjugated with the chemotherapeutic drug paclitaxel. We found that the peptide-drug conjugate is dramatically more effective than paclitaxel alone at inhibiting tumor growth in a prostate cancer xenograft model, delivering significantly higher levels of drug to the tumor site. We believe these studies open the way to the development of a new class of therapeutic compounds that exploit the EphA2 receptor for drug delivery to cancer cells. PMID:22329578

  15. Expression of Plant Receptor Kinases in E. coli.

    PubMed

    Agha, Moneeza Akbar; Lightfoot, David; Afzal, Ahmed Jawaad

    2017-01-01

    Plant receptor kinases play diverse signaling roles in disease resistance and plant development. They represent a large plant gene family with over 600 members in Arabidopsis thaliana. While the functions of several members of the receptor kinase family have now been elucidated, a great proportion still remains uncharacterized. The structural and functional characterization of such plant receptor kinases may entail biochemical approaches that require access to purified protein, which can be made possible through heterologous protein expression. This chapter describes a strategy for expressing plant receptor kinases in E. coli, a bacterial host that has successfully been used to express and purify certain plant receptor kinase domains, some of which were subsequently used for biochemical assays. As full-length receptor-like kinases may be difficult to express, it is suggested to clone and express domains separately, after having identified domain borders using bioinformatics tools. A detailed cloning protocol is provided, as well as advice for testing expression efficiency and handling of expressed protein ending up in inclusion bodies.

  16. Potentiation of cytokine induction of group IIA phospholipase A(2) in rat mesangial cells by ATP and adenosine via the A2A adenosine receptor.

    PubMed

    Scholz-Pedretti, K; Pfeilschifter, J; Kaszkin, M

    2001-01-01

    1. In rat mesangial cells extracellular nucleotides were found to increase arachidonic acid release by a cytosolic phospholipase A(2) through the P2Y(2) purinergic receptor. 2. In this study we investigated the effects of ATP and UTP on interleukin-1ss (IL-1ss)-induced mRNA expression and activity of group IIA phospholipase A(2) (sPLA(2)-IIA) in rat mesangial cells. 3. Treatment of cells for 24 h with extracellular ATP potentiated IL-1ss-stimulated sPLA(2)-IIA induction, whereas UTP had no effect. 4. We obtained the following evidence that the P2Y(2) receptor is not involved in the potentiation of sPLA(2)-IIA induction: (i) ATP-gamma-S had no enhancing effect; (ii) suramin, a P(2) receptor antagonist, did not inhibit ATP-mediated potentiation; (iii) inhibition of degradation of extracellular nucleotides by the 5'-ectonucleotidase inhibitor AOPCP did not enhance sPLA(2)-IIA induction and (iv) adenosine deaminase treatment completely abolished the ATP-mediated potentiation of sPLA(2)-IIA induction. 5. In contrast, treatment of mesangial cells with adenosine or the A2A receptor agonist CGS 21680 mimicked the effects of ATP in enhancing IL-1ss-stimulated sPLA(2)-IIA induction, whereas the specific A2A receptor antagonist ZM 241385 completely abolished the potentiating effect of ATP or adenosine. 6. The protein kinase A inhibitor Rp-8-Br-cyclic AMPS dose-dependently inhibited the enhancing effect of ATP or adenosine indicating the participation of an adenosine receptor-mediated cyclic AMP-dependent signalling pathway. 7. These data indicate that ATP mediates proinflammatory long-term effects in rat mesangial cells via its degradation product adenosine through the A2A receptor resulting in potentiation of sPLA(2)-IIA induction.

  17. EphA2-receptor deficiency exacerbates myocardial infarction and reduces survival in hyperglycemic mice.

    PubMed

    DuSablon, Augustin; Kent, Susan; Coburn, Anita; Virag, Jitka

    2014-08-13

    We have previously shown that EphrinA1/EphA expression profile changes in response to myocardial infarction (MI), exogenous EphrinA1-Fc administration following MI positively influences wound healing, and that deletion of the EphA2 Receptor (EphA2-R) exacerbates injury and remodeling. To determine whether or not ephrinA1-Fc would be of therapeutic value in the hyperglycemic infarcted heart, it is critical to evaluate how ephrinA1/EphA signaling changes in the hyperglycemic myocardium in response to MI. Streptozotocin (STZ)-induced hyperglycemia in wild type (WT) and EphA2-receptor mutant (EphA2-R-M) mice was initiated by an intraperitoneal injection of STZ (150 mg/kg) 10 days before surgery. MI was induced by permanent ligation of the left anterior descending coronary artery and analyses were performed at 4 days post-MI. ANOVAs with Student-Newman Keuls multiple comparison post-hoc analysis illustrated which groups were significantly different, with significance of at least p < 0.05. Both WT and EphA2-R-M mice responded adversely to STZ, but only hyperglycemic EphA2-R-M mice had lower ejection fraction (EF) and fractional shortening (FS). At 4 days post-MI, we observed greater post-MI mortality in EphA2-R-M mice compared with WT and this was greater still in the EphA2-R-M hyperglycemic mice. Although infarct size was greater in hyperglycemic WT mice vs normoglycemic mice, there was no difference between hyperglycemic EphA2-R-M mice and normoglycemic EphA2-R-M mice. The hypertrophic response that normally occurs in viable myocardium remote to the infarct was noticeably absent in epicardial cardiomyocytes and cardiac dysfunction worsened in hyperglycemic EphA2-R-M hearts post-MI. The characteristic interstitial fibrotic response in the compensating myocardium remote to the infarct also did not occur in hyperglycemic EphA2-R-M mouse hearts to the same extent as that observed in the hyperglycemic WT mouse hearts. Differences in neutrophil and pan

  18. LASSBio-897 Reduces Lung Injury Induced by Silica Particles in Mice: Potential Interaction with the A2A Receptor

    PubMed Central

    Carvalho, Vinicius F.; Ferreira, Tatiana P. T.; de Arantes, Ana C. S.; Noël, François; Tesch, Roberta; Sant’Anna, Carlos M. R.; Barreiro, Eliezer J. L.; Fraga, Carlos A. M.; Rodrigues e Silva, Patrícia M.; Martins, Marco A.

    2017-01-01

    Silicosis is a lethal fibro-granulomatous pulmonary disease highly prevalent in developing countries, for which no proper therapy is available. Among a small series of N-acylhydrazones, the safrole-derived compound LASSBio-897 (3-thienylidene-3, 4-methylenedioxybenzoylhydrazide) raised interest due to its ability to bind to the adenosine A2A receptor. Here, we evaluated the anti-inflammatory and anti-fibrotic potential of LASSBio-897, exploring translation to a mouse model of silicosis and the A2A receptor as a site of action. Pulmonary mechanics, inflammatory, and fibrotic changes were assessed 28 days after intranasal instillation of silica particles in Swiss–Webster mice. Glosensor cAMP HEK293G cells, CHO cells stably expressing human adenosine receptors and ligand binding assay were used to evaluate the pharmacological properties of LASSBio-897 in vitro. Molecular docking studies of LASSBio-897 were performed using the genetic algorithm software GOLD 5.2. We found that the interventional treatment with the A2A receptor agonist CGS 21680 reversed silica particle-induced airway hyper-reactivity as revealed by increased responses of airway resistance and lung elastance following aerosolized methacholine. LASSBio-897 (2 and 5 mg/kg, oral) similarly reversed pivotal lung pathological features of silicosis in this model, reducing levels of airway resistance and lung elastance, granuloma formation and collagen deposition. In competition assays, LASSBio-897 decreased the binding of the selective A2A receptor agonist [3H]-CGS21680 (IC50 = 9.3 μM). LASSBio-897 (50 μM) induced modest cAMP production in HEK293G cells, but it clearly synergized the cAMP production by adenosine in a mechanism sensitive to the A2A antagonist SCH 58261. This synergism was also seen in CHO cells expressing the A2A, but not those expressing A2B, A1 or A3 receptors. Based on the evidence that LASSBio-897 binds to A2A receptor, molecular docking studies were performed using the A2A receptor

  19. Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC.

    PubMed

    Brand, Frank; Klutz, Athena M; Jacobson, Kenneth A; Fredholm, Bertil B; Schulte, Gunnar

    2008-08-20

    G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.

  20. Adenosine A2A Receptor Signaling in the Immunopathogenesis of Experimental Autoimmune Encephalomyelitis

    PubMed Central

    Rajasundaram, Skanda

    2018-01-01

    Our increasing appreciation of adenosine as an endogenous signaling molecule that terminates inflammation has generated excitement regarding the potential to target adenosine receptors (ARs) in the treatment of multiple sclerosis (MS), a disease of chronic neuroinflammation. Of the four G protein-coupled ARs, A2ARs are the principal mediator of adenosine’s anti-inflammatory effects and accordingly, there is a growing body of evidence surrounding the role of A2ARs in experimental autoimmune encephalomyelitis (EAE), the dominant animal model of MS. Such evidence points to a complex, often paradoxical role for A2ARs in the immunopathogenesis of EAE, where they have the ability to both exacerbate and alleviate disease severity. This review seeks to interpret these paradoxical findings and evaluate the therapeutic promise of A2ARs. In essence, the complexities of A2AR signaling arise from two properties. Firstly, A2AR signaling downregulates the inflammatory potential of TH lymphocytes whilst simultaneously facilitating the recruitment of these cells into the CNS. Secondly, A2AR expression by myeloid cells – infiltrating macrophages and CNS-resident microglia – has the capacity to promote both tissue injury and repair in chronic neuroinflammation. Consequently, the therapeutic potential of targeting A2ARs is greatly undermined by the risk of collateral tissue damage in the periphery and/or CNS. PMID:29559972

  1. The adenosine a2a receptor inhibits matrix-induced inflammation in a novel fashion.

    PubMed

    Scheibner, Kara A; Boodoo, Sada; Collins, Samuel; Black, Katharine E; Chan-Li, Yee; Zarek, Paul; Powell, Jonathan D; Horton, Maureen R

    2009-03-01

    Endogenous mediators within the inflammatory milieu play a critical role in directing the scope, duration, and resolution of inflammation. High-molecular-weight extracellular matrix hyaluronan (HA) helps to maintain homeostasis. During inflammation, hyaluronan is broken down into fragments that induce chemokines and cytokines, thereby augmenting the inflammatory response. Tissue-derived adenosine, released during inflammation, inhibits inflammation via the anti-inflammatory A2 adenosine receptor (A2aR). We demonstrate that adenosine modulates HA-induced gene expression via the A2aR. A2aR stimulation inhibits HA fragment-induced pro-fibrotic genes TNF-alpha, keratinocyte chemoattractant (KC), macrophage inflammatory protein (MIP)-2, and MIP-1alpha while simultaneously synergizing with hyaluronan fragments to up-regulate the TH1 cytokine IL-12. Interestingly, A2aR stimulation mediates these affects via the novel cAMP-activated guanine nucleotide exchange factor EPAC. In addition, A2aR-null mice are more susceptible to bleomycin-induced lung injury, consistent with a role for endogenous adenosine in inhibiting the inflammation that may lead to fibrosis. Indeed, the bleomycin treated A2aR-null mice demonstrate increased lung inflammation, HA accumulation, and histologic damage. Overall, our data elucidate the opposing roles of tissue-derived HA fragments and adenosine in regulating noninfectious lung inflammation and support the pursuit of A2aR agonists as a means of pharmacologically inhibiting inflammation that may lead to fibrosis.

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

    PubMed

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

    2017-07-01

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

  3. Cloning and expression of a novel neuropeptide Y receptor.

    PubMed

    Weinberg, D H; Sirinathsinghji, D J; Tan, C P; Shiao, L L; Morin, N; Rigby, M R; Heavens, R H; Rapoport, D R; Bayne, M L; Cascieri, M A; Strader, C D; Linemeyer, D L; MacNeil, D J

    1996-07-12

    The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

  4. Expression of Neurotrophins and their Receptors in Human Bone Marrow

    PubMed Central

    Labouyrie, Eric; Dubus, Pierre; Groppi, Alexis; Mahon, François Xavier; Ferrer, Jacky; Parrens, Marie; Reiffers, Josy; de Mascarel, Antoine; Merlio, Jean Philippe

    1999-01-01

    The expression of neurotrophins and their receptors, the low-affinity nerve growth factor receptor (p75LNGFR) and the Trk receptors (TrkA, TrkB, and TrkC), was investigated in human bone marrow from 16 weeks fetal age to adulthood. Using reverse transcription-polymerase chain reaction, all transcripts encoding for catalytic and truncated human TrkB or TrkC receptors were detected together with trkAI transcripts, whereas trkAII transcripts were found only in control nerve tissues. Transcripts for the homologue of the rat truncated TrkC(ic113) receptor were identified for the first time in human tissue. Stromal adventitial reticular cells were found immunoreactive for all neutrophin receptors. In contrast, hematopoietic cell types were not immunoreactive for p75LNGFR but showed immunoreactivity for one or several Trk receptors. TrkA immunoreactivity was found in immature erythroblasts. Catalytic TrkB immunoreactivity was observed in eosinophilic metamyelocytes and polymorphonuclear cells. Truncated TrkB immunoreactivity was found in erythroblasts and megacaryocytes. Immunoreactivity for both catalytic and truncated TrkC receptor was observed in promyelocytes, myelocytes, some polymorphonuclear cells and megacaryocytes. Neutrophin transcript levels appeared higher at fetal than at adult stages, no variation in Trk family transcript levels was observed. The local expression of neurotrophin genes suggests a wide range of paracrine and/or autocrine mode of action through their corresponding receptors within the bone marrow. PMID:10027399

  5. Increased Expression of Cannabinoid CB1 Receptors in Achilles Tendinosis

    PubMed Central

    Björklund, Emmelie; Forsgren, Sture; Alfredson, Håkan; Fowler, Christopher J.

    2011-01-01

    Background The endogenous cannabinoid system is involved in the control of pain. However, little is known as to the integrity of the cannabinoid system in human pain syndromes. Here we investigate the expression of the cannabinoid receptor 1 (CB1) in human Achilles tendons from healthy volunteers and from patients with Achilles tendinosis. Methodology Cannabinoid CB1 receptor immunoreactivity (CB1IR) was evaluated in formalin-fixed biopsies from individuals suffering from painful Achilles tendinosis in comparison with healthy human Achilles tendons. Principal Findings CB1IR was seen as a granular pattern in the tenocytes. CB1IR was also observed in the blood vessel wall and in the perineurium of the nerve. Quantification of the immunoreactivity in tenocytes showed an increase of CB1 receptor expression in tendinosis tissue compared to control tissue. Conclusion Expression of cannabinoid receptor 1 is increased in human Achilles tendinosis suggesting that the cannabinoid system may be dysregulated in this disorder. PMID:21931835

  6. Endothelin-receptor gene-expression in rat endotoxemia.

    PubMed

    Bucher, Michael; Taeger, Kai

    2002-05-01

    The reduced vascular response to endothelin-1 has focused interest onto the regulation of the endothelin-receptor subtypes ET(A) and ET(B) during severe sepsis. Prospective animal trial followed by a controlled cell culture study in the laboratory of the Department of Anesthesiology. Male Sprague-Dawley rats weighing 200-250 g, aortic vascular smooth muscle cell line A7r5. Rats were injected with lipopolysaccharide to induce severe experimental endotoxemia. ET(A)/ET(B) receptor gene expression was investigated by specific RNase protection assay, and abundance of tumor necrosis factor alpha was determined in the lung and kidney. Aortic vascular smooth muscle cells were incubated with the proinflammatory cytokines interleukin-1beta, tumor necrosis factor alpha, and interferon gamma or with the nitric oxide donor S-nitroso- N-acetylpenicillamine to investigate the regulation of ET(A) receptor gene expression during severe inflammation. ET(A)/ET(B) receptor gene expression was markedly downregulated in the lung but was unchanged in the kidney during endotoxemia. ET(A) receptor gene expression was downregulated in aortic vascular smooth muscle cells by tumor necrosis factor alpha but not by interleukin 1beta, interferon gamma, or nitric oxide. In vivo there seems to be a correlation between the tissue concentration of tumor necrosis factor alpha and gene expression of ET(A) receptors in the lung and kidney. Our data show that sepsis causes downregulation of ET(A) receptors at the level of gene expression, and provide correlative evidence that this effect can be mediated by tumor necrosis factor alpha. This downregulation of ET(A) receptors possibly contributes to the attenuated vascular response to endothelin-1 in the pulmonary circulation.

  7. 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

  8. Expression cloning of an ATP receptor from mouse neuroblastoma cells.

    PubMed Central

    Lustig, K D; Shiau, A K; Brake, A J; Julius, D

    1993-01-01

    Extracellular ATP activates cell-surface metabotropic and ionotropic nucleotide (P2) receptors in vascular, neural, connective, and immune tissues. These P2 receptors mediate a wealth of physiological processes, including nitric oxide-dependent vasodilation of vascular smooth muscle and fast excitatory neurotransmission in sensory afferents. Although ATP is now recognized as a signaling molecule, the cellular and molecular mechanisms underlying its actions have been difficult to study due to the absence of selective P2 receptor antagonists and cloned receptor genes. Nonetheless, five mammalian P2 receptor subtypes have been tentatively assigned based solely on agonist specificity and signaling properties. Here we report the cloning of a mouse cDNA encoding a P2 receptor that shares striking homology with several G protein-coupled peptide receptors. When expressed in Xenopus laevis oocytes, the cloned receptor resembles a metabotropic P2U receptor; activation by either ATP or UTP elicits the mobilization of intracellular calcium. mRNA encoding the P2U purinergic receptor is found in neural and nonneural tissues. Images Fig. 3 Fig. 5 PMID:7685114

  9. Expression and structure of the human NGF receptor

    SciTech Connect

    Johnson, D.E.

    1988-01-01

    Nerve growth factor (NGF) plays an essential role in the development and maintenance of sensory and sympathetic neurons of the peripheral nervous system. Recent evidence indicates that NGF also acts in the central nervous system. We have used fragments of the human NGF receptor gene to isolate human NGF receptor cDNA clones. The cDNA clones have been sequenced, and the organization of the NGF receptor mRNA as well as the primary structure of the receptor protein have been determined. The message contains a short 5{prime} nontranslated region and a long 3{prime} nontranslated region. The receptor protein contains 399 amino acids,more » is cysteine-rich, and spans the membrane once. A full-length NGF receptor cDNA was cloned into a mammalian expression vector and transfected into NGFR-mouse L cells. Binding of {sup 125}I-NGF to transfectant cell lines was specific and saturable, verifying that the cloned protein represents an authentic NGF receptor. Transfectant cell lines overexpressing the human receptor were used to immunize syngenic animals and polyclonal antisera specific for the human NGF receptor were isolated. A series of NGF receptor mutants with truncated carboxyl termini were constructed and introduced into mouse L cells. Mutant proteins which contain intact transmembrane domains retain NGF binding activity and are localized to the plasma membrane.« less

  10. Characterization of the A2B adenosine receptor from mouse, rabbit, and dog.

    PubMed

    Auchampach, John A; Kreckler, Laura M; Wan, Tina C; Maas, Jason E; van der Hoeven, Dharini; Gizewski, Elizabeth; Narayanan, Jayashree; Maas, Garren E

    2009-04-01

    We have cloned and pharmacologically characterized the A(2B) adenosine receptor (AR) from the dog, rabbit, and mouse. The full coding regions of the dog and mouse A(2B)AR were obtained by reverse transcriptase-polymerase chain reaction, and the rabbit A(2B)AR cDNA was obtained by screening a rabbit brain cDNA library. It is noteworthy that an additional clone was isolated by library screening that was identical in sequence to the full-length rabbit A(2B)AR, with the exception of a 27-base pair deletion in the region encoding amino acids 103 to 111 (A(2B)AR(103-111)). This 9 amino acid deletion is located in the second intracellular loop at the only known splice junction of the A(2B)AR and seems to result from the use of an additional 5' donor site found in the rabbit and dog but not in the human, rat, or mouse sequences. [(3)H]3-Isobutyl-8-pyrrolidinoxanthine and 8-[4-[((4-cyano-[2,6-(3)H]-phenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine ([(3)H]MRS 1754) bound with high affinity to membranes prepared from human embryonic kidney (HEK) 293 cells expressing mouse, rabbit, and dog A(2B)ARs. Competition binding studies performed with a panel of agonist (adenosine and 2-amino-3,5-dicyano-4-phenylpyridine analogs) and antagonist ligands identified similar potency orders for the A(2B)AR orthologs, although most xanthine antagonists displayed lower binding affinity for the dog A(2B)AR compared with A(2B)ARs from rabbit and mouse. No specific binding could be detected with membranes prepared from HEK 293 cells expressing the rabbit A(2B)AR(103-111) variant. Furthermore, the variant failed to stimulate adenylyl cyclase or calcium mobilization. We conclude that significant differences in antagonist pharmacology of the A(2B)AR exist between species and that some species express nonfunctional variants of the A(2B)AR due to "leaky" splicing.

  11. CGRP receptor activity in mice with global expression of human receptor activity modifying protein 1.

    PubMed

    Bohn, Keegan J; Li, Baolin; Huang, Xiaofang; Mason, Bianca N; Wattiez, Anne-Sophie; Kuburas, Adisa; Walker, Christopher S; Yang, Peiyi; Yu, Jianliang; Heinz, Beverly A; Johnson, Kirk W; Russo, Andrew F

    2017-06-01

    CGRP is a potent vasodilator and nociceptive neuropeptide linked to migraine. CGRP receptors are heterodimers of receptor activity modifying protein 1 (RAMP1) and either calcitonin receptor-like receptor (CLR; forms canonical CGRP receptor) or calcitonin receptor (CT receptor; forms AMY 1 receptor). The goal of this study was to test whether transgenic mice globally expressing human RAMP1 have increased CGRP receptor activity and whether the receptors are sensitive to human selective antagonist telcagepant. cAMP production was measured in primary cultures of aortic smooth muscle and trigeminal ganglia neurons from global hRAMP1 mice and non-transgenic littermates. Functional activity and inhibition were compared with clonal cell lines expressing combinations of CLR or CT receptors with RAMP1. Cultured smooth muscle from global hRAMP1 mice had a 10-fold greater CGRP-induced cAMP maximal response (Rmax) than non-transgenic littermates, with similar EC 50 s. In contrast, cultured trigeminal ganglia from global hRAMP1 mice had a 40-fold leftward shift of the EC 50 , with similar Rmax values as littermates. In both hRAMP1 cultures, telcagepant blocked CGRP-induced cAMP production, but was not effective in non-transgenic cultures. IC 50 values were closer to those observed for CT receptor/hRAMP1 than CLR/hRAMP1 in clonal cell lines. Overexpression of hRAMP1 increases CGRP signalling by changing the maximal response or ligand sensitivity, depending on tissue type. Furthermore, telcagepant inhibited transgenic hRAMP1 CGRP receptors, but the degree of inhibition suggests that the transgenic mice are only partially humanized or both canonical CGRP and AMY 1 receptors are functional in trigeminal ganglia neurons and vascular smooth muscle. © 2017 The British Pharmacological Society.

  12. Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

    PubMed

    Peñas-Cazorla, Raúl; Vilaró, M Teresa

    2015-11-01

    Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists.

  13. The orphan nuclear receptor Nr5a2 is essential for luteinization in the female mouse ovary.

    PubMed

    Bertolin, Kalyne; Gossen, Jan; Schoonjans, Kristina; Murphy, Bruce D

    2014-05-01

    In the ovary, the follicular granulosa cells express the nuclear receptor Nr5a2 (nuclear receptor subfamily 5 group A member 2), also known as liver receptor homolog-1, and after ovulation, Nr5a2 expression persists in the corpus luteum. Previous studies demonstrated that Nr5a2 is required for both ovulation and luteal steroid synthesis. Our objectives were to analyze the temporal sequence in the regulatory effects of Nr5a2 in the ovary, with focus on its contribution to luteal function. We developed a female mouse model of granulosa-specific targeted disruption from the formation of the antral follicles forward (genotype Nr5a2(Cyp19-/-)). Mice lacking Nr5a2 in granulosa cells of antral follicles are infertile. Although their cumulus cells undergo expansion after gonadotropin stimulation, ovulation is disrupted in those mice, at least in part, due to the down-regulation of the progesterone receptor (Pgr) gene. The depletion of Nr5a2 in antral follicles permits formation of luteal-like structures but not functional corpora lutea, as evidenced by reduced progesterone levels and failure to support pseudopregnancy. Progesterone synthesis is affected by depletion of Nr5a2 due to, among others, defects in the transport of cholesterol, evidenced by down-regulation of Scarb1, Ldlr, and Star. Comparison of this mouse line with the models in which Nr5a2 is depleted from the primary follicle forward (genotype Nr5a2(Amhr2-/-)) and after the ovulatory signal (genotype Nr5a2(Pgr-/-)) demonstrates that Nr5a2 differentially regulates female fertility across the trajectory of follicular development.

  14. [The expression of triggering receptor expressed on myeloid cells receptor-1 in Aspergillus infected mice].

    PubMed

    Cui, N; Wang, H; Su, L X; Zhang, J H; Liu, D W

    2017-08-01

    Objective: To investigate the expression of triggering receptor expressed on myeloid cells receptor-1 (TREM-1) in plasma and bronchoalveolar lavage fluid (BALF) and its correlation with Galactomannan, IFNγ, IL-6 and IL-10 in Aspergillus infected mice. Methods: Cyclophosphamide(CTX) was intraperitoneally injected and fumigatus spore suspension was inhaled by nose to establish the immunocompromised invasive pulmonary aspergillosis(IPA) mouse model.Healthy controls, immunocompromised only and IPA only groups were also established. Each group had 6 mice. After inoculation, mice were sacrificed. Lung tissue specimens, BALF, and plasma samples were collected. Plasma and BALF soluble TREM-1 (sTREM-1), Galactomannan, IFNγ, IL-6, and IL-10 were detected by ELISA. Results: Positive Aspergillus fumigatus was found by tissue culture in the lung. Infiltration of inflammatory cells, blood congestion and interstitial lung tissue injury were observed in histological sections of both IPA and immunocompromised IPA mice. Compared to IPA group [(453.78±74.18) ng/L, P <0.001; (10.21±1.46) ng/L, P <0.001] and control group [(245.16±65.85) ng/L, P <0.001; (6.60±3.74) ng/L, P <0.001], the plasma and BALF sTREM-1 significantly increased in immunocompromised IPA group [(1 537.64±359.52) ng/L; (20.12±2.72) ng/L]. Compared to control group, both the BALF sTREM-1 in IPA group ( P =0.041) and the plasma and BALF Galactomannan, IFNγ, IL-6, and IL-10 levels in IPA and immunocompromised IPA groups were significantly higher ( P <0.01). Pearson correlation analysis showed that plasma and BALF sTREM-1 were significantly correlated with Galactomannan ( r =0.83, P <0.001; r =0.82, P <0.001), IFNγ ( r =0.79, P <0.001; r =0.61, P <0.01), IL-6 ( r =0.81, P <0.001; r =0.66, P <0.01), and IL-10 ( r =0.70, P =0.001; r =0.54, P =0.02). Conclusions: Plasma and BALF sTREM-1 appears highly expressed in Aspergillus infected mice. sTREM-1 in mice plasma and BALF is closely correlated with Galactomannan

  15. A 2A adenosine receptor regulates glia proliferation and pain after peripheral nerve injury.

    PubMed

    Bura, S Andreea; Nadal, Xavier; Ledent, Catherine; Maldonado, Rafael; Valverde, Olga

    2008-11-15

    Peripheral nerve injury produces a persistent neuropathic pain state characterized by spontaneous pain, allodynia and hyperalgesia. In this study, we evaluated the possible involvement of A 2ARs in the development of neuropathic pain and the expression of microglia and astrocytes in the spinal cord after sciatic nerve injury. For this purpose, partial ligation of the sciatic nerve was performed in A 2A knockout mice and wild-type littermates. The development of mechanical and thermal allodynia, as well as thermal hyperalgesia was evaluated by using the von Frey filament model, the cold-plate test and the plantar test, respectively. In wild-type animals, sciatic nerve injury led to a neuropathic pain syndrome that was revealed in these three nociceptive behavioural tests. However, a significant decrease of the mechanical allodynia and a suppression of thermal hyperalgesia and allodynia were observed in A 2AR deficient mice. The expression of microglia and astrocytes was enhanced in wild-type mice exposed to sciatic nerve injury and this response was attenuated in knockout animals. Taken together, our results demonstrate the involvement of A 2ARs in the control of neuropathic pain and propose this receptor as an interesting target for the development of new drugs for the management of this clinical syndrome.

  16. 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

  17. Prolactin receptor antagonism in mouse anterior pituitary: effects on cell turnover and prolactin receptor expression.

    PubMed

    Ferraris, Jimena; Boutillon, Florence; Bernadet, Marie; Seilicovich, Adriana; Goffin, Vincent; Pisera, Daniel

    2012-02-01

    Since anterior pituitary expresses prolactin receptors, prolactin secreted by lactotropes could exert autocrine or paracrine actions on anterior pituitary cells. In fact, it has been observed that prolactin inhibits its own expression by lactotropes. Our hypothesis is that prolactin participates in the control of anterior pituitary cell turnover. In the present study, we explored the action of prolactin on proliferation and apoptosis of anterior pituitary cells and its effect on the expression of the prolactin receptor. To determine the activity of endogenous prolactin, we evaluated the effect of the competitive prolactin receptor antagonist Δ1-9-G129R-hPRL in vivo, using transgenic mice that constitutively and systemically express this antagonist. The weight of the pituitary gland and the anterior pituitary proliferation index, determined by BrdU incorporation, were higher in transgenic mice expressing the antagonist than in wild-type littermates. In addition, blockade of prolactin receptor in vitro by Δ1-9-G129R-hPRL increased proliferation and inhibited apoptosis of somatolactotrope GH3 cells and of primary cultures of male rat anterior pituitary cells, including lactotropes. These results suggest that prolactin acts as an autocrine/paracrine antiproliferative and proapoptotic factor in the anterior pituitary gland. In addition, anterior pituitary expression of the long isoform of the prolactin receptor, measured by real-time PCR, increased about 10-fold in transgenic mice expressing the prolactin receptor antagonist, whereas only a modest increase in the S3 short-isoform expression was observed. These results suggest that endogenous prolactin may regulate its own biological actions in the anterior pituitary by inhibiting the expression of the long isoform of the prolactin receptor. In conclusion, our observations suggest that prolactin is involved in the maintenance of physiological cell renewal in the anterior pituitary. Alterations in this physiological

  18. Functional histamine H3 and adenosine A2A receptor heteromers in recombinant cells and rat striatum.

    PubMed

    Márquez-Gómez, Ricardo; Robins, Meridith T; Gutiérrez-Rodelo, Citlaly; Arias, Juan-Manuel; Olivares-Reyes, Jesús-Alberto; van Rijn, Richard M; Arias-Montaño, José-Antonio

    2018-03-01

    In the striatum, histamine H 3 receptors (H 3 Rs) are co-expressed with adenosine A 2A receptors (A 2A Rs) in the cortico-striatal glutamatergic afferents and the GABAergic medium-sized spiny neurons that originate the indirect pathway of the basal ganglia. This location allows H 3 Rs and A 2A Rs to regulate the striatal GABAergic and glutamatergic transmission. However, whether these receptors can physically interact has not yet been assessed. To test this hypothesis, a heteromer-selective in vitro assay was used to detect functional complementation between a chimeric A 2A R 302 -Gα qi4 and wild-type H 3 Rs in transfected HEK-293T cells. H 3 R activation with the agonist RAMH resulted in Ca 2+ mobilization (pEC 50 7.31 ± 0.23; maximal stimulation, Emax 449 ± 25% of basal) indicative of receptor heterodimerization. Functional H 3 R-A 2A R heteromers were confirmed by co-immunoprecipitation and observations of differential cAMP signaling when both receptors were co-expressed in the same cells. In membranes from rat striatal synaptosomes, H 3 R activation decreased A 2A R affinity for the agonist CGS-21680 (pKi values 8.10 ± 0.04 and 7.70 ± 0.04). Moreover, H 3 Rs and A 2A Rs co-immunoprecipitated in protein extracts from striatal synaptosomes. These results support the existence of a H 3 R-A 2A R heteromer with possible physiological implications for the modulation of the intra-striatal transmission. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Links Between Insulin Resistance, Adenosine A2B Receptors, and Inflammatory Markers in Mice and Humans

    PubMed Central

    Figler, Robert A.; Wang, Guoquan; Srinivasan, Susseela; Jung, Dae Young; Zhang, Zhiyou; Pankow, James S.; Ravid, Katya; Fredholm, Bertil; Hedrick, Catherine C.; Rich, Stephen S.; Kim, Jason K.; LaNoue, Kathryn F.; Linden, Joel

    2011-01-01

    OBJECTIVE To determine the mechanisms by which blockade of adenosine A2B receptors (A2BRs) reduces insulin resistance. RESEARCH DESIGN AND METHODS We investigated the effects of deleting or blocking the A2BR on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A2BR transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ∼42 kb encompassing the A2BR gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation. RESULTS Treatment of mice with the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A2BR deletion or blockade and minimally affected by deletion of A1Rs or A2ARs. During hyperinsulinemic-euglycemic clamp of diabetic KKAY mice, A2BR antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A2BR mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status. CONCLUSIONS Diabetes affects the production of adenosine and the expression of A2BRs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A2BR signaling in diabetes

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

    PubMed

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

    2015-07-01

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

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

    PubMed Central

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

    2015-01-01

    Background Eph receptors and ephrin ligands have several functions including angiogenesis, cell migration, axon guidance, fluid homeostasis, oncogenesis, inflammation and injury repair. The EphA2 receptor potentially mediates the regulation of vascular permeability and inflammation in response to lung injury. Methods Mice were divided into 3 experimental groups to study the role of EphA2 signaling in the lipopolysaccharide (LPS)-induced lung injury model i.e., IgG+phosphate-buffered saline (PBS) group (IgG instillation before PBS exposure), IgG+LPS group (IgG instillation before LPS exposure) and EphA2 monoclonal antibody (mAb)+LPS group (EphA2 mAb pretreatment before LPS exposure). Results EphA2 and ephrinA1 were upregulated in LPS-induced lung injury. The lung injury score of the EphA2 mAb+LPS group was lower than that of the IgG+LPS group (4.30±2.93 vs. 11.45±1.20, respectively; p=0.004). Cell counts (EphA2 mAb+LPS: 11.33×104±8.84×104 vs. IgG+LPS: 208.0×104±122.6×104; p=0.018) and total protein concentrations (EphA2 mAb+LPS: 0.52±0.41 mg/mL vs. IgG+LPS: 1.38±1.08 mg/mL; p=0.192) were decreased in EphA2 mAb+LPS group, as compared to the IgG+LPS group. In addition, EphA2 antagonism reduced the expression of phospho-p85, phosphoinositide 3-kinase 110γ, phospho-Akt, nuclear factor κB, and proinflammatory cytokines. Conclusion This results of the study indicated a role for EphA2-ephrinA1 signaling in the pathogenesis of LPS-induced lung injury. Furthermore, EphA2 antagonism inhibits the phosphoinositide 3-kinase-Akt pathway and attenuates inflammation. PMID:26175775

  2. Ligand-dependent oligomerization of dopamine D(2) and adenosine A(2A) receptors in living neuronal cells.

    PubMed

    Vidi, Pierre-Alexandre; Chemel, Benjamin R; Hu, Chang-Deng; Watts, Val J

    2008-09-01

    Adenosine A(2A) and dopamine D(2) receptors (A(2A) and D(2)) associate in homo- and heteromeric complexes in the striatum, providing a structural basis for their mutual antagonism. At the cellular level, the portion of receptors engaging in homo- and heteromers, as well as the effect of persistent receptor activation or antagonism on the cell oligomer repertoire, are largely unknown. We have used bimolecular fluorescence complementation (BiFC) to visualize A(2A) and D(2) oligomerization in the Cath.a differentiated neuronal cell model. Receptor fusions to BiFC fluorescent protein fragments retained their function when expressed alone or in A(2A)/A(2A), D(2)/D(2), and A(2A)/D(2) BiFC pairs. Robust fluorescence complementation reflecting A(2A)/D(2) heteromers was detected at the cell membrane as well as in endosomes. In contrast, weaker BiFC signals, largely confined to intracellular domains, were detected with A(2A)/dopamine D(1) BiFC pairs. Multicolor BiFC was used to simultaneously visualize A(2A) and D(2) homo- and heteromers in living cells and to examine drug-induced changes in receptor oligomers. Prolonged D(2) stimulation with quinpirole lead to the internalization of D(2)/D(2) and A(2A)/D(2) oligomers and resulted in decreased A(2A)/D(2) relative to A(2A)/A(2A) oligomer formation. Opposing effects were observed in cells treated with D(2) antagonists or with the A(2A) agonist 5'-N-methylcarboxamidoadenosine (MECA). Subsequent radioreceptor binding analysis indicated that the drug-induced changes in oligomer formation were not readily explained by alterations in receptor density. These observations support the hypothesis that long-term drug exposure differentially alters A(2A)/D(2) receptor oligomerization and provide the first demonstration for the use of BiFC to monitor drug-modulated GPCR oligomerization.

  3. 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

  4. Developmental changes in NMDA receptor expression in the platyfish brain

    NASA Technical Reports Server (NTRS)

    Flynn, K. M.; Schreibman, M. P.; Magliulo-Cepriano, L.

    1997-01-01

    We have examined the distribution of the N-methyl-D-aspartate (NMDA) receptor in the brain of a freshwater teleost using an antibody against the R1 subunit of the receptor (NMDAR1). The primary site of localization was the nucleus olfactoretinalis (NOR), a significant gonadotropin releasing hormone (GnRH)-containing brain nucleus. The number of cells expressing NMDAR1 in this nucleus was dependent upon developmental stage, with pubescent and mature animals displaying significantly more stained cells than immature and senescent animals. This is the first reported observation of age- and maturity-related NMDA receptor association with GnRH-containing brain areas.

  5. Beta-adrenergic receptors are expressed across diverse cancers.

    PubMed

    Rains, Steven L; Amaya, Clarissa N; Bryan, Brad A

    2017-07-01

    Based largely on retrospective analyses and a handful of prospective case reports, pharmacological inhibition of the beta adrenergic receptors using beta blockers has shown clinical anti-cancer efficacy in reproductive cancers, as well as angiosarcoma and multiple myeloma. Because of the potential promise of beta blockers as an adjunct to standard anti-cancer therapy, it is imperative to identify other tumor types expressing beta adrenergic (β-AR) receptors so future preclinical and clinical studies can be directed at the most promising tumor targets. We performed immunohistochemical detection of β1-AR, β2-AR, and β3-AR across 29 of the most common human cancer types (389 tissues total) and 19 matching non-diseased controls (100 tissues total). Our analysis revealed all three β-AR receptors were expressed most strongly in melanoma relative to other cancer types. Other malignancies that revealed relatively higher levels of β-AR receptors were esophagus, pancreas, kidney, and lung cancers. Moreover, particular β-AR receptors exhibited significant overexpression in tumor tissue relative to their matching normal tissue in urogenital/reproductive malignancies including breast, endometrium, ovarian, and urothelial cancer, as well as colon, lung, and thyroid cancer. This study identifies several cancer types expressing the β-AR receptors which should be evaluated in future studies for susceptibility to beta blockade.

  6. Elevated hepatic glucocorticoid receptor expression during liver regeneration in rats.

    PubMed

    Karabélyos, C; Dobozy, O; Szalai, C; Klenjánszki, K; Varjú, K; Hadházi, A; Kiss, A; Fülöp, A K; Madarász, B; Falus, A

    1999-01-01

    In rats within the first week of partial hepatectomy reconstruction of the normal histological structure of the liver already starts. To approach the possible role of endogenous glucocorticoids in the process of regeneration we measured the changes in the expression of steroid glucocorticoid receptor gene after various regeneration intervals. After partial hepatectomy, between 0.5 168 hours from the surgery, the gene expression (mRNA) of glucocorticoid receptor was determined by reverse transcription followed by PCR and normalized to that of glycerolphoshate dehydrogenase. Two peaks of glucocorticoid receptor mRNA were detected first, between 3 and 6 hours (first peak) and a second between 24 and 36 hours. Immunoreactive glucocorticoid receptor was detected by immunohistochemistry using monoclonal anti-glucocorticoid receptor. Three days after the surgery immunohistochemical studies showed substantially more immunoreactive GcR protein in the regenerated liver than in the controls. These semiquantitative data provide evidence suggesting elevation of glucocorticoid receptor expression during regeneration of liver at mRNA and protein levels.

  7. Expression of hormone receptors in oropharyngeal squamous cell carcinoma.

    PubMed

    Mohamed, Hesham; Aro, Katri; Jouhi, Lauri; Mäkitie, Antti; Remes, Satu; Haglund, Caj; Atula, Timo; Hagström, Jaana

    2018-03-26

    Hormone receptors play an important role in many types of cancers. Alongside factors associated with human papillomavirus (HPV) infection, hormonal receptors may impact the tumorigenesis of oropharyngeal cancer. This study consists of 199 consecutive oropharyngeal squamous cell carcinoma (OPSCC) patients diagnosed and treated with a curative intent. We examined androgen (AR), estrogen (ER; both alpha and beta), and progesterone receptor (PR) expressions using immunohistochemistry comparing tumor and patient characteristics. AR was expressed in 16%, PR in 27% and ER-beta in 63% of the tumors. HPV- and p16-positive tumors expressed more AR and less PR than their negative counterparts. High PR expression was associated with poor disease-specific and locoregional recurrence-free survival. AR, PR, and ER-beta are expressed in OPSCC, and AR and PR expressions are associated with HPV and p16 status. Furthermore, PR appears to have prognostic significance. This may allow us to investigate the role of anti-hormone receptors in the treatment of OPSCC.

  8. Multiple melanocortin receptors are expressed in bone cells

    NASA Technical Reports Server (NTRS)

    Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

    2005-01-01

    Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

  9. TAM Receptors in Leukemia: Expression, Signaling, and Therapeutic Implications

    PubMed Central

    Brandão, Luis; Migdall-Wilson, Justine; Eisenman, Kristen; Graham, Douglas K.

    2016-01-01

    In the past 30 years there has been remarkable progress in the treatment of leukemia and lymphoma. However, current treatments are largely ineffective against relapsed leukemia and, in the case of pediatric patients, are often associated with severe long-term toxicities. Thus, there continues to be a critical need for the development of effective biologically targeted therapies. The TAM family of receptor tyrosine kinases—Tyro3, Axl, and Mer—plays an important role in normal hematopoiesis, including natural killer cell maturation, macrophage function, and platelet activation and signaling. Furthermore, TAM receptor activation leads to upregulation of pro-survival and proliferation signaling pathways, and aberrant TAM receptor expression contributes to cancer development, including myeloid and lymphoid leukemia. This review summarizes the role of TAM receptors in leukemia. We outline TAM receptor expression patterns in different forms of leukemia, describe potential mechanisms leading to their overexpression, and delineate the signaling pathways downstream of receptor activation that have been implicated in leukemogenesis. Finally, we discuss the current research focused on inhibitors against these receptors in an effort to develop new therapeutic strategies for leukemia. PMID:22150307

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

    PubMed Central

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

    2016-01-01

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

  11. Regulation of mu opioid receptor expression in developing T cells.

    PubMed

    Zhang, Lily; Belkowski, Judith Sliker; Briscoe, Tammi; Rogers, Thomas J

    2012-12-01

    We have previously reported that functionally active μ-opioid receptors (MOR) are constitutively expressed at relatively low levels by developing T cells in the thymus. However, very little is known about the regulation of MOR expression by immature T cells. In this report, we first attempted to determine the effect of T cell receptor-induced T cell activation on the expression of MOR. We activated T cells with either the combination of anti-CD3 and CD28, or with superantigen, and observed a substantial increase in MOR transcript expression. We also chose to examine the effect of cytokine-mediated T cell activation on the expression of this opioid receptor. We selected certain cytokines that play a role in T cell development and are known to be present at functional levels in the thymus gland. Our results show that interferon γ (IFNγ), IL-1β, and IL-2, and in particular transforming growth factor-β (TGFβ), all induced significant increases in MOR transcript expression. On the other hand, both TNFα and IL-7 exhibited much weaker effects on MOR expression. These results show that MOR expression by developing T cells is strongly regulated by several cytokines involved in T cell development in the thymus gland.

  12. Hormone receptor expression in patients with dermatofibrosarcoma protuberans.

    PubMed

    Kreicher, Kathryn L; Honda, Kord S; Kurlander, David E; Bordeaux, Jeremy S

    2016-12-01

    Dermatofibrosarcoma protuberans (DFSP) is a rare cutaneous sarcoma for which the exact etiology is unknown. Case reports exist of DFSP appearing and growing rapidly during pregnancy, suggesting a hormonal role. Our goal was to determine the expression of estrogen receptors (ERs) and progesterone receptors (PRs) in patients with DFSP. Archived formalin-fixed, paraffin-embedded tissue from patients with DFSP in the past 20 years at a single institution were analyzed for ER and PR using immunohistochemistry. A semiquantitative scoring method was used to evaluate the expression as positive or negative. Analysis was used to determine whether there was an association between receptor positivity and tumor site, age at diagnosis, sex, race, or disease recurrence. Forty-four patients with DFSP were included in the study. Tumors were 22.7% ER + /PR + , 34.1% ER + /PR - , 9.1% ER - /PR + , and 34.1% ER - /PR - . There was no significant association between expression of ER and PR and sex, age at diagnosis, race, or tumor location. Loss of receptor expression was observed in all recurrent tumors. This study is limited by a lack of follow-up and a new scoring system. The data presented warrant additional study to determine hormone receptor function and the potential efficacy of antihormone therapies for the treatment of patients with DFSP. Copyright © 2016 American Academy of Dermatology, Inc. Published by Elsevier Inc. All rights reserved.

  13. An update on adenosine A2A receptors as drug target in Parkinson's disease.

    PubMed

    Vallano, Antoni; Fernandez-Duenas, Victor; Pedros, Consuelo; Arnau, Josep Maria; Ciruela, Francisco

    2011-09-01

    Adenosine receptors are G protein-coupled receptors (GPCRs) that mediate the physiological functions of adenosine. In the central nervous system adenosine A(2A) receptors (A(2A)Rs) are highly enriched in striatopallidal neurons where they form functional oligomeric complexes with other GPCRs such us the dopamine D(2) receptor (D(2)R). Furthermore, it is assumed that the formation of balanced A(2A)R/D(2)R receptor oligomers are essential for correct striatal function as the allosteric receptor-receptor interactions established within the oligomer are needed for properly sensing adenosine and dopamine. Interestingly, A(2A)R activation reduces the affinity of striatal D(2)R for dopamine and the blockade of A(2A)R with specific antagonists facilitates function of the D(2)R. Thus, it may be postulated that A(2A)R antagonists are pro-dopaminergic agents. Therefore, A(2A)R antagonists will potentially reduce the effects associated with dopamine depletion in Parkinson's disease (PD). Accordingly, this class of compounds have recently attracted considerable attention as potential therapeutic agents for PD pharmacotherapy as they have shown potential effectiveness in counteracting motor dysfunctions and also displayed neuroprotective and anti-inflammatory effects in animal models of PD. Overall, we provide here an update of the current state of the art of these A(2A)R-based approaches that are under clinical study as agents devoted to alleviate PD symptoms.

  14. Repeated exposure to morphine alters surface expression of AMPA receptors in the rat medial prefrontal cortex.

    PubMed

    Mickiewicz, Amanda L; Napier, T Celeste

    2011-01-01

    Behavioral sensitization describes the intensification of motor activity that results from repeated exposure to drugs of misuse, and the underlying neuronal adaptations are hypothesized to model aspects of the brain changes that occur in humans misusing such drugs. The α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor is an ionotropic glutamate receptor involved in the neuroplasticity that accompanies acute and repeated drug administration. Changing surface expression is one means to regulate AMPA receptor function, and the present study tested the hypothesis that behavioral sensitization to the μ-opioid receptor agonist morphine is accompanied by changes in the subcellular distribution of AMPA receptors in limbic brain regions. To test this hypothesis, we used a protein cross-linking assay to assess cell surface and intracellular levels of GluA1 and GluA2 subunits in the nucleus accumbens, medial prefrontal cortex and ventral pallidum. Repeated morphine treatment decreased surface expression of GluA1 in the medial prefrontal cortex without affecting levels of GluA2. In contrast, surface levels of GluA1 or GluA2 were unchanged in the nucleus accumbens and ventral pallidum, demonstrating that although AMPA receptors in accumbal and pallidal regions are critical mediators of behaviors induced by repeated opiate exposure, these effects are not accompanied by changes in surface expression. The findings reveal that the involvement of AMPA receptor trafficking in opiate-induced behavioral sensitization is relegated to selective regions and that AMPA receptors in the medial prefrontal cortex may be particularly sensitive to these actions. © 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

  15. Aggregation Limits Surface Expression of Homomeric GluA3 Receptors*

    PubMed Central

    Coleman, Sarah K.; Hou, Ying; Willibald, Marina; Semenov, Artur; Möykkynen, Tommi; Keinänen, Kari

    2016-01-01

    AMPA receptors are glutamate-gated cation channels assembled from GluA1–4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway. PMID:26912664

  16. 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.

  17. Evolution of odorant receptors expressed in mammalian testes.

    PubMed

    Branscomb, A; Seger, J; White, R L

    2000-10-01

    About 10% of mammalian odorant receptors are transcribed in testes, and odorant-receptor proteins have been detected on mature spermatozoa. Testis-expressed odorant receptors (TORs) are hypothesized to play roles in sperm chemotaxis, but they might also be ordinary nasal odorant receptors (NORs) that are expressed gratuitously in testes. Under the sperm-chemotaxis hypothesis, TORs should be subject to intense sexual selection and therefore should show higher rates of amino acid substitution than NORs, but under the gratuitous-expression hypothesis, TORs are misidentified NORs and therefore should evolve like other NORs. To test these predictions, we estimated synonymous and nonsynonymous divergences of orthologous NOR and TOR coding sequences from rat and mouse. Contrary to both hypotheses, TORs are on average more highly conserved than NORs, especially in certain domains of the OR protein. This pattern suggests that some TORs might perform internal nonolfactory functions in testes; for example, they might participate in the regulation of sperm development. However, the pattern is also consistent with a modified gratuitous-expression model in which NORs with specialized ligand specificities are both more highly conserved than typical NORs and more likely to be expressed in testes.

  18. Functional Expression of FSH Receptor in Endometriotic Lesions.

    PubMed

    Ponikwicka-Tyszko, Donata; Chrusciel, Marcin; Stelmaszewska, Joanna; Bernaczyk, Piotr; Sztachelska, Maria; Sidorkiewicz, Iwona; Doroszko, Milena; Tomaszewski, Jakub; Tapanainen, Juha S; Huhtaniemi, Ilpo; Wolczynski, Slawomir; Rahman, Nafis A

    2016-07-01

    FSH receptor (FSHR), besides being expressed in gonads, is also expressed in some extragonadal tissues at low levels. We examined the functional expression of FSHR in different types of endometriotic lesions. Extensive studies were carried out to detect functional FSHR expression and FSH-stimulated estrogen production in ovarian endometriomas and recto-vaginal endometriotic nodules (RVEN). Normal endometrium, ovary, and myometrium tissues from nonpregnant cycling women served as controls. This laboratory-based study was carried out on tissue specimens from patients with endometriosis and healthy donors. Endometriotic lesions and normal secretory-phase endometrium showed FSHR expression at both mRNA and protein level. RVEN and ovarian endometrioma demonstrated up-regulated CYP19A1, dependent on the activation of CYP19A1 proximal promoter II. Estrogen receptor-β (ESR2) expression was significantly increased in RVEN vs normal endometrium. Recombinant human FSH stimulation of RVEN explants significantly increased estradiol production and CYP19A1 and ESR2 expression. FSHR was up-regulated in recombinant human FSH-stimulated endometrial and decidualized stromal cells with increased CYP19A1 expression. We described a novel functional FSHR expression, where FSH-stimulated CYP19A1 expression and estrogen production in RVEN are demonstrated. This locally FSH-induced estrogen production may contribute to the pathology, development, progression, and severity of RVEN.

  19. Changes in spinal delta and kappa opioid systems in mice deficient in the A2A receptor gene.

    PubMed

    Bailey, Alexis; Ledent, Catherine; Kelly, Mary; Hourani, Susanna M O; Kitchen, Ian

    2002-11-01

    A large body of evidence indicates important interactions between the adenosine and opioid systems in regulating pain at both the spinal and supraspinal level. Mice lacking the A(2A) receptor gene have been developed successfully, and these animals were shown to be hypoalgesic. To investigate whether there are any compensatory alterations in opioid systems in mutant animals, we have performed quantitative autoradiographic mapping of mu, delta, kappa, and opioid receptor-like (ORL1) opioid receptors in the brains and spinal cords of wild-type and homozygous A(2A) receptor knock-out mice. In addition, mu-, delta-, and kappa-mediated antinociception using the tail immersion test was tested in wild-type and homozygous A(2A) receptor knock-out mice. A significant reduction in [3H]deltorphin-I binding to delta receptors and a significant increase in [3H]CI-977 binding to kappa receptors was detected in the spinal cords but not in the brains of the knock-out mice. Mu and ORL1 receptor expression were not altered significantly. Moreover, a significant reduction in delta-mediated antinociception and a significant increase in kappa-mediated antinociception were detected in mutant mice, whereas mu-mediated antinociception was unaffected. Comparison of basal nociceptive latencies showed a significant hypoalgesia in knock-out mice when tested at 55 degrees C but not at 52 degrees C. The results suggest a functional interaction between the spinal delta and kappa opioid and the peripheral adenosine system in the control of pain pathways.

  20. 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.

  1. A2B adenosine receptor induces protective antihelminth type 2 immune responses.

    PubMed

    Patel, Nirav; Wu, Wenhui; Mishra, Pankaj K; Chen, Fei; Millman, Ariel; Csóka, Balázs; Koscsó, Balázs; Eltzschig, Holger K; Haskó, György; Gause, William C

    2014-03-12

    The type 2 immune response evoked by intestinal nematode parasites contributes to worm expulsion and tolerance to associated tissue damage. We investigated whether this host response is affected by blocking signaling by the putative endogenous danger signal adenosine, which can be released during inflammation and host cell damage. Specific blockade of the A2B adenosine receptor (A2BAR) inhibited worm elimination and the development of innate and adaptive components of the type 2 primary and memory response. Infected mice lacking A2BAR exhibited decreased M2 macrophage and eosinophil recruitment and reduced IL-4 and IL-13 cytokine production. Additionally, shortly after infection, upregulation of the alarmin IL-33, which drives type 2 immunity, and activation of innate lymphoid type 2 (ILC2) cells was inhibited, while exogenous IL-33 restored ILC2 cell activation and type 2 cytokine expression. Thus, adenosine acts as a danger-associated molecular pattern (DAMP) that initiates helminth-induced type 2 immune responses through A2BAR. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. The plant hormone zeatin riboside inhibits T lymphocyte activity via adenosine A2A receptor activation.

    PubMed

    Lappas, Courtney M

    2015-01-01

    Cytokinins are plant hormones that play an integral role in multiple aspects of plant growth and development. The biological functions of cytokinins in mammalian systems are, however, largely uncharacterized. The naturally occurring cytokinin zeatin riboside has recently been demonstrated to activate the mammalian adenosine A(2A) receptor, which is broadly expressed by various cell types including immune system cells, with the activation of the A(2A)R playing a role in the regulation of cells involved in both innate and adaptive immunity. We show for the first time that zeatin riboside modulates mammalian immune system activity via an A(2A)R-dependent mechanism. Specifically, zeatin riboside treatment induces the production of cyclic adenosine monophosphate (cAMP) by T lymphocytes and inhibits the production by CD3(+)CD4(+) T cells of interferon (IFN)-γ, IL-2, tumor-necrosis factor (TNF)-α, IL-4 and IL-13, and the production by CD3(+)CD8(+) T cells of IFN-γ, IL-2 and TNF-α. Additionally, the upregulation of CD25, CD69 and CD40L by activated T lymphocytes is modulated by zeatin riboside. Zeatin riboside treatment also potently inhibits thioglycollate-induced peritoneal leukocytosis. The immunomodulatory activities of zeatin riboside are blocked by co-treatment with the selective A(2A)R antagonist ZM241385. These data suggest that zeatin riboside possesses therapeutic potential as a mammalian immunomodulatory agent.

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

    PubMed Central

    Mundell, SJ; Matharu, A-L; Nisar, S; Palmer, TM; Benovic, JL; Kelly, E

    2010-01-01

    Background and purpose: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A2B adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5′-(N-ethylcarboxamido)-adenosine. Experimental approach: The trafficking of the wild type A2B adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. Key results: The wild type A2B adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln325-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu330-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln325-stop, Ser326-stop and Phe328-stop receptors. Following internalization, the wild type A2B adenosine receptor recycled rapidly to the cell surface, whereas the Gln325-stop receptor did not recycle. Conclusions and implications: Deletion of the COOH-terminus of the A2B adenosine receptor beyond Leu330 switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A2B adenosine receptor following prolonged agonist addition. PMID:20128803

  4. Molecular Imaging and Quantitation of EphA2 Expression in Xenograft Models with 89Zr-DS-8895a.

    PubMed

    Burvenich, Ingrid J G; Parakh, Sagun; Gan, Hui K; Lee, Fook-Thean; Guo, Nancy; Rigopoulos, Angela; Lee, Sze-Ting; Gong, Sylvia; O'Keefe, Graeme J; Tochon-Danguy, Henri; Kotsuma, Masakatsu; Hasegawa, Jun; Senaldi, Giorgio; Scott, Andrew M

    2016-06-01

    Subtype A2 of the erythropoietin-producing hepatocellular tyrosine kinase (EphA2) cell surface receptor is expressed in a range of epithelial cancers. This study evaluated the molecular imaging of EphA2 expression in vivo in mouse tumor models using SPECT/MR and PET/MR and a humanized anti-EphA2 antibody, DS-8895a. DS-8895a was labeled with (111)In, (125)I, and (89)Zr and assessed for radiochemical purity, immunoreactivity (Lindmo analysis), antigen-binding affinity (Scatchard analysis), and serum stability in vitro. In vivo biodistribution, imaging, and pharmacokinetic studies were performed with SPECT/MR and PET/MR. A dose-escalation study was also performed to determine EphA2 receptor saturability through tissue and imaging quantitative analysis. All conjugates demonstrated good serum stability and specific binding to EphA2-expressing cells in vitro. In vivo biodistribution studies showed high uptake of (111)In-CHX-A″-DTPA-DS-8895a and (89)Zr-Df-Bz-NCS-DS-8895a in EphA2-expressing xenograft models, with no specific uptake in normal tissues. In comparison, retention of (125)I-DS-8895a in tumors was lower because of internalization of the radioconjugate and dehalogenation. These results were confirmed by SPECT/MR and PET/MR. EphA2 receptor saturation was observed at the 30 mg/kg dose. Molecular imaging of tumor uptake of DS-8895a allows noninvasive measurement of EphA2 expression in tumors in vivo and determination of receptor saturation. (89)Zr-Df-Bz-NCS-DS-8895a is suited for human bioimaging trials on the basis of superior imaging characteristics and will inform DS-8895a dose assessment and patient response evaluation in clinical trials. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

  5. Modulation of adenosine A1 and A2A receptors in C6 glioma cells during hypoxia: involvement of endogenous adenosine.

    PubMed

    Castillo, Carlos A; León, David; Ruiz, María Angeles; Albasanz, José Luis; Martín, Mairena

    2008-06-01

    During hypoxia, extracellular adenosine levels are increased to prevent cell damage, playing a neuroprotective role mainly through adenosine A(1) receptors. The aim of the present study was to analyze the effect of hypoxia in both adenosine A(1) and A(2A) receptors endogenously expressed in C6 glioma cells. Two hours of hypoxia (5% O(2)) caused a significant decrease in adenosine A(1) receptors. The same effect was observed at 6 h and 24 h of hypoxia. However, adenosine A(2A) receptors were significantly increased at the same times. These effects were not due to hypoxia-induced alterations in cells number or viability. Changes in receptor density were not associated with variations in the rate of gene expression. Furthermore, hypoxia did not alter HIF-1alpha expression in C6 cells. However, HIF-3alpha, CREB and CREM were decreased. Adenosine A(1) and A(2A) receptor density in normoxic C6 cells treated with adenosine for 2, 6 and 24 h was similar to that observed in cells after oxygen deprivation. When C6 cells were subjected to hypoxia in the presence of adenosine deaminase, the density of receptors was not significantly modulated. Moreover, DPCPX, an A(1) receptor antagonist, blocked the effects of hypoxia on these receptors, while ZM241385, an A(2A) receptor antagonist, was unable to prevent these changes. These results suggest that moderate hypoxia modulates adenosine receptors and cAMP response elements in glial cells, through a mechanism in which endogenous adenosine and tonic A(1) receptor activation is involved.

  6. Early Expression of Odorant Receptors Distorts the Olfactory Circuitry

    PubMed Central

    Nguyen, Minh Q.; Marks, Carolyn A.; Belluscio, Leonardo; Ryba, Nicholas J. P.

    2010-01-01

    The odor response properties of a mammalian olfactory sensory neuron (OSN) are determined by the tightly regulated expression of a single member of a very large family of odorant receptors (ORs). The OR also plays an important role in focusing the central projections of all OSNs expressing that particular receptor to a pair of stereotypic locations (glomeruli) in each olfactory bulb (OB), thus creating a spatial map of odor responses in the brain. Here we show that when initiated late in neural development, transgenic expression of one OR in almost all OSNs has little influence on the architecture of the OB. In contrast, early OR-transgene expression (mediated by the Gγ8-promoter) in 50–70% of OSNs grossly distorts the morphology of glomeruli and alters the projection patterns of many residual OSNs not expressing the transgene. Interestingly, this disruption of targeting persists in adult animals despite down-regulation of Gγ8 and transgenic OR expression that occurs as olfactory neurogenesis declines. Indeed, functional imaging studies reveal a dramatic decrease in the complexity of responses to odorants in adult Gγ8-transgenic OR mice. Thus, we show that initiation of transgenic OR-expression early in the development of OSNs, rather than just the extent of transgene expression, determines its effectiveness at modifying OB anatomy and function. Taken together these data imply that OR-expression timing needs to be very tightly controlled to achieve the precise wiring and function of the mammalian olfactory system. PMID:20610762

  7. CHOLESTEROL 27-HYDROXYLASE BUT NOT APOLIPOPROTEIN apoE CONTRIBUTES TO A2A ADENOSINE RECEPTOR STIMULATED REVERSE CHOLESTEROL TRANSPORT

    PubMed Central

    Bingham, Taiese Crystal; Parathath, Saj; Tian, Heather; Reiss, Allison; Chan, Edwin; Fisher, Edward A.; Cronstein, Bruce N.

    2011-01-01

    Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A2A receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A2A receptors on foam cell formation in vitro are mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p< 0.0002, IFNγ CGS vs. IFNγ alone, n= 4) CGS-21680, an adenosine A2A receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4±2% p<0.5113 inhibition vs. IFNγ alone n= 4). Previously we reported the A2A agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly 2-fold in wildtype macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A2A receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1. PMID:21258856

  8. Cholesterol 27-hydroxylase but not apolipoprotein apoE contributes to A2A adenosine receptor stimulated reverse cholesterol transport.

    PubMed

    Bingham, Taiese Crystal; Parathath, Saj; Tian, Heather; Reiss, Allison; Chan, Edwin; Fisher, Edward A; Cronstein, Bruce N

    2012-02-01

    Movement of free cholesterol between the cellular compartment and acceptor is governed by cholesterol gradients that are determined by several enzymes and reverse cholesterol transport proteins. We have previously demonstrated that adenosine A(2A) receptors inhibit foam cell formation and stimulate production of cholesterol 27-hydroxylase (CYP27A1), an enzyme involved in the conversion of cholesterol to oxysterols. We therefore asked whether the effect of adenosine A(2A) receptors on foam cell formation in vitro is mediated by CYP27A1 or apoE, a carrier for cholesterol in the serum. We found that specific lentiviral siRNA infection markedly reduced apoE or 27-hydroxylase mRNA in THP-1 cells. Despite diminished apoE expression (p < 0.0002, interferon-gamma (IFNγ) CGS vs. IFNγ alone, n=4), CGS-21680, an adenosine A(2A) receptor agonist, inhibits foam cell formation. In contrast, CGS-21680 had no effect on reducing foam cell formation in CYP27A1 KD cells (4 ± 2%; p<0.5113, inhibition vs. IFNγ alone, n=4). Previously, we reported the A(2A) agonist CGS-21680 increases apoAI-mediated cholesterol efflux nearly twofold in wild-type macrophages. Adenosine receptor activation had no effect on cholesterol efflux in CYP27A1 KD cells but reduced efflux in apoE KD cells. These results demonstrate that adenosine A(2A) receptor occupancy diminishes foam cell formation by increasing expression and function of CYP27A1.

  9. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  10. Prolactin receptor expression in gynaecomastia and male breast carcinoma.

    PubMed

    Ferreira, M; Mesquita, M; Quaresma, M; André, S

    2008-07-01

    Despite the well-established function of prolactin (PRL) in normal breast development, its role in breast cancer pathogenesis is still controversial. PRL activity is dependent on the activation of a transmembrane protein, the PRL receptor (PRLR). The aim was to evaluate and compare PRLR expression in gynaecomastia and male breast carcinoma (MBC). PRLR expression was detected immunohistochemically in 30 cases of gynaecomastia and 30 cases of MBC. The whole series was also assessed for oestrogen receptors (ER), progesterone receptors (PR) and androgen receptors (AR). A cut-off of 10% was used as the criterion for positivity. Histological type and tumour differentiation were evaluated. Pathological stage was assessed [Tumour Node Metastasis (TNM)-International Union Against Cancer system]. Statistical analysis was performed with Fisher's exact test. PRLR positivity was seen in 20% of gynaecomastia cases and in 60% of MBC cases (P = 0.003). In gynaecomastia immunoreactivity was predominantly observed in luminal cell borders, whereas in MBC the reactivity was heterogeneous and mainly cytoplasmic. There was no statistically significant correlation between PRLR expression and ER, PR, AR, pTNM, or histological grade. PRLR is significantly more expressed in MBC than in gynaecomastia, and with different patterns of reactivity, suggesting a role for PRL in male breast carcinogenesis.

  11. 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.

  12. Integrated olfactory receptor and microarray gene expression databases

    PubMed Central

    Liu, Nian; Crasto, Chiquito J; Ma, Minghong

    2007-01-01

    Background Gene expression patterns of olfactory receptors (ORs) are an important component of the signal encoding mechanism in the olfactory system since they determine the interactions between odorant ligands and sensory neurons. We have developed the Olfactory Receptor Microarray Database (ORMD) to house OR gene expression data. ORMD is integrated with the Olfactory Receptor Database (ORDB), which is a key repository of OR gene information. Both databases aim to aid experimental research related to olfaction. Description ORMD is a Web-accessible database that provides a secure data repository for OR microarray experiments. It contains both publicly available and private data; accessing the latter requires authenticated login. The ORMD is designed to allow users to not only deposit gene expression data but also manage their projects/experiments. For example, contributors can choose whether to make their datasets public. For each experiment, users can download the raw data files and view and export the gene expression data. For each OR gene being probed in a microarray experiment, a hyperlink to that gene in ORDB provides access to genomic and proteomic information related to the corresponding olfactory receptor. Individual ORs archived in ORDB are also linked to ORMD, allowing users access to the related microarray gene expression data. Conclusion ORMD serves as a data repository and project management system. It facilitates the study of microarray experiments of gene expression in the olfactory system. In conjunction with ORDB, ORMD integrates gene expression data with the genomic and functional data of ORs, and is thus a useful resource for both olfactory researchers and the public. PMID:17603910

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  15. Glycine receptor subunits expression in the developing rat retina.

    PubMed

    Sánchez-Chávez, Gustavo; Velázquez-Flores, Miguel Ángel; Ruiz Esparza-Garrido, Ruth; Salceda, Rocío

    2017-09-01

    Glycine receptor (GlyR) consists of two α (1-4) and three β subunits. Considerable evidence indicates that the adult retina expresses the four types of α subunits; however, the proportion of these subunits in adult and immature retina is almost unknown. In this report we have studied mRNA and the protein expression of GlyR subunits in the retina during postnatal rat development by Real-Time qRT-PCR and western blot. mRNA and protein expression indicated a gradual increase of the α1, α3, α4 and β GlyR subunits during postnatal ages tested. The mRNA β subunit showed higher expression levels (∼3 fold) than those observed for the α1 and α3 subunits. Very interestingly, the α2 GlyR subunit had the highest expression in the retina, even in the adult. These results revealed the expression of GlyR at early postnatal ages, supporting its role in retina development. In addition, our results indicated that the adult retina expressed a high proportion of the α2 subunit, suggesting the expression of monomeric and/or heteromeric receptors. A variety of studies are needed to further characterize the role of the specific subunits in both adult and immature retina. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Adenosine A2A Receptor Up-Regulates Retinal Wave Frequency via Starburst Amacrine Cells in the Developing Rat Retina

    PubMed Central

    Huang, Pin-Chien; Hsiao, Yu-Tien; Kao, Shao-Yen; Chen, Ching-Feng; Chen, Yu-Chieh; Chiang, Chung-Wei; Lee, Chien-fei; Lu, Juu-Chin; Chern, Yijuang; Wang, Chih-Tien

    2014-01-01

    Background Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A2A receptor (A2AR) regulates retinal waves and whether A2AR regulation of retinal waves acts via presynaptic SACs. Methodology/Principal Findings We showed that A2AR was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A2AR decreased the frequency of spontaneous Ca2+ transients, suggesting that endogenous A2AR may up-regulate wave frequency. To investigate whether A2AR acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca2+ transient frequency was increased by expressing wild-type A2AR (A2AR-WT) in SACs, suggesting that A2AR may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A2AR-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A2AR may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A2AR mutant (A2AR-ΔC) in SACs, the wave frequency was reduced compared to the A2AR-WT, but was similar to the control, suggesting that the full-length A2AR in SACs is required for A2AR up-regulation of retinal waves. Conclusions/Significance A2AR up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full

  17. Adenosine A(2A) receptor up-regulates retinal wave frequency via starburst amacrine cells in the developing rat retina.

    PubMed

    Huang, Pin-Chien; Hsiao, Yu-Tien; Kao, Shao-Yen; Chen, Ching-Feng; Chen, Yu-Chieh; Chiang, Chung-Wei; Lee, Chien-Fei; Lu, Juu-Chin; Chern, Yijuang; Wang, Chih-Tien

    2014-01-01

    Developing retinas display retinal waves, the patterned spontaneous activity essential for circuit refinement. During the first postnatal week in rodents, retinal waves are mediated by synaptic transmission between starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). The neuromodulator adenosine is essential for the generation of retinal waves. However, the cellular basis underlying adenosine's regulation of retinal waves remains elusive. Here, we investigated whether and how the adenosine A(2A) receptor (A(2A)R) regulates retinal waves and whether A(2A)R regulation of retinal waves acts via presynaptic SACs. We showed that A(2A)R was expressed in the inner plexiform layer and ganglion cell layer of the developing rat retina. Knockdown of A(2A)R decreased the frequency of spontaneous Ca²⁺ transients, suggesting that endogenous A(2A)R may up-regulate wave frequency. To investigate whether A(2A)R acts via presynaptic SACs, we targeted gene expression to SACs by the metabotropic glutamate receptor type II promoter. Ca²⁺ transient frequency was increased by expressing wild-type A(2A)R (A2AR-WT) in SACs, suggesting that A(2A)R may up-regulate retinal waves via presynaptic SACs. Subsequent patch-clamp recordings on RGCs revealed that presynaptic A(2A)R-WT increased the frequency of wave-associated postsynaptic currents (PSCs) or depolarizations compared to the control, without changing the RGC's excitability, membrane potentials, or PSC charge. These findings suggest that presynaptic A(2A)R may not affect the membrane properties of postsynaptic RGCs. In contrast, by expressing the C-terminal truncated A(2A)R mutant (A(2A)R-ΔC) in SACs, the wave frequency was reduced compared to the A(2A)R-WT, but was similar to the control, suggesting that the full-length A(2A)R in SACs is required for A(2A)R up-regulation of retinal waves. A(2A)R up-regulates the frequency of retinal waves via presynaptic SACs, requiring its full-length protein structure. Thus, by

  18. Expression of epidermal growth factor receptor in breast carcinoma.

    PubMed Central

    Lewis, S; Locker, A; Todd, J H; Bell, J A; Nicholson, R; Elston, C W; Blamey, R W; Ellis, I O

    1990-01-01

    A series of 90 patients with primary operable breast cancer and with up to 36 months of follow up were investigated for expression of epidermal growth factor receptor (EGFR) in their tumours by immunocytochemical staining with the monoclonal antibody EGFR 1. Tumour samples were snap frozen in liquid nitrogen immediately after resection and subsequently stained using a standard indirect immunocytochemical method. Tumour staining was assessed by two observers and scored on a four point scale (0-3). Thirteen (14%) tumours showed positive immunoreactivity. A strong correlation between distinct EGFR expression and short disease free interval was observed. Significant correlations were also shown with oestrogen and progesterone receptor expression and tumour nuclear size. No significant association was found with tumour size, lymph node stage, and histological grade. The association with disease free interval remained significant in multivariate analysis. Images PMID:2370306

  19. Myeloid-derived suppressor cells contribute to A2B adenosine receptor-induced VEGF production and angiogenesis in a mouse melanoma model.

    PubMed

    Sorrentino, Claudia; Miele, Lucio; Porta, Amalia; Pinto, Aldo; Morello, Silvana

    2015-09-29

    Vascular endothelial growth factor (VEGF) is an angiogenic factor critically involved in tumor progression. Adenosine A2B receptor plays a pivotal role in promoting tumor growth. The aim of this study was to investigate the role of myeloid-derived suppressor cells (MDSCs) in the pro-angiogenic effects of A2B and to determine whether A2B blockade could enhance the effectiveness of anti-VEGF treatment. Mice treated with Bay60-6583, a selective A2B receptor agonist, showed enhanced tumor VEGF-A expression and vessel density. This effect was associated with accelerated tumor growth, which could be reversed with anti-VEGF treatment. Bay60-6583 increased the accumulation of tumor CD11b+Gr1+ cells. Depletion of MDSCs in mice significantly reduced A2B-induced VEGF production. However, A2B receptor stimulation did not directly regulate VEGF expression in isolated tumor myeloid cells. Mechanistically, Bay60-6583-treated melanoma tissues showed increased STAT3 activation. Inhibition of STAT3 significantly decreased the pro-tumor activity of Bay60-6583 and reduced tumor VEGF expression. Pharmacological blockade of A2B receptor with PSB1115 significantly reduced tumor growth by inhibiting tumor angiogenesis and increasing T cells numbers within the tumor microenvironment. These effects are, at least in part, dependent on impaired tumor accumulation of Gr1+ cells upon A2B receptor blockade. PSB1115 increased the effectiveness of anti-VEGF treatment.

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

    PubMed

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

    2018-04-01

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

  1. Tropomyosin Receptor Kinase A Expression on Merkel Cell Carcinoma Cells.

    PubMed

    Wehkamp, Ulrike; Stern, Sophie; Krüger, Sandra; Hauschild, Axel; Röcken, Christoph; Egberts, Friederike

    2017-11-01

    Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor frequently associated with the Merkel cell polyomavirus. Immune checkpoint therapy showed remarkable results, although not all patients are responsive to this therapy. Anti-tropomyosin receptor kinase A (TrkA)-targeted treatment has shown promising results in several tumor entities. To determine TrkA expression in MCC as a rationale for potential targeted therapy. This case series study investigated the MCC specimens of 55 patients treated at the Department of Dermatology, University Hospital of Schleswig-Holstein, Kiel, Germany, from January 1, 2005, through December 31, 2015. Thirty-nine of the 55 samples were suitable for further histopathologic examination. Expression of TrkA was explored by immunohistochemical analysis. Diagnosis of MCC was confirmed by staining positive for cytokeratin 20 (CK20) and synaptophysin. Expression of TrkA on the tumor cells. Specimens of 39 patients (21 women and 18 men; mean [SD] age, 75.0 [7.8] years) underwent immunohistochemical investigation. Thirty-eight of 38 specimens expressed CK20 and synaptophysin on the MCC tumor cells (100% expression). Merkel cell polyomavirus was detected in 32 of 38 specimens (84%). Tropomyosin receptor kinase A was found in all 36 evaluable specimens on the tumor cells; 34 (94%) showed a weak and 2 (6%) showed a strong cytoplasmic expression. In addition, strongly positive perinuclear dots were observed in 30 of 36 specimens (83%). Tropomyosin receptor kinase A was expressed on MCC tumor cells in 100% of evaluable specimens. This result may lead to the exploration of new targeted treatment options in MCC, especially for patients who do not respond to anti-programmed cell death protein 1 treatment.

  2. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster.

    PubMed

    Cheung, Samantha K; Scott, Kristin

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation.

  3. GABAA receptor-expressing neurons promote consumption in Drosophila melanogaster

    PubMed Central

    Cheung, Samantha K.

    2017-01-01

    Feeding decisions are highly plastic and bidirectionally regulated by neurons that either promote or inhibit feeding. In Drosophila melanogaster, recent studies have identified four GABAergic interneurons that act as critical brakes to prevent incessant feeding. These GABAergic neurons may inhibit target neurons that drive consumption. Here, we tested this hypothesis by examining GABA receptors and neurons that promote consumption. We find that Resistance to dieldrin (RDL), a GABAA type receptor, is required for proper control of ingestion. Knockdown of Rdl in a subset of neurons causes overconsumption of tastants. Acute activation of these neurons is sufficient to drive consumption of appetitive substances and non-appetitive substances and acute silencing of these neurons decreases consumption. Taken together, these studies identify GABAA receptor-expressing neurons that promote Drosophila ingestive behavior and provide insight into feeding regulation. PMID:28362856

  4. Up-regulation of striatal adenosine A2A receptors with iron deficiency in rats. Effects on locomotion and cortico-striatal neurotransmission

    PubMed Central

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-01-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A2A receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A2A receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A2A receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A2A receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A2A receptors was found in rats fed during three weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A2A receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A2A receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A2A receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A2A receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS. PMID:20385128

  5. Up-regulation of striatal adenosine A(2A) receptors with iron deficiency in rats: effects on locomotion and cortico-striatal neurotransmission.

    PubMed

    Quiroz, César; Pearson, Virginia; Gulyani, Seema; Allen, Richard; Earley, Christopher; Ferré, Sergi

    2010-07-01

    Brain iron deficiency leads to altered dopaminergic function in experimental animals, which can provide a mechanistic explanation for iron deficiency-related human sensory-motor disorders, such as Restless Legs Syndrome (RLS). However, mechanisms linking both conditions have not been determined. Considering the strong modulation exerted by adenosine on dopamine signaling, one connection could involve changes in adenosine receptor expression or function. In the striatum, presynaptic A(2A) receptors are localized in glutamatergic terminals contacting GABAergic dynorphinergic neurons and their function can be analyzed by the ability of A(2A) receptor antagonists to block the motor output induced by cortical electrical stimulation. Postsynaptic A(2A) receptors are localized in the dendritic field of GABAergic enkephalinergic neurons and their function can be analyzed by studying the ability of A(2A) receptor antagonists to produce locomotor activity and to counteract striatal ERK1/2 phosphorylation induced by cortical electrical stimulation. Increased density of striatal A(2A) receptors was found in rats fed during 3 weeks with an iron-deficient diet during the post-weaning period. In iron-deficient rats, the selective A(2A) receptor antagonist MSX-3, at doses of 1 and 3 mg/kg, was more effective at blocking motor output induced by cortical electrical stimulation (presynaptic A(2A) receptor-mediated effect) and at enhancing locomotor activation and blocking striatal ERK phosphorylation induced by cortical electrical stimulation (postsynaptic A(2A) receptor-mediated effects). These results indicate that brain iron deficiency induces a functional up-regulation of both striatal pre- and postsynaptic A(2A) receptor, which could be involved in sensory-motor disorders associated with iron deficiency such as RLS. Copyright 2010. Published by Elsevier Inc.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28338017

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

    PubMed

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

    2018-03-01

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

  9. Pharmacological evidence for different populations of postsynaptic adenosine A2A receptors in the rat striatum

    PubMed Central

    Orrú, Marco; Quiroz, César; Guitart, Xavier; Ferré, Sergi

    2011-01-01

    Adenosine A2A receptors (A2ARs) are highly concentrated in the striatum. Two pharmacological different functional populations of A2ARs have been recently described based on their different affinities for the A2AR antagonist SCH-442416. This compound has high affinity for A2ARs not forming heteromers or forming heteromers with adenosine A1 receptors (A1Rs) while showing very low affinity for A2ARs forming heteromers with dopamine D2 receptors (D2Rs). It has been widely described that striatal A1R-A2AR heteromers are preferentially localized presynaptically in the glutamatergic terminals that contact GABAergic dynorphinergic neurons, and that A2AR-D2R heteromers are localized postsynaptically in GABAergic enkephalinergic neurons. In the present study we provide evidence suggesting that SCH-442416 also targets postsynaptic A2AR not forming heteromers with D2R, which are involved in the motor depressant effects induced by D2R antagonists. SCH-442416 counteracted motor depression in rats induced by the D2R antagonist raclopride at a dose that did not produce motor activation or that blocked motor depression induced by an A2AR agonist. Furthermore, we re-evaluated the recently suggested key role of cannabinoid CB1 receptors (CB1Rs) in the effects of A2AR antagonists acting at postsynaptic A2ARs. By recording locomotor activity and monitoring striatal glutamate release induced by cortical electrical stimulation in rats after administration of A2AR and CB1R antagonists, we did not find evidence for any significant role of endocannabinoids in the post- or presynaptic effects of A2AR antagonists. The present results further suggest the existence of at least two functionally and pharmacologically different populations of striatal postsynaptic A2ARs. PMID:21752341

  10. 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.

  11. 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

  12. Toll-Like Receptor 4 Expression in Human Breast Implant Capsules: Localization and Correlation with Estrogen Receptors.

    PubMed

    Segreto, Francesco; Carotti, Simone; Tosi, Daniele; Pendolino, Alfonso Luca; Marangi, Giovanni Francesco; Morini, Sergio; Persichetti, Paolo

    2016-03-01

    Capsular contracture is the most common complication following breast augmentation and reconstruction. Myofibroblasts, which are specialized fibroblasts with contractile activity, are involved in its pathogenesis. Toll-like receptor 4 stimulation in fibroblasts induces transcription of genes involved in extracellular matrix remodeling and tissue repair; furthermore, it enhances sensitivity to transforming growth factor-β1 and promotes transition to myofibroblasts. 17β-Estradiol, by binding to its main receptors, α and/or β, increases the expression of toll-like receptor 4 and the production of proinflammatory mediators by macrophages; moreover, it promotes extracellular matrix production and myofibroblasts contraction and differentiation. The aim of the study was to investigate the expression of toll-like receptor 4 in breast implant capsules and its relationship with estrogen receptors. The study enrolled 30 women who underwent expander removal following breast reconstruction. Specimens were stained with hematoxylin and eosin, Masson trichrome, immunohistochemistry, and immunofluorescence for toll-like receptor 4, α-smooth muscle actin (a marker of myofibroblasts), estrogen receptor-α, and estrogen receptor-β. Toll-like receptor 4 was expressed by fibroblasts and myofibroblasts of capsular tissue. Its expression positively correlated with estrogen receptorexpression (p = 0.012). A positive correlation was found between estrogen receptor-β and α-smooth muscle actin expression (p = 0.037). This study demonstrates the expression of toll-like receptor 4 in myofibroblasts of capsular tissue and its correlation with estrogen receptor-β positivity. Activation of toll-like receptor 4 and estrogen receptor-β, and their interplay, may be involved in myofibroblast differentiation and in the profibrotic pathogenic process underlying capsular contracture.

  13. Therapeutic Opportunities for Caffeine and A2A Receptor Antagonists in Retinal Diseases.

    PubMed

    Boia, Raquel; Ambrósio, António Francisco; Santiago, Ana Raquel

    2016-01-01

    Caffeine, the major component of coffee, is the most consumed psychostimulant in the world. Caffeine is an adenosine analog and acts as a nonselective adenosine receptor antagonist. The majority of the effects of caffeine are mainly mediated by the blockade of adenosine receptors, and the proved neuroprotective effects of caffeine in brain disorders have been mimicked by the blockade of adenosine A2A receptor (A2AR). A growing body of evidence demonstrates that microglia-mediated neuroinflammation plays a key role in the pathophysiology of brain and retinal diseases. Moreover, the control of microglia reactivity by blocking A2AR has been proposed to be the mechanism underlying the observed protective effects of caffeine. Hence, it is conceivable that caffeine and A2AR antagonists offer therapeutic value for the treatment of retinal diseases, mainly those involving microglia-mediated neuroinflammation. © 2016 S. Karger AG, Basel.

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

    ERIC Educational Resources Information Center

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

    2017-01-01

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

  15. 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.

  16. Adenosine A2A receptors are necessary and sufficient to trigger memory impairment in adult mice

    PubMed Central

    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

    2015-01-01

    Background and Purpose Caffeine (a non-selective adenosine receptor antagonist) prevents memory deficits in aging and Alzheimer’s disease, an effect mimicked by adenosine A2A 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. Experimental Approach We determined whether A2A receptors are necessary for the emergence of memory impairments induced by scopolamine and whether A2A 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. Key Results 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 A2A 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 A2A 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. Conclusions and Implications These results show that A2A 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. PMID:25939452

  17. Ephrin Ligands and Eph Receptors Show Regionally Restricted Expression in the Developing Palate and Tongue.

    PubMed

    Xavier, Guilherme M; Miletich, Isabelle; Cobourne, Martyn T

    2016-01-01

    The Eph family receptor-interacting (ephrin) ligands and erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest known family of receptor tyrosine kinases. Ephrin ligands and their receptors form an important cell communication system with widespread roles in normal physiology and disease pathogenesis. In order to investigate potential roles of the ephrin-Eph system during palatogenesis and tongue development, we have characterized the cellular mRNA expression of family members EphrinA1-A3, EphA1-A8, and EphrinB2, EphB1, EphB4 during murine embryogenesis between embryonic day 13.5-16.5 using radioactive in situ hybridization. With the exception of EphA6 and ephrinA3, all genes were regionally expressed during the process of palatogenesis, with restricted and often overlapping domains. Transcripts were identified in the palate epithelium, localized at the tip of the palatal shelves, in the mesenchyme and also confined to the medial epithelium seam. Numerous Eph transcripts were also identified during tongue development. In particular, EphA1 and EphA2 demonstrated a highly restricted and specific expression in the tongue epithelium at all stages examined, whereas EphA3 was strongly expressed in the lateral tongue mesenchyme. These results suggest regulatory roles for ephrin-EphA signaling in development of the murine palate and tongue.

  18. Ephrin Ligands and Eph Receptors Show Regionally Restricted Expression in the Developing Palate and Tongue

    PubMed Central

    Xavier, Guilherme M.; Miletich, Isabelle; Cobourne, Martyn T.

    2016-01-01

    The Eph family receptor-interacting (ephrin) ligands and erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest known family of receptor tyrosine kinases. Ephrin ligands and their receptors form an important cell communication system with widespread roles in normal physiology and disease pathogenesis. In order to investigate potential roles of the ephrin-Eph system during palatogenesis and tongue development, we have characterized the cellular mRNA expression of family members EphrinA1-A3, EphA1–A8, and EphrinB2, EphB1, EphB4 during murine embryogenesis between embryonic day 13.5–16.5 using radioactive in situ hybridization. With the exception of EphA6 and ephrinA3, all genes were regionally expressed during the process of palatogenesis, with restricted and often overlapping domains. Transcripts were identified in the palate epithelium, localized at the tip of the palatal shelves, in the mesenchyme and also confined to the medial epithelium seam. Numerous Eph transcripts were also identified during tongue development. In particular, EphA1 and EphA2 demonstrated a highly restricted and specific expression in the tongue epithelium at all stages examined, whereas EphA3 was strongly expressed in the lateral tongue mesenchyme. These results suggest regulatory roles for ephrin-EphA signaling in development of the murine palate and tongue. PMID:26941654

  19. Differential estrogen receptor expression in autoimmune myasthenia gravis.

    PubMed

    Nancy, Patrice; Berrih-Aknin, Sonia

    2005-05-01

    Myasthenia gravis (MG) is an autoimmune disease associated with thymic hyperplasia and is much more prevalent in women than men. In this study we investigated potential changes in estrogen receptor (ER) expression in thymic hyperplasia. We first quantified by real-time PCR the relative expression of ER alpha and ER beta in normal thymus and found that the ER beta to ER alpha ratio was inverted in thymocytes (8.6 +/- 1.2), compared with thymic epithelial cells (0.18 +/- 0.05). The ER transcript number gradually decreased in thymic epithelial cells during culture, indicating that the thymic environment influences ER expression. CD4+ helper T cells expressed higher level of ERs, compared with CD8+ cells, as assessed by flow cytometry in thymocytes and peripheral blood mononuclear cells. In MG patients, we found an increased expression of ER alpha on thymocytes and both ERs on T cells from peripheral blood mononuclear cells, indicating that the signals provided by thymic and peripheral microenvironments are distinct. Finally, activation of normal thymocytes by proinflammatory cytokines induced increased expression of ERs especially in the CD4+ subset, suggesting that an excess of proinflammatory cytokines could explain the increase of ERs expression on MG lymphocytes. The dysregulation of ER expression in MG lymphocytes could affect the maintenance of the homeostatic conditions and might influence the progression of the autoimmune response.

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

    PubMed Central

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

    2013-01-01

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

  1. Bile acid receptor agonist GW4064 regulates PPARγ coactivator-1α expression through estrogen receptor-related receptor α.

    PubMed

    Dwivedi, Shailendra Kumar Dhar; Singh, Nidhi; Kumari, Rashmi; Mishra, Jay Sharan; Tripathi, Sarita; Banerjee, Priyam; Shah, Priyanka; Kukshal, Vandana; Tyagi, Abdul Malik; Gaikwad, Anil Nilkanth; Chaturvedi, Rajnish Kumar; Mishra, Durga Prasad; Trivedi, Arun Kumar; Sanyal, Somali; Chattopadhyay, Naibedya; Ramachandran, Ravishankar; Siddiqi, Mohammad Imran; Bandyopadhyay, Arun; Arora, Ashish; Lundåsen, Thomas; Anakk, Sayee Priyadarshini; Moore, David D; Sanyal, Sabyasachi

    2011-06-01

    Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is induced in energy-starved conditions and is a key regulator of energy homeostasis. This makes PGC-1α an attractive therapeutic target for metabolic syndrome and diabetes. In our effort to identify new regulators of PGC-1α expression, we found that GW4064, a widely used synthetic agonist for the nuclear bile acid receptor [farnesoid X receptor (FXR)] strongly enhances PGC-1α promoter reporter activity, mRNA, and protein expression. This induction in PGC-1α concomitantly enhances mitochondrial mass and expression of several PGC-1α target genes involved in mitochondrial function. Using FXR-rich or FXR-nonexpressing cell lines and tissues, we found that this effect of GW4064 is not mediated directly by FXR but occurs via activation of estrogen receptor-related receptor α (ERRα). Cell-based, biochemical and biophysical assays indicate GW4064 as an agonist of ERR proteins. Interestingly, FXR disruption alters GW4064 induction of PGC-1α mRNA in a tissue-dependent manner. Using FXR-null [FXR knockout (FXRKO)] mice, we determined that GW4064 induction of PGC-1α expression is not affected in oxidative soleus muscles of FXRKO mice but is compromised in the FXRKO liver. Mechanistic studies to explain these differences revealed that FXR physically interacts with ERR and protects them from repression by the atypical corepressor, small heterodimer partner in liver. Together, this interplay between ERRα-FXR-PGC-1α and small heterodimer partner offers new insights into the biological functions of ERRα and FXR, thus providing a knowledge base for therapeutics in energy balance-related pathophysiology.

  2. Mycobacterium tuberculosis interferes with the response to infection by inducing the host EphA2 receptor.

    PubMed

    Khounlotham, Manirath; Subbian, Selvakumar; Smith, Roger; Cirillo, Suat L G; Cirillo, Jeffrey D

    2009-06-15

    Mycobacterium tuberculosis is an unusual pathogen, persisting for years in infected persons despite an immune response. Erythropoietin-producing hepatoma (Eph) receptors are critical for tissue organization. One hallmark of tuberculosis is the presence of granulomas consisting of organized immune cells. The importance of granuloma structure makes it likely that Eph receptors play a role in immunity to tuberculosis. We infected mice with low doses of M. tuberculosis by the aerosol method and examined the effects on ephA gene expression, pathology, composition of lymphocytes in the lungs (by flow cytometry), migration of CD4+ and CD8+ T cells, and numbers of cytokine-expressing cells. Mice infected with M. tuberculosis displayed higher expression of ephA1 and ephA2 as well as ephrinA1, which encodes the ligand for EphA1 and EphA2. Interestingly, ephA2-/- mice displayed greater pathology, greater accumulation of T cells and dendritic cells, and higher levels of proinflammatory cytokines than did normal C57BL/6 mice. Furthermore, T cells from ephA2-/- mice migrated more efficiently than did those from C57BL/6 mice. These observations suggest that ephA-related genes may provide a mechanism that M. tuberculosis uses to circumvent the host response, given that accumulation of T cells appears to be due to the inhibition of immune cell migration by EphA2. Ultimately, the absence of ephA2 results in greater clearance of M. tuberculosis during the chronic phase of infection, suggesting that induction of ephA2 is important for the survival of M. tuberculosis during latency.

  3. Endothelin-1 downregulates Mas receptor expression in human cardiomyocytes.

    PubMed

    Chen, Zhiheng; Tang, Yamei; Yang, Zuocheng; Liu, Shaojun; Liu, Yong; Li, Yan; He, Wei

    2013-09-01

    Endothelin-1 (ET-1) and the renin-angiotensin system (RAS) are involved in the pathogenesis of cardiac dysfunction. The Mas receptor is a functional binding site for angiotensin (Ang)‑(1-7), which is now considered a critical component of the RAS and exerts cardioprotective effects. To the best of our knowledge, the present study aimed to examine, for the first time, the effects of ET-1 on Mas expression in cultured human cardiomyocytes. Human cardiomyocytes were treated with ET-1 at different concentrations (1, 5, 10, 20 and 30 nM) for varied time periods (0.5, 1.5, 3, 4.5 or 6 h) with or without the transcription inhibitor actinomycin D, endothelin A (ETA) receptor blocker BQ123 and ETB receptor blocker BQ788, or different kinase inhibitors. ET-1 decreased the Mas mRNA level in a statistically significant dose- and time-dependent manner within 4.5 h, which was reflected in the dose-dependent downregulation of Mas promoter activity, Mas protein levels and Ang-(1-7) binding on the cell membrane. Actinomycin D (1 mg/ml), BQ123 (1 µM), p38 mitogen-activated protein kinase (MAPK) siRNA and inhibitor PD169316 (25 µM), completely eliminated the inhibitory effects of ET-1 on Mas expression in human cardiomyocytes. In conclusion, the present study demonstrated that ET-1 downregulates Mas expression at the transcription level in human cardiomyocytes via the ETA receptor by a p38 MAPK‑dependent mechanism. This study provides novel insights into the function of ET-1 and the Ang‑(1-7)/Mas axis in cardiac pathophysiology.

  4. Anxious behavior induces elevated hippocampal Cb2 receptor gene expression.

    PubMed

    Robertson, James M; Achua, Justin K; Smith, Justin P; Prince, Melissa A; Staton, Clarissa D; Ronan, Patrick J; Summers, Tangi R; Summers, Cliff H

    2017-06-03

    Anxiety is differentially expressed across a continuum of stressful/fearful intensity, influenced by endocannabinoid systems and receptors. The hippocampus plays important roles in the regulation of affective behavior, emotion, and anxiety, as well as memory. Location of Cb 1 /Cb 2 receptor action could be important in determining emotional valence, because while the dorsal hippocampus is involved in spatial memory and cognition, the ventral hippocampus has projections to the PFC, BNST, amygdala, and HPA axis, and is important for emotional responses to stress. During repeated social defeat in a Stress-Alternatives Model arena (SAM; an oval open field with escape portals only large enough for smaller mice), smaller C57BL6/N mice are subject to fear conditioning (tone=CS), and attacked by novel larger aggressive CD1 mice (US) over four daily (5min) trials. Each SAM trial presents an opportunity for escape or submission, with stable behavioral responses established by the second day of interaction. Additional groups had access to a running wheel. Social aggression plus fear conditioning stimulates enhanced Cb 2 receptor gene expression in the dorsal CA 1 , dorsal and ventral dentate gyrus subregions in animals displaying a submissive behavioral phenotype. Escape behavior is associated with reduced Cb 2 expression in the dorsal CA 1 region, with freezing and escape latency correlated with mRNA levels. Escaping and submitting animals with access to running wheels had increased Cb 2 mRNA in dorsal DG/CA 1 . These results suggest that the Cb 2 receptor system is rapidly induced during anxiogenic social interactions plus fear conditioning or exercise; with responses potentially adaptive for coping mechanisms. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. The regulation of oxytocin receptor gene expression during adipogenesis.

    PubMed

    Yi, K J; So, K H; Hata, Y; Suzuki, Y; Kato, D; Watanabe, K; Aso, H; Kasahara, Y; Nishimori, K; Chen, C; Katoh, K; Roh, S G

    2015-05-01

    Although it has been reported that oxytocin stimulates lipolysis in adipocytes, changes in the expression of oxytocin receptor (OTR) mRNA in adipogenesis are still unknown. The present study aimed to investigate the expression of OTR mRNA during adipocyte differentiation and fat accumulation in adipocytes. OTR mRNA was highly expressed in adipocytes prepared from mouse adipose tissues compared to stromal-vascular cells. OTR mRNA expression was increased during the adipocyte differentiation of 3T3-L1 cells. OTR expression levels were higher in subcutaneous and epididymal adipose tissues of 14-week-old male mice compared to 7-week-old male mice. Levels of OTR mRNA expression were higher in adipose tissues at four different sites of mice fed a high-fat diet than in those of mice fed a normal diet. The OTR expression level was also increased by refeeding for 4 h after fasting for 16 h. Oxytocin significantly induced lipolysis in 3T3-L1 adipocytes. In conclusion, a new regulatory mechanism is demonstrated for oxytocin to control the differentiation and fat accumulation in adipocytes via activation of OTR as a part of the hypothalamic-pituitary-adipose axis. © 2015 British Society for Neuroendocrinology.

  6. Differential expression of ryanodine receptor isoforms after spinal cord injury.

    PubMed

    Pelisch, Nicolas; Gomes, Cynthia; Nally, Jacqueline M; Petruska, Jeffrey C; Stirling, David P

    2017-11-01

    Ryanodine receptors (RyRs) are highly conductive intracellular Ca 2+ release channels and are widely expressed in many tissues, including the central nervous system. RyRs have been implicated in intracellular Ca 2+ overload which can drive secondary damage following traumatic injury to the spinal cord (SCI), but the spatiotemporal expression of the three isoforms of RyRs (RyR1-3) after SCI remains unknown. Here, we analyzed the gene and protein expression of RyR isoforms in the murine lumbar dorsal root ganglion (DRG) and the spinal cord lesion site at 1, 2 and 7 d after a mild contusion SCI. Quantitative RT PCR analysis revealed that RyR3 was significantly increased in lumbar DRGs and at the lesion site at 1 and 2 d post contusion compared to sham (laminectomy only) controls. Additionally, RyR2 expression was increased at 1 d post injury within the lesion site. RyR2 and -3 protein expression was localized to lumbar DRG neurons and their spinal projections within the lesion site acutely after SCI. In contrast, RyR1 expression within the DRG and lesion site remained unaltered following trauma. Our study shows that SCI initiates acute differential expression of RyR isoforms in DRG and spinal cord. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Interleukin 2 receptor expression by macrophages in inflammatory bowel disease.

    PubMed Central

    Mahida, Y R; Patel, S; Wu, K; Jewell, D P

    1988-01-01

    The expression of interleukin 2 receptor by macrophages from normal and inflamed terminal ileum and colon has been studied by using two monoclonal antibodies. In tissue sections from normal ileum and colon, scattered positive lymphocytes and only occasional weakly positive macrophages were seen. In ileal and colonic Crohn's disease or ulcerative colitis many positive macrophages and lymphocytes were seen in the lamina propria. These findings were confirmed by staining cytospin preparations of isolated intestinal mononuclear cells. The isolated macrophages were able to phagocytose opsonized zymosan and the majority were able to undergo a respiratory burst when triggered with opsonized zymosan or phorbol myristate acetate (PMA), suggesting that they were activated. Stimulation with interferon-gamma or lipopolysaccharide did not increase the number of macrophages staining with the antibodies to the interleukin 2 receptor. Therefore we postulate that a large majority of the macrophages expressing interleukin 2 receptor in inflammatory bowel disease are a recently recruited population of cells. Images Fig. 1 Fig. 2 Figure 3 PMID:3266118

  8. Somatostatin receptor subtype expression in human thyroid tumours.

    PubMed

    Klagge, A; Krause, K; Schierle, K; Steinert, F; Dralle, H; Fuhrer, D

    2010-04-01

    Somatostatin receptors (SSTR) are expressed in various endocrine tumours. The expression of SSTR at the tumour cell surface confers the possibility for diagnostic imaging and therapy of tumours using radiolabeled somatostatin analogues. The majority of currently available somatostatin analogues show a higher binding affinity for the SSTR2 subtype. To date, the precise expression pattern of the SSTR subtypes 1-5 in thyroid epithelial tumours remains to be determined. We investigated the mRNA expression of SSTR1-5 in benign and malignant epithelial thyroid tumours [20 cold thyroid nodules (CTNs), 20 toxic thyroid nodules (TTNs), 20 papillary, 20 follicular, and 5 anaplastic carcinomas (PTCs, FTCs, ATCs, respectively)] and compared them to normal surrounding thyroid tissues. Four out of five SSTR subtypes were detected in malignant thyroid tumours, benign neoplasia, and normal surrounding tissue with a predominant expression of SSTR2 and SSTR5, and a weak expression of SSTR1 and SSTR3. Weak SSTR4 mRNA expression was detected in some PTCs. Compared to normal thyroid tissue, SSTR2 was significantly upregulated in PTC and ATC. In addition significant upregulation of SSTR3 was found in PTC. SSTR5 mRNA expression was increased in PTC and FTC and significantly decreased in CTN and TTN compared to normal thyroid tissue. SSTR2 is the predominant subtype in thyroid epithelial tumours with a high expression pattern, in particular, in PTC . Perspectively, the expression of distinct SSTR in thyroid epithelial tumours might represent a promising avenue for diagnostics and therapy of advanced thyroid cancer with somatostatin analogues. Georg Thieme Verlag KG Stuttgart New York.

  9. Design and Synthesis of Potent Bivalent Peptide Agonists Targeting the EphA2 Receptor

    PubMed Central

    2013-01-01

    Designing potent and selective peptides and small molecules that target Eph receptor tyrosine kinases remains a challenge, and new strategies are needed for developing novel and potent ligands for these receptors. In this study, we performed a structure–activity relationship study of a previously identified 12 amino acid-long peptide, SWL, by alanine scanning to identify residues important for receptor binding. To further enhance and optimize the receptor binding affinity of the SWL peptide, we applied the concept of bivalent ligand design to synthesize several SWL-derived dimeric peptides as novel ligands capable of binding simultaneously to two EphA2 receptor molecules. The dimeric peptides possess higher receptor binding affinity than the original monomeric SWL peptide, consistent with bivalent binding. The most potent dimeric peptide, a SWL dimer with a six-carbon linker, has about 13-fold increased potency as compared to SWL. Furthermore, similar to SWL, the dimeric peptide is an agonist and can promote EphA2 tyrosine phosphorylation (activation) in cultured cells. PMID:24167659

  10. Expression pattern of asialoglycoprotein receptor in human testis.

    PubMed

    Sun, Pingnan; Zheng, Junhong; She, Guizhou; Wei, Xiujing; Zhang, Xiaoyu; Shi, Haijun; Zhou, Xiaoling

    2013-06-01

    During acute or chronic hepatitis B virus (HBV) infection, the virus can invade the male reproductive system, pass through the blood-testis barrier and integrate into the germ line, resulting in abnormal spermatozoa. However, the pathway remains unclear. The asialoglycoprotein receptor (ASGR), a potential receptor for HBV, is mainly distributed in hepatocytes. We have examined the distribution of ASGR in human testis and found it in the seminiferous tubules and interstitial region but its enrichment in human testis is much lower than that in liver. By multiple immunoenzyme histochemistry staining, ASGR was precisely co-localized with vimentin (Sertoli cell marker) but not proliferating cell nuclear antigen (spermatogonial cell marker) in testis tissue. ASGR was expressed in human Leydig cells, stromal cells in the seminiferous tubules and Sertoli cells but seldom in spermatogonial cells. Therefore, ASGR could provide HBV with access to the luminal compartment of human testis. The mechanism by which HBV invades germ cells remains unknown.

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

    PubMed

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

    2016-12-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Eph-A2 and Eph-A4 expression in human benign and malignant thyroid lesions: An immunohistochemical study

    PubMed Central

    Karidis, Nikolaos P.; Giaginis, Constantinos; Tsourouflis, Gerasimos; Alexandrou, Paraskevi; Delladetsima, Ioanna; Theocharis, Stamatios

    2011-01-01

    Summary Background Ephrin receptors (Ephs) are frequently overexpressed in a wide variety of human malignant tumors, being associated with tumor growth, invasion, metastasis and angiogenesis. The aim of the present study was to evaluate the clinical significance of Eph-A2 and Eph-A4 expression in human benign and malignant thyroid lesions. Material/Methods Eph-A2 and Eph-A4 protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 131 patients with benign and malignant lesions. Results Eph-A2 was significantly overexpressed in malignant compared to benign thyroid lesions (p<0.001). Papillary carcinoma cases presented significantly increased Eph-A2 expression compared to those with hyperplasia nodules (p<0.001). Eph-A4 expression was not differentiated between cases with malignant or benign thyroid lesions. Papillary carcinoma cases presented significantly increased Eph-A4 expression compared to those with hyperplasia nodules (p=0.006). In the subgroup of malignant thyroid lesions, Eph-A2 and Eph-A4 expression was not associated with TNM stage, capsular, lymphatic or vascular invasion. Conclusions The present data suggest that Eph-A2, but not Eph-A4, overexpression may be associated with the malignant transformation of thyroid neoplasia. Further studies conducted on cohorts including a higher proportion of patients with advanced nodal and metastatic disease are recommended to draw definite conclusions on the clinical significance of Eph proteins in thyroid neoplasia. PMID:21873938

  14. Eph-A2 and Eph-A4 expression in human benign and malignant thyroid lesions: an immunohistochemical study.

    PubMed

    Karidis, Nikolaos P; Giaginis, Constantinos; Tsourouflis, Gerasimos; Alexandrou, Paraskevi; Delladetsima, Ioanna; Theocharis, Stamatios

    2011-09-01

    Ephrin receptors (Ephs) are frequently overexpressed in a wide variety of human malignant tumors, being associated with tumor growth, invasion, metastasis and angiogenesis. The aim of the present study was to evaluate the clinical significance of Eph-A2 and Eph-A4 expression in human benign and malignant thyroid lesions. Eph-A2 and Eph-A4 protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 131 patients with benign and malignant lesions. Eph-A2 was significantly overexpressed in malignant compared to benign thyroid lesions (p<0.001). Papillary carcinoma cases presented significantly increased Eph-A2 expression compared to those with hyperplasia nodules (p<0.001). Eph-A4 expression was not differentiated between cases with malignant or benign thyroid lesions. Papillary carcinoma cases presented significantly increased Eph-A4 expression compared to those with hyperplasia nodules (p=0.006). In the subgroup of malignant thyroid lesions, Eph-A2 and Eph-A4 expression was not associated with TNM stage, capsular, lymphatic or vascular invasion. The present data suggest that Eph-A2, but not Eph-A4, overexpression may be associated with the malignant transformation of thyroid neoplasia. Further studies conducted on cohorts including a higher proportion of patients with advanced nodal and metastatic disease are recommended to draw definite conclusions on the clinical significance of Eph proteins in thyroid neoplasia.

  15. Molecular Cooperativity Governs Diverse and Monoallelic Olfactory Receptor Expression

    NASA Astrophysics Data System (ADS)

    Xing, Jianhua; Tian, Xiaojun; Zhang, Hang; Sannerud, Jens

    Multiple-objective optimization is common in biological systems. In the mammalian olfactory system, each sensory neuron stochastically expresses only one out of up to thousands of olfactory receptor (OR) gene alleles; at organism level the types of expressed ORs need to be maximized. The molecular mechanism of this Nobel-Prize winning puzzle remains unresolved after decades of extensive studies. Existing models focus only on monoallele activation, and cannot explain recent observations in mutants, especially the reduced global diversity of expressed ORs in G9a/GLP knockouts. In this work we integrated existing information on OR expression, and proposed an evolutionarily optimized three-layer regulation mechanism, which includes zonal segregation, epigenetic and enhancer competition coupled to a negative feedback loop. This model not only recapitulates monoallelic OR expression, but also elucidates how the olfactory system maximizes and maintains the diversity of OR expression. The model is validated by several experimental results, and particularly underscores cooperativity and synergy as a general design principle of multi-objective optimization in biology. The work is supported by the NIGMS/DMS Mathematical Biology program.

  16. Modulation of Progesterone Receptor Isoform Expression in Pregnant Human Myometrium

    PubMed Central

    2017-01-01

    Background. Regulation of myometrial progesterone receptor (PR) expression is an unresolved issue central to understanding the mechanism of functional progesterone withdrawal and initiation of labor in women. Objectives. To determine whether pregnant human myometrium undergoes culture-induced changes in PR isoform expression ex situ and, further, to determine if conditions approaching the in vivo environment stabilise PR isoform expression in culture. Methods. Term nonlaboring human myometrial tissues were cultured under specific conditions: serum supplementation, steroids, stretch, cAMP, PMA, PGF2α, NF-κB inhibitors, or TSA. Following 48 h culture, PR-T, PR-A, and PR-B mRNA levels were determined using qRT-PCR. PR-A/PR-B ratios were calculated. Results. PR-T and PR-A expression and the PR-A/PR-B ratio significantly increased in culture. Steroids prevented the culture-induced increase in PR-T and PR-A expression. Stretch blocked the effects of steroids on PR-T and PR-A expression. PMA further increased the PR-A/PR-B ratio, while TSA blocked culture-induced increases of PR-A expression and the PR-A/PR-B ratio. Conclusion. Human myometrial tissue in culture undergoes changes in PR gene expression consistent with transition toward a laboring phenotype. TSA maintained the nonlaboring PR isoform expression pattern. This suggests that preserving histone and/or nonhistone protein acetylation is critical for maintaining the progesterone dependent quiescent phenotype of human myometrium in culture. PMID:28540297

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

    PubMed

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

    2012-01-04

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

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

    PubMed Central

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

    2012-01-01

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

  19. Activation of the adenosine A2A receptor attenuates experimental autoimmune encephalomyelitis and is associated with increased intracellular calcium levels.

    PubMed

    Liu, Yumei; Zou, Haifeng; Zhao, Ping; Sun, Bo; Wang, Jinghua; Kong, Qingfei; Mu, Lili; Zhao, Sihan; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Zhao, Jiaying; Yin, Pengqi; Liu, Lei; Zhao, Xiuli; Li, Hulun

    2016-08-25

    Multiple sclerosis (MS) is a common autoimmune disease that inevitably causes inflammatory nerve demyelination. However, an effective approach to prevent its course is still lacking and urgently needed. Recently, the adenosine A2A receptor (A2AR) has emerged as a novel inflammation regulator. Manipulation of A2AR activity may suppress the MS process and protect against nerve damage. To test this hypothesis, we treated murine experimental autoimmune encephalomyelitis (EAE), a model for MS, with the selective A2AR agonist, CGS21680 (CGS). We evaluated the effects of CGS on the pathological features of EAE progression, including CNS cellular infiltration, inflammatory cytokine expression, lymphocyte proliferation, and cell surface markers. Treatment with CGS significantly suppressed specific lymphocyte proliferation, reduced infiltration of CD4(+) T lymphocytes, and attenuated the expression of inflammatory cytokines, which in turn inhibited the EAE progression. For the first time, we demonstrate that CGS can increase the intracellular calcium concentration ([Ca(2+)]i) in murine lymphocytes, which may be the mechanism underlying the suppressive effects of CGS-induced A2AR activation on EAE progression. Our findings strongly suggest that A2AR is a potential therapeutic target for MS and provide insight into the mechanism of action of A2AR agonists, which may offer a therapeutic option for this disease. Copyright © 2016. Published by Elsevier Ltd.

  20. Amylase expression in taste receptor cells of rat circumvallate papillae.

    PubMed

    Merigo, Flavia; Benati, Donatella; Cecchini, Maria Paola; Cristofoletti, Mirko; Osculati, Francesco; Sbarbati, Andrea

    2009-06-01

    The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.

  1. Differential expression of mesotocin receptors in the uterus and ovary of the pregnant tammar wallaby.

    PubMed

    Siebel, Andrew L; Bathgate, Ross A D; Parry, Laura J

    2005-05-01

    Mesotocin, an oxytocin-like peptide, is released in highest concentrations during parturition in macropodid marsupials. In late pregnant wallabies, uterine sensitivity to mesotocin increases markedly in the myometrium of the gravid uterus. This coincides with a significant increase in myometrial mesotocin receptor concentrations 3-4 days before term. To date, there is no information on mesotocin receptor gene expression in female wallaby reproductive tissues. This study aimed to examine mesotocin receptor gene expression in the uterus and ovaries of pregnant tammar wallabies, and to localise mesotocin receptors within the uterus. An RT-PCR strategy produced a consensus nucleotide sequence of 834 bp, which encoded 278 amino acids of transmembrane domains I to VI. This protein sequence has approximately 80% homology with the bovine and rat oxytocin receptor exon 2 region. Only one mesotocin receptor was detected in the tammar genome. The myometrium and mammary gland both expressed a 4.1 kb mesotocin receptor gene transcript. Myometrial mesotocin receptor gene expression increased on day 22 of the 26-day gestation and was significantly higher in the gravid than the non-gravid uterus in late pregnancy. This pattern of mesotocin receptor gene expression paralleled mesotocin receptor concentrations. Mesotocin binding sites were localised only to the myometrium, the highest densities being observed in the gravid uterus. Finally, this study showed high expression of mesotocin receptors in the corpus luteum. The pattern of luteal mesotocin receptor expression differed from the myometrium, with a decrease in mesotocin receptors occurring on the day of expected births.

  2. NH125 reduces the level of CPEB3, an RNA binding protein, to promote synaptic GluA2 expression.

    PubMed

    Bender, Crhistian L; Yang, Qian; Sun, Lu; Liu, Siqiong June

    2016-02-01

    Neuronal activity can alter the phosphorylation state of eukaryotic elongation factor 2 (eEF2) and thereby regulates protein synthesis. This is thought to be the underlying mechanism for a form of synaptic plasticity that involves changes in the expression of synaptic AMPA type glutamate receptors. Phosphorylation of eEF2 by Ca/calmodulin-dependent eEF2 kinase reduces the activity of eEF2, and this is prevented by a commonly used eEF2 kinase inhibitor, NH125. Here we show that 10 μM NH125 increased the expression of synaptic GluA2-containing receptors in mouse cerebellar stellate cells and this was prevented by a protein synthesis inhibitor. However NH125 at 10 μM also reduced the level of CPEB3, a protein that is known to bind to GluA2 mRNA and suppress GluA2 (also known as GluR2) synthesis. In contrast, a low concentration of NH125 lowered the peEF2 level, but did not alter CPEB3 expression and also failed to increase synaptic GluA2 receptors. A selective eEF2 kinase inhibitor, A-484954, decreased the level of peEF2, without changing the expression of CPEB3. This suggests that reducing peEF2 does not lead to a decrease in CPEB3 levels and is not sufficient to increase GluA2 synthesis. Thus NH125 at 10 μM reduced the level of CPEB3, and promoted GluA2 translation via a mechanism independent of inhibition of eEF2 kinase. Therefore NH125 does not always alter protein synthesis via selective inhibition of eEF2 kinase and the effects of NH125 on translation of mRNAs should be interpreted with caution. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. GLP-1 Receptor Expression Within the Human Heart.

    PubMed

    Baggio, Laurie L; Yusta, Bernardo; Mulvihill, Erin E; Cao, Xiemin; Streutker, Catherine J; Butany, Jagdish; Cappola, Thomas P; Margulies, Kenneth B; Drucker, Daniel J

    2018-04-01

    Glucagonlike peptide-1 receptor (GLP-1R) agonists, which are used to treat type 2 diabetes and obesity, reduce the rates of myocardial infarction and cardiovascular death. GLP-1R has been localized to the human sinoatrial node; however, its expression in ventricular tissue remains uncertain. Here we studied GLP-1R expression in the human heart using GLP-1R-directed antisera, quantitative polymerase chain reaction (PCR), reverse transcription PCR to detect full-length messenger RNA (mRNA) transcripts, and in situ hybridization (ISH). GLP1R mRNA transcripts, encompassing the entire open reading frame, were detected in all four cardiac chambers from 15 hearts at levels approximating those detected in human pancreas. In contrast, cardiac GLP2R expression was relatively lower, and cardiac GCGR expression was sporadic and not detected in the left ventricle. GLP1R mRNA transcripts were not detected in RNA from human cardiac fibroblasts, coronary artery endothelial, or vascular smooth muscle cells. Human Brunner glands and pancreatic islets exhibited GLP-1R immunopositivity and abundant expression of GLP1R mRNA transcripts by ISH. GLP1R transcripts were also detected by ISH in human cardiac sinoatrial node tissue. However, definitive cellular localization of GLP1R mRNA transcripts or immunoreactive GLP-1R protein within human cardiomyocytes or cardiac blood vessels remained elusive. Moreover, validated GLP-1R antisera lacked sufficient sensitivity to detect expression of the endogenous islet or cardiac GLP-1R by Western blotting. Hence, although human cardiac ventricles express the GLP1R, the identity of one or more ventricular cell type(s) that express a translated GLP1R protein requires further clarification with highly sensitive methods of detection.

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

    PubMed

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

    2016-08-08

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

  5. The collagen receptor DDR2 is expressed during early cardiac development.

    PubMed

    Goldsmith, Edie C; Zhang, Xiadong; Watson, James; Hastings, Josh; Potts, Jay D

    2010-05-01

    Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase which has been shown to regulate cell migration upon binding its ligand, collagen. Expression studies determined that DDR2 mRNA and protein are present in the atrioventricular canal during epithelial-mesenchymal transformation (EMT) and the receptor is expressed in both activated endothelial and migrating mesenchymal cells in vivo.

  6. NTS adenosine A2a receptors inhibit the cardiopulmonary chemoreflex control of regional sympathetic outputs via a GABAergic mechanism

    PubMed Central

    Minic, Zeljka; O'Leary, Donal S.

    2015-01-01

    Adenosine is a powerful central neuromodulator acting via opposing A1 (inhibitor) and A2a (activator) receptors. However, in the nucleus of the solitary tract (NTS), both adenosine receptor subtypes attenuate cardiopulmonary chemoreflex (CCR) sympathoinhibition of renal, adrenal, and lumbar sympathetic nerve activity and attenuate reflex decreases in arterial pressure and heart rate. Adenosine A1 receptors inhibit glutamatergic transmission in the CCR pathway, whereas adenosine A2a receptors most likely facilitate release of an unknown inhibitory neurotransmitter, which, in turn, inhibits the CCR. We hypothesized that adenosine A2a receptors inhibit the CCR via facilitation of GABA release in the NTS. In urethane-chloralose-anesthetized rats (n = 51), we compared regional sympathetic responses evoked by stimulation of the CCR with right atrial injections of the 5-HT3 receptor agonist phenylbiguanide (1–8 μg/kg) before and after selective stimulation of NTS adenosine A2a receptors [microinjections into the NTS of CGS-21680 (20 pmol/50 nl)] preceded by blockade of GABAA or GABAB receptors in the NTS [bicuculline (10 pmol/100 nl) or SCH-50911 (1 nmol/100 nl)]. Blockade of GABAA receptors virtually abolished adenosine A2a receptor-mediated inhibition of the CCR. GABAB receptors had much weaker but significant effects. These effects were similar for the different sympathetic outputs. We conclude that stimulation of NTS adenosine A2a receptors inhibits CCR-evoked hemodynamic and regional sympathetic reflex responses via a GABA-ergic mechanism. PMID:25910812

  7. Clinical significance of farnesoid X receptor expression in thyroid neoplasia.

    PubMed

    Giaginis, Constantinos; Tsoukalas, Nikolaos; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Dana, Eugene; Delladetsima, Ioanna; Patsouris, Efstratios; Theocharis, Stamatios

    2017-08-01

    To evaluate the clinical significance of farnesoid X receptor (FXR) in thyroid neoplasia. FXR expression was assessed immunohistochemically on 88 thyroid neoplastic tissues (benign = 44, malignant = 44). Enhanced FXR was more frequently observed in papillary carcinomas compared with hyperplastic nodules (p = 0.0489). In malignant lesions, elevated FXR was associated with capsular (p = 0.0004) and vascular invasion (p = 0.0056) and increased follicular cells' proliferative rate (p < 0.0001). Elevated FXR expression was also associated with larger tumor size (p = 0.0086), presence of lymph node metastases (p = 0.0239) and lymphatic invasion (p = 0.0086) and increased recurrence rate risk (p = 0.0239). FXR may be associated with tumor aggressiveness that affects patients' survival in thyroid neoplasia.

  8. Allosteric mechanisms within the adenosine A2A-dopamine D2 receptor heterotetramer

    PubMed Central

    Ferré, Sergi; Bonaventura, Jordi; Tomasi, Dardo; Navarro, Gemma; Moreno, Estefanía; Cortés, Antonio; Lluís, Carme; Casadó, Vicent; Volkow, Nora D.

    2017-01-01

    The structure constituted by a G protein coupled receptor (GPCR) homodimer and a G protein provides a main functional unit and oligomeric entities can be viewed as multiples of dimers. For GPCR heteromers, experimental evidence supports a tetrameric structure, comprised of two different homodimers, each able to signal with its preferred G protein. GPCR homomers and heteromers can act as the conduit of allosteric interactions between orthosteric ligands. The well-known agonist/agonist allosteric interaction in the adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromer, by which A2AR agonists decrease the affinity of D2R agonists, gave the first rationale for the use of A2AR antagonists in Parkinson’s disease. We review new pharmacological findings that can be explained in the frame of a tetrameric structure of the A2AR-D2R heteromer: first, ligand-independent allosteric modulations by the D2R that result in changes of the binding properties of A2AR ligands; second, differential modulation of the intrinsic efficacy of D2R ligands for G protein-dependent and independent signaling; third, the canonical antagonistic Gs-Gi interaction within the frame of the heteromer; and fourth, the ability of A2AR antagonists, including caffeine, to also exert the same allosteric modulations of D2R ligands than A2AR agonists, while A2AR agonists and antagonists counteract each other’s effects. These findings can have important clinical implications when evaluating the use of A2AR antagonists. They also call for the need of monitoring caffeine intake when evaluating the effect of D2R ligands, when used as therapeutic agents in neuropsychiatric disorders or as probes in imaging studies. PMID:26051403

  9. Reduced adenosine A2a receptor-mediated efferent arteriolar vasodilation contributes to diabetes-induced glomerular hyperfiltration.

    PubMed

    Persson, Patrik; Hansell, Peter; Palm, Fredrik

    2015-01-01

    Diabetes is associated with increased risk for development of kidney disease, and an increased glomerular filtration rate is an early indication of altered kidney function. Here we determine whether reduced adenosine A2a receptor-mediated vasodilation of the efferent arteriole contributes to the increased glomerular filtration rate in diabetes. The glomerular filtration rate, renal blood flow, and proximal tubular stop flow pressure were investigated in control and streptozotocin-diabetic rats during baseline and after administration of the adenosine A2a receptor antagonist ZM241385 or the adenosine A2a receptor agonist CGS21680. The diabetes-induced glomerular hyperfiltration was reduced by 24% following A2a receptor stimulation but was unaffected by A2a receptor inhibition. Contrarily, glomerular filtration rate in controls increased by 22% after A2a receptor inhibition and was unaffected by A2a stimulation. The increased glomerular filtration rate after A2a receptor inhibition in controls and decreased glomerular filtration rate after A2a receptor activation in diabetics were caused by increased and decreased stop flow pressure, respectively. None of the interventions affected renal blood flow. Thus, the normal adenosine A2a receptor-mediated tonic vasodilation of efferent arterioles is abolished in the diabetic kidney. This causes increased efferent arteriolar resistance resulting in increased filtration fraction and hyperfiltration.

  10. Adenosine receptor A2b on hematopoietic cells mediates LPS-induced migration of PMNs into the lung interstitium.

    PubMed

    Konrad, Franziska M; Witte, Esther; Vollmer, Irene; Stark, Stefanie; Reutershan, Jörg

    2012-09-01

    Uncontrolled transmigration of polymorphonuclear leukocytes (PMNs) into the different compartments of the lungs (intravascular, interstitial, alveolar) is a critical event in the early stage of acute lung injury and acute respiratory distress syndrome. Adenosine receptor A(2b) is highly expressed in the inflamed lungs and has been suggested to mediate cell trafficking. In a murine model of LPS-induced lung inflammation, we investigated the role of A(2b) on migration of PMNs into the different compartments of the lung. In A(2b)(-/-) mice, LPS-induced accumulation of PMNs was significantly higher in the interstitium, but not in the alveolar space. In addition, pulmonary clearance of PMNs was delayed in A(2b)(-/-) mice. Using chimeric mice, we identified A(2b) on hematopoietic cells as crucial for PMN migration. A(2b) did not affect the release of relevant chemokines into the alveolar space. LPS-induced microvascular permeability was under the control of A(2b) on both hematopoietic and nonhematopoietic cells. Activation of A(2b) on endothelial cells also reduced formation of LPS-induced stress fibers, highlighting its role for endothelial integrity. A specific A(2b) agonist (BAY 60-6583) was effective in decreasing PMN migration into the lung interstitium and microvascular permeability. In addition, in vitro transmigration of human PMNs through a layer of human endothelial or epithelial cells was A(2b) dependent. Activation of A(2b) on human PMNs reduced oxidative burst activity. Together, our results demonstrate anti-inflammatory effects of A(2b) on two major characteristics of acute lung injury, with a distinct role of hematopoietic A(2b) for cell trafficking and endothelial A(2b) for microvascular permeability.

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

    PubMed Central

    Wykosky, Jill; Debinski, Waldemar

    2013-01-01

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

  12. The EphA2 receptor and ephrinA1 ligand in solid tumors: function and therapeutic targeting.

    PubMed

    Wykosky, Jill; Debinski, Waldemar

    2008-12-01

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

  13. Structure-Activity Relationships of the Sustained Effects of Adenosine A2A Receptor Agonists Driven by Slow Dissociation Kinetics

    PubMed Central

    Hothersall, J. Daniel; Guo, Dong; Sarda, Sunil; Sheppard, Robert J.; Chen, Hongming; Keur, Wesley; Waring, Michael J.; IJzerman, Adriaan P.; Hill, Stephen J.; Dale, Ian L.

    2017-01-01

    The duration of action of adenosine A2A receptor (A2A) agonists is critical for their clinical efficacy, and we sought to better understand how this can be optimized. The in vitro temporal response profiles of a panel of A2A agonists were studied using cAMP assays in recombinantly (CHO) and endogenously (SH-SY5Y) expressing cells. Some agonists (e.g., 3cd; UK-432,097) but not others (e.g., 3ac; CGS-21680) demonstrated sustained wash-resistant agonism, where residual receptor activation continued after washout. The ability of an antagonist to reverse pre-established agonist responses was used as a surrogate read-out for agonist dissociation kinetics, and together with radioligand binding studies suggested a role for slow off-rate in driving sustained effects. One compound, 3ch, showed particularly marked sustained effects, with a reversal t1/2 > 6 hours and close to maximal effects that remained for at least 5 hours after washing. Based on the structure-activity relationship of these compounds, we suggest that lipophilic N6 and bulky C2 substituents can promote stable and long-lived binding events leading to sustained agonist responses, although a high compound logD is not necessary. This provides new insight into the binding interactions of these ligands and we anticipate that this information could facilitate the rational design of novel long-acting A2A agonists with improved clinical efficacy. PMID:27803241

  14. Overexpression of Ephrin A2 receptors in cancer stromal cells is a prognostic factor for the relapse of gastric cancer.

    PubMed

    Kikuchi, Shojiro; Kaibe, Nobuaki; Morimoto, Koji; Fukui, Hirokazu; Niwa, Hirotaka; Maeyama, Yoshihiro; Takemura, Masashi; Matsumoto, Masaki; Nakamori, Shoji; Miwa, Hiroto; Hirota, Seiichi; Sasako, Mitsuru

    2015-07-01

    Microenvironments control cancer growth and progression. We explored the prognostic impact of stromal reaction and cancer stromal cells on relapse risk and survival after curative gastrectomy in gastric cancer patients. Tissue samples were obtained from 107 patients with gastric adenocarcinoma who underwent curative (R0) gastrectomy. Primary stromal cells isolated from gastric cancer tissue (GCSC) and normal gastric tissue (Gastric stromal cell: GSC) in each patient were cultured and subjected to comprehensive proteome (LC-MS/MS) and real-time RT-PCR analysis. Expression of Ephrin A2 receptors (EphA2) in cancers and GCSC was evaluated immunohistochemically. Intermingling of EphA2-positive cancer cells and GCSC (IC/A2+) and overexpression of EphA2 in cancer cells (Ca/A2+) in invasive parts of tumors were assessed, as were relationships of IC/A2+, Ca/A2+, and clinicopathological factors with relapse-free survival and overall survival. Proteome analysis showed that EphA2 expression was significantly higher in GCSC than GSC. Real-time RT-PCR analysis showed that levels of EphA1/A2/A3/A5 and EphB2/B4 were ≥2.0-fold higher in GCSC than GSC. Ca/A2 and IC/A2 were positive in 65 (60.7 %) and 26 (24.3 %) patients, respectively. Relapse was significantly more frequent in IC/A2-positive than in IC/A2-negative (HR, 2.12; 95 % CI, 1.16-5.41; p = 0.0207) patients. Among the 54 patients who received S-1 adjuvant chemotherapy, relapse-free survival (RFS) was significantly shorter in those who were IC/A2-positive than in those who were IC/A2-negative and Ca/A2-negative (HR, 2.83; 95 % CI, 1.12-12.12; p = 0.0339). Multivariable analysis indicated that pathological stage (p = 0.010) and IC/A2+ (p = 0.008) were independent risk factors for recurrence. IC/A2+ was predictive of relapse after curative (R0) gastrectomy.

  15. 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

  16. EphA2: expression in the renal medulla and regulation by hypertonicity and urea stress in vitro and in vivo.

    PubMed

    Xu, Hongshi; Tian, Wei; Lindsley, Jessie N; Oyama, Terry T; Capasso, Juan M; Rivard, Christopher J; Cohen, Herbert T; Bagnasco, Serena M; Anderson, Sharon; Cohen, David M

    2005-04-01

    EphA2, a member of the large family of Eph receptor tyrosine kinases, is highly expressed in epithelial tissue and has been implicated in cell-cell and cell-matrix interactions, as well as cell growth and survival. Expression of EphA2 mRNA and protein was markedly upregulated by both hypertonic stress and by elevated urea concentrations in cells derived from the murine inner medullary collecting duct. This upregulation likely required transactivation of the epidermal growth factor (EGF) receptor tyrosine kinase and metalloproteinase-dependent ectodomain cleavage of an EGF receptor ligand, based on pharmacological inhibitor studies. A human EphA2 promoter fragment spanning nucleotides -4030 to +21 relative to the putative EphA2 transcriptional start site was responsive to tonicity but insensitive to urea. A promoter fragment spanning -1890 to +128 recapitulated both tonicity- and urea-dependent upregulation of expression, consistent with transcriptional activation. Neither the bona fide p53 response element at approximately -1.5 kb nor a pair of putative TonE elements at approximately -3 kb conferred the tonicity responsiveness. EphA2 mRNA and protein were expressed at low levels in rat renal cortex but at high levels in the collecting ducts of the renal medulla and papilla. Water deprivation in rats increased EphA2 expression in renal papilla, whereas dietary supplementation with 20% urea increased EphA2 expression in outer medulla. These data indicate that transcription and expression of the EphA2 receptor tyrosine kinase are regulated by tonicity and urea in vitro and suggest that this phenomenon is also operative in vivo. Renal medullary EphA2 expression may represent an adaptive response to medullary hypertonicity or urea exposure.

  17. 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.

  18. Expression of urokinase receptors by human trophoblast. A histochemical and ultrastructural analysis.

    PubMed

    Multhaupt, H A; Mazar, A; Cines, D B; Warhol, M J; McCrae, K R

    1994-09-01

    Through their ability to invade endometrium, remodel the uterine spiral arteries, and sustain placental blood fluidity, trophoblast cells play a central role in establishing and maintaining the integrity of the uteroplacental vasculature. The expression of urokinase receptors by trophoblast may facilitate these processes by focusing plasminogen activator activity to discrete sites on the cell surface and promoting the activation of cell-bound plasminogen. However, although urokinase receptors are expressed by cultured trophoblast, the expression of these receptors by trophoblast in vivo has not been examined. Immunohistochemistry and immunoelectron microscopy were used to characterize the expression of urokinase receptors by villous and extravillous trophoblast at several points in gestation. Urokinase receptors were expressed in a polarized fashion at the leading edge of migrating extravillous trophoblast cells. Receptors were also abundantly expressed during the first and second trimesters of gestation by villous trophoblast, where they were located on apical villous projections and within intracellular vacuoles, a subset of which were lysosomes. The polarized expression of urokinase receptors by invasive extravillous trophoblast cells is consistent with a role for these receptors in mediating the extent and directionality of trophoblast migration. In contrast, the expression of urokinase receptors by villous trophoblast, which are not actively invasive in vivo, may serve to facilitate the generation of plasmin at the interface of these cells with maternal plasma, thereby limiting the deposition of fibrin within the placental intervillous spaces. Diminished urokinase receptor expression by villous trophoblast at term may represent a physiologic adaptation to diminish local fibrinolysis and limit hemorrhage at parturition.

  19. Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

    PubMed Central

    Yu, J. C.; Pickard, J. D.; Davenport, A. P.

    1995-01-01

    151242 competed for the binding of [125I]-ET-1 monophasically and analysis of the competition curves indicated that a one-site fit was preferred over a two-site model, implying that the cultures express mainly ETA receptors. 6. Although messenger RNA encoding both ETA and ETB receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ETA receptor protein. Antagonism of this sub-type may be necessary to block the actions of ET-1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage. Images Figure 1 Figure 2 PMID:8581282

  20. Cocaine decreases expression of neurogranin via alterations in thyroid receptor/retinoid X receptor signaling

    PubMed Central

    Kovalevich, Jane; Corley, Gladys; Yen, William; Kim, Jae; Rawls, Scott M.; Langford, Dianne

    2013-01-01

    Mounting evidence suggests a potential link between cocaine abuse, disruptions in hypothalamic-pituitary-thyroid axis signaling, and neuroplasticity, but molecular mechanisms remain unknown. Neurogranin (Ng) is a gene containing a thyroid hormone-responsive element within its first intron that is involved in synaptic plasticity. Transcriptional activation requires heterodimerization of thyroid hormone receptor (TR) and retinoid X receptor (RXR) bound by their respective ligands, tri-iodothryonine and 9-cis-retinoic acid (9-cis-RA), and subsequent binding of this complex to the thyroid hormone-responsive element of the Ng gene. In this study, the effects of chronic cocaine abuse on Ng expression in euthyroid and hypothyroid mice were assessed. In cocaine-treated mice, decreased Ng expression was observed in the absence of changes in levels of thyroid hormones or other hypothalamic-pituitary-thyroid signaling factors. Therefore, we hypothesized that cocaine decreases Ng expression via alterations in 9-cis-RA availability and TR/RXR signaling. In support of this hypothesis, RXR-γ was significantly decreased in brains of cocaine-treated mice while CYP26A1, the main enzyme responsible for neuronal RA degradation, was significantly increased. Results from this study provide the first evidence for a direct effect of cocaine abuse on TR/RXR signaling, RA metabolism, and transcriptional regulation of Ng, a gene essential for adult neuroplasticity. PMID:22300446

  1. Novel Alexa Fluor-488 labeled antagonist of the A(2A) adenosine receptor: Application to a fluorescence polarization-based receptor binding assay.

    PubMed

    Kecskés, Miklós; Kumar, T Santhosh; Yoo, Lena; Gao, Zhan-Guo; Jacobson, Kenneth A

    2010-08-15

    Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A(2A) adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A(2A)AR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a K(i) value of 111+/-16nM in radioligand binding using [(3)H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A(2A)AR. In a cyclic AMP functional assay, MRS5346 was shown to be an A(2A)AR antagonist. MRS5346 did not show any effect on A(1) and A(3) ARs in binding or the A(2B)AR in a cyclic AMP assay at 10microM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal following A(2A)AR binding. The FP signal was optimal with 20nM MRS5346 and 150microg protein/mL HEK293 membranes. The association and dissociation kinetic parameters were readily determined using this FP assay. The K(d) value of MRS5346 calculated from kinetic parameters was 16.5+/-4.7nM. In FP competition binding experiments using MRS5346 as a tracer, K(i) values of known AR agonists and antagonists consistently agreed with K(i) values from radioligand binding. Thus, this FP assay, which eliminates using radioisotopes, appears to be appropriate for both routine receptor binding and high-throughput screening with respect to speed of analysis, displaceable signal and precision. The approach used in the present study could be generally applicable to other GPCRs. Published by Elsevier Inc.

  2. Functional importance of GLP-1 receptor species and expression levels in cell lines.

    PubMed

    Knudsen, Lotte Bjerre; Hastrup, Sven; Underwood, Christina Rye; Wulff, Birgitte Schjellerup; Fleckner, Jan

    2012-04-10

    Of the mammalian species, only the GLP-1 receptors of rat and human origin have been described and characterized. Here, we report the cloning of the homologous GLP-1 receptors from mouse, rabbit, pig, cynomolgus monkey and chimp. The GLP-1 receptor is highly conserved across species, thus underlining the physiological importance of the peptide hormone and its receptor across a wide range of mammals. We expressed the receptors by stable transfection of BHK cells, both in cell lines with high expression levels of the cloned receptors, as well as in cell lines with lower expression levels, more comparable to endogenous expression of these receptors. High expression levels of cloned GLP-1 receptors markedly increased the potency of GLP-1 and other high affinity ligands, whereas the K(d) values were not affected. For a low affinity ligand like the ago-allosteric modulator Compound 2, expression levels of the human GLP-1 receptor were important for maximal efficacy as well as potency. The two natural metabolites of GLP-1, GLP-1(9-37) and GLP-1(9-36)amide were agonists when tested on a cell line with high expression of the recombinant human GLP-1 receptor, whereas they behaved as (low potent) antagonists on a cell line that expressed the receptor endogenously, as well as cells expressing a moderate level of the recombinant human GLP-1 receptor. The amide form was a more potent agonist than the free acid from. In conclusion, receptor expression level is an important parametre for selecting cell lines with cloned GLP-1 receptors for functional characterization of physiological and pharmaceutical ligands. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum

    SciTech Connect

    Voith, G.; Dingermann, T.

    1995-11-01

    We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I{gamma} promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I{gamma} promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays. 36 refs., 4 figs.

  4. Nuclear receptor expression in human differentiated thyroid tumors.

    PubMed

    Mond, Michael; Alexiadis, Maria; Eriksson, Natalie; Davis, Melissa J; Muscat, George E O; Fuller, Peter J; Gilfillan, Christopher

    2014-06-01

    Nuclear receptors (NRs) play a key role in endocrine signaling and metabolism and are important therapeutic targets in a number of hormone-dependent malignancies. Studies on the role of NRs in thyroid cancer are limited. The objective of the study was to examine systematically the expression of the 48 human NRs in a series of benign and malignant thyroid tissues. Within the papillary carcinoma cohort, we sought to determine if NR expression differed significantly by BRAF mutation status. RNA was isolated from multinodular goiter (MNG; n=6), papillary carcinoma (PTC, n=14), follicular carcinoma (FC; n=5), and Hürthle cell carcinoma (HCC; n=7). The 48 human NRs were profiled in this panel by quantitative real time polymerase chain reaction. Protein expression for selected NRs (Rev-erbα and LXR-β) was examined by immunohistochemistry (IHC) on tissue microarrays comprising benign and malignant thyroid tissues. Across all groups of benign and malignant thyroid tissue, there was prominent expression of LXR-β and ROR-γ. Key findings in PTC were marked overexpression of RXR-γ and Rev-erbα compared to MNG. Within the PTC cohort, when BRAF(V600E) tumors were compared with wild type BRAF, there was relative upregulation of RXR-γ and Rev-erbα and downregulation of AR, ERR-γ, and ROR-γ. In FC, EAR-2 was overexpressed, while PPAR-α and PPAR-δ were underexpressed compared to MNG. The NR expression profile of HCC was distinct, characterized by significant downregulation of a wide range of NRs. IHC for Rev-erbα and LXR-β localized protein expression to the tumor cells. Moderate to strong Rev-erbα immunostaining was seen in 22 out of 23 PTC, and, overall, staining was stronger than in the benign group. These results represent the first systematic examination of NR expression in thyroid cancer. Our finding of tumor-specific patterns of NR expression, as well as significant differences in NR expression between BRAF(V600E) and wild type BRAF PTC, provides a basis for

  5. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease

    PubMed Central

    Karmouty-Quintana, Harry; Zhong, Hongyan; Acero, Luis; Weng, Tingting; Melicoff, Ernestina; West, James D.; Hemnes, Anna; Grenz, Almut; Eltzschig, Holger K.; Blackwell, Timothy S.; Xia, Yang; Johnston, Richard A.; Zeng, Dewan; Belardinelli, Luiz; Blackburn, Michael R.

    2012-01-01

    Development of pulmonary hypertension is a common and deadly complication of interstitial lung disease. Little is known regarding the cellular and molecular mechanisms that lead to pulmonary hypertension in patients with interstitial lung disease, and effective treatment options are lacking. The purpose of this study was to examine the adenosine 2B receptor (A2BR) as a regulator of vascular remodeling and pulmonary hypertension secondary to pulmonary fibrosis. To accomplish this, cellular and molecular changes in vascular remodeling were monitored in mice exposed to bleomycin in conjunction with genetic removal of the A2BR or treatment with the A2BR antagonist GS-6201. Results demonstrated that GS-6201 treatment or genetic removal of the A2BR attenuated vascular remodeling and hypertension in our model. Furthermore, direct A2BR activation on vascular cells promoted interleukin-6 and endothelin-1 release. These studies identify a novel mechanism of disease progression to pulmonary hypertension and support the development of A2BR antagonists for the treatment of pulmonary hypertension secondary to interstitial lung disease.—Karmouty-Quintana, H., Zhong, H., Acero, L., Weng, T., Melicoff, E., West, J. D., Hemnes, A., Grenz, A., Eltzschig, H. K., Blackwell, T. S., Xia, Y., Johnston, R. A., Zeng, D., Belardinelli, L., Blackburn, M. R. The A2B adenosine receptor modulates pulmonary hypertension associated with interstitial lung disease. PMID:22415303

  6. Striatal adenosine A2A receptor neurons control active-period sleep via parvalbumin neurons in external globus pallidus

    PubMed Central

    Qu, Wei-Min; Yang, Su-Rong; Cherasse, Yoan; Lazarus, Michael; Schiffmann, Serge N; d'Exaerde, Alban de Kerchove

    2017-01-01

    Dysfunction of the striatum is frequently associated with sleep disturbances. However, its role in sleep-wake regulation has been paid little attention even though the striatum densely expresses adenosine A2A receptors (A2ARs), which are essential for adenosine-induced sleep. Here we showed that chemogenetic activation of A2AR neurons in specific subregions of the striatum induced a remarkable increase in non-rapid eye movement (NREM) sleep. Anatomical mapping and immunoelectron microscopy revealed that striatal A2AR neurons innervated the external globus pallidus (GPe) in a topographically organized manner and preferentially formed inhibitory synapses with GPe parvalbumin (PV) neurons. Moreover, lesions of GPe PV neurons abolished the sleep-promoting effect of striatal A2AR neurons. In addition, chemogenetic inhibition of striatal A2AR neurons led to a significant decrease of NREM sleep at active period, but not inactive period of mice. These findings reveal a prominent contribution of striatal A2AR neuron/GPe PV neuron circuit in sleep control. PMID:29022877

  7. Estrogen receptor gene expression in relation to neuropsychiatric disorders.

    PubMed

    Ostlund, Hanna; Keller, Eva; Hurd, Yasmin L

    2003-12-01

    Compelling evidence now exists for estrogen's involvement in the regulation of mood and cognitive functions. Serum estrogen levels have been shown to play an important role in the expression of psychiatric disorders such as depression and schizophrenia. We have characterized the distribution of the estrogen receptors, ERalpha and ERbeta, in the human brain and showed a preferential limbic-related expression pattern for these transcripts. The ERalpha mRNA dominates in the amygdala and hypothalamus, suggesting estrogen modulation of autonomic and neuroendocrine as well as emotional functions. In contrast, the hippocampal formation, entorhinal cortex, and thalamus appear to be ERbeta-dominant areas, suggesting a role for ERbeta in cognition, non-emotional memory, and motor functions. The role of estradiol can also be examined in regard to its relationship to other neurotransmitter systems known to be linked to specific psychiatric disorders. Estradiol has been shown to regulate the serotonin (5-HT) system, which has been strongly implicated in affective disorders. We have studied a genetic animal model of depression, and found altered 5-HT receptor mRNA levels in discrete brain regions; many of the abnormalities are reversed by estradiol treatment, especially for the 5-HT(2A) receptor subtype. The norepinephrine (NE) system is, similar to serotonin, a target for antidepressant drugs, and projects to mesocorticolimbic structures implicated in mood disorders. We have recently observed that NE neurons in the human locus coeruleus (LC) express moderate levels of both ER transcripts. The possibility of estrogen's regulating LC function has been documented in animal studies. Results from our preliminary experiments have revealed that the ERbeta mRNA is decreased in persons committing suicide, a cause of death that is highly linked to affective disorder. Follow-up studies are currently under way with a much larger population to validate these results. Overall, the discrete

  8. Establishing and maintaining gene expression patterns: insights from sensory receptor patterning

    PubMed Central

    Rister, Jens; Desplan, Claude; Vasiliauskas, Daniel

    2013-01-01

    In visual and olfactory sensory systems with high discriminatory power, each sensory neuron typically expresses one, or very few, sensory receptor genes, excluding all others. Recent studies have provided insights into the mechanisms that generate and maintain sensory receptor expression patterns. Here, we review how this is achieved in the fly retina and compare it with the mechanisms controlling sensory receptor expression patterns in the mouse retina and in the mouse and fly olfactory systems. PMID:23293281

  9. Constitutive androstane receptor activation evokes the expression of glycolytic genes.

    PubMed

    Yarushkin, Andrei A; Kazantseva, Yuliya A; Prokopyeva, Elena A; Markova, Diana N; Pustylnyak, Yuliya A; Pustylnyak, Vladimir O

    2016-09-23

    It is well-known that constitutive androstane receptor (CAR) activation by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) increases the liver-to-body weight ratio. CAR-mediated liver growth is correlated with increased expression of the pleiotropic transcription factor cMyc, which stimulates cell cycle regulatory genes and drives proliferating cells into S phase. Because glycolysis supports cell proliferation and cMyc is essential for the activation of glycolytic genes, we hypothesized that CAR-mediated up-regulation of cMyc in mouse livers might play a role in inducing the expression of glycolytic genes. The aim of the present study was to examine the effect of long-term CAR activation on glycolytic genes in a mouse model not subjected to metabolic stress. We demonstrated that long-term CAR activation by TCPOBOP increases expression of cMyc, which was correlated with reduced expression of gluconeogenic genes and up-regulation of glucose transporter, glycolytic and mitochondrial pyruvate metabolising genes. These changes in gene expression after TCPOBOP treatment were strongly correlated with changes in levels of glycolytic intermediates in mouse livers. Moreover, we demonstrated a significant positive regulatory effect of TCPOBOP-activated CAR on both mRNA and protein levels of Pkm2, a master regulator of glucose metabolism and cell proliferation. Thus, our findings provide evidence to support the conclusion that CAR activation initiates a transcriptional program that facilitates the coordinated metabolic activities required for cell proliferation. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Nicotinic acetylcholine receptor expression by directionally selective ganglion cells.

    PubMed

    Strang, Christianne E; Renna, Jordan M; Amthor, Franklin R; Keyser, Kent T

    2007-01-01

    Acetylcholine (ACh) enhances the preferred direction responses of directionally selective ganglion cells (DS GCs; Ariel & Daw, 1982; Ariel & Adolph, 1985) through the activation of nicotinic acetylcholine receptors (nAChRs; Ariel & Daw, 1982; Massey et al., 1997; Kittila & Massey, 1997). DS GCs appear to express at least two types of nAChRs, those that are sensitive to the partially subtype-specific antagonist methyllycaconitine (MLA), and those that are MLA-insensitive (Reed et al., 2002). Our purpose was to confirm the expression of alpha7 nAChRs by DS GCs and to assess the contributions of other nAChR subtypes to DS GC responses. Using choline as a nAChR partially subtype-specific agonist, we found that the majority of DS GCs demonstrated responses to choline while under synaptic blockade. The blockade or reduction of choline-induced responses by bath application of nanomolar (nM) concentrations of MLA provided direct evidence that the choline responses were mediated by alpha7 nAChRs. Because choline is a partial agonist for alpha3beta4 nAChRs (Alkondon et al., 1997), the residual choline responses are consistent with mediation by alpha3beta4 nAChRs. Additionally, a subset of DS GCs responded to nicotine but not to choline, indicating the expression of a third nAChR subtype. The pharmacological results were supported by single cell reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry experiments. The expression of alpha7 and specific non-alpha7 nAChR subtypes was correlated with the preferred direction. This indicates the possibility of differential responses to ACh depending on the direction of movement. This is the first description of differential expression of multiple nAChR subtypes by DS GCs.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2016-10-01

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

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

    PubMed Central

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

    2012-01-01

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

  14. Acetate supplementation modulates brain adenosine metabolizing enzymes and adenosine A2A receptor levels in rats subjected to neuroinflammation

    PubMed Central

    2014-01-01

    Background Acetate supplementation reduces neuroglia activation and pro-inflammatory cytokine expression in rat models of neuroinflammation and Lyme neuroborreliosis. Because single-dose glyceryl triacetate (GTA) treatment increases brain phosphocreatine and reduces brain AMP levels, we postulate that GTA modulates adenosine metabolizing enzymes and receptors, which may be a possible mechanism to reduce neuroinflammation. Methods To test this hypothesis, we quantified the ability of GTA to alter brain levels of ecto-5’-nucleotidase (CD73), adenosine kinase (AK), and adenosine A2A receptor using western blot analysis and CD73 activity by measuring the rate of AMP hydrolysis. Neuroinflammation was induced by continuous bacterial lipopolysaccharide (LPS) infusion in the fourth ventricle of the brain for 14 and 28 days. Three treatment strategies were employed, one and two where rats received prophylactic GTA through oral gavage with LPS infusion for 14 or 28 days. In the third treatment regimen, an interventional strategy was used where rats were subjected to 28 days of neuroinflammation, and GTA treatment was started on day 14 following the start of the LPS infusion. Results We found that rats subjected to neuroinflammation for 28 days had a 28% reduction in CD73 levels and a 43% increase in AK levels that was reversed with prophylactic acetate supplementation. CD73 activity in these rats was increased by 46% with the 28-day GTA treatment compared to the water-treated rats. Rats subjected to neuroinflammation for 14 days showed a 50% increase in levels of the adenosine A2A receptor, which was prevented with prophylactic acetate supplementation. Interventional GTA therapy, beginning on day 14 following the induction of neuroinflammation, resulted in a 67% increase in CD73 levels and a 155% increase in adenosine A2A receptor levels. Conclusion These results support the hypothesis that acetate supplementation can modulate brain CD73, AK and adenosine A2A receptor

  15. Role of Microglia Adenosine A2A Receptors in Retinal and Brain Neurodegenerative Diseases

    PubMed Central

    Santiago, Ana R.; Baptista, Filipa I.; Santos, Paulo F.; Cristóvão, Gonçalo; Ambrósio, António F.; Cunha, Rodrigo A.; Gomes, Catarina A.

    2014-01-01

    Neuroinflammation mediated by microglial cells in the brain has been commonly associated with neurodegenerative diseases. Whether this microglia-mediated neuroinflammation is cause or consequence of neurodegeneration is still a matter of controversy. However, it is unequivocal that chronic neuroinflammation plays a role in disease progression and halting that process represents a potential therapeutic strategy. The neuromodulator adenosine emerges as a promising targeting candidate based on its ability to regulate microglial proliferation, chemotaxis, and reactivity through the activation of its G protein coupled A2A receptor (A2AR). This is in striking agreement with the ability of A2AR blockade to control several brain diseases. Retinal degenerative diseases have been also associated with microglia-mediated neuroinflammation, but the role of A2AR has been scarcely explored. This review aims to compare inflammatory features of Parkinson's and Alzheimer's diseases with glaucoma and diabetic retinopathy, discussing the therapeutic potential of A2AR in these degenerative conditions. PMID:25132733

  16. Genetic modification of cytotoxic T lymphocytes to express cytokine receptors.

    PubMed

    Perna, Serena K; Savoldo, Barbara; Dotti, Gianpietro

    2014-01-01

    Adoptive transfer of tumor-infiltrating lymphocytes (TIL) or antigen-specific cytotoxic T lymphocytes (CTL) is safe and can be effective in cancer patients. Achievement of clinical responses in these patients is associated with the in vivo expansion and persistence of the transferred T lymphocytes. For this reason, recombinant human interleukin-2 (IL-2) is frequently used to support the in vivo survival of T lymphocytes infused into patients. However, IL-2 also causes important side effects. Thus, alternative strategies are highly demanded to limit cytokine-related off-target effects and to redirect the responsiveness of specific T-cell subsets to selected cytokines. Interleukin-7 (IL-7) is a promising alternative cytokine as it possesses the above mentioned properties. However, because its receptor is downregulated in ex vivo-expanded T cells, methods are required to restore their responsiveness to this homeostatic cytokine. In this chapter, we describe the methodology to obtain the ectopic expression of IL-7 receptor alpha (IL-7Rα) in antigen-specific CTL, using Epstein-Barr virus-specific CTL (EBV-CTL), as a model.

  17. Key Modulatory Role of Presynaptic Adenosine A2A Receptors in Cortical Neurotransmission to the Striatal Direct Pathway

    PubMed Central

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N.; Borycz, Janusz; Kachroo, Anil; Canas, Paula M.; Orru, Marco; Schwarzschild, Michael A.; Rosin, Diane L.; Kreitzer, Anatol C.; Cunha, Rodrigo A.; Watanabe, Masahiko; Ferré, Sergi

    2009-01-01

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders. PMID:19936569

  18. Key modulatory role of presynaptic adenosine A2A receptors in cortical neurotransmission to the striatal direct pathway.

    PubMed

    Quiroz, César; Luján, Rafael; Uchigashima, Motokazu; Simoes, Ana Patrícia; Lerner, Talia N; Borycz, Janusz; Kachroo, Anil; Canas, Paula M; Orru, Marco; Schwarzschild, Michael A; Rosin, Diane L; Kreitzer, Anatol C; Cunha, Rodrigo A; Watanabe, Masahiko; Ferré, Sergi

    2009-11-18

    Basal ganglia processing results from a balanced activation of direct and indirect striatal efferent pathways, which are controlled by dopamine D1 and D2 receptors, respectively. Adenosine A2A receptors are considered novel antiparkinsonian targets, based on their selective postsynaptic localization in the indirect pathway, where they modulate D2 receptor function. The present study provides evidence for the existence of an additional, functionally significant, segregation of A2A receptors at the presynaptic level. Using integrated anatomical, electrophysiological, and biochemical approaches, we demonstrate that presynaptic A2A receptors are preferentially localized in cortical glutamatergic terminals that contact striatal neurons of the direct pathway, where they exert a selective modulation of corticostriatal neurotransmission. Presynaptic striatal A2A receptors could provide a new target for the treatment of neuropsychiatric disorders.

  19. Transactivation of the Receptor-tyrosine Kinase Ephrin Receptor A2 Is Required for the Low Molecular Weight Hyaluronan-mediated Angiogenesis That Is implicated in Tumor Progression*

    PubMed Central

    Lennon, Frances E; Mirzapoiazova, Tamara; Mambetsariev, Nurbek; Mambetsariev, Bolot; Salgia, Ravi; Singleton, Patrick A.

    2014-01-01

    Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression. PMID:25023279

  20. The regulation of acetylcholine receptor expression in mammalian muscle.

    PubMed

    Merlie, J P; Sebbane, R; Gardner, S; Olson, E; Lindstrom, J

    1983-01-01

    completion of this process. alpha-Subunits that do not undergo conformational maturation are degraded rapidly (t1/2 = 0.5 hr) ( Merlie et al. 1982). Assembly of alpha- and beta-subunits synthesized during a 5-minute pulse labeling lags for approximately 30 minutes and is not complete until 90 minutes. Finally, assembled receptors are transported to the surface and appear in the plasma membrane. These processes occur during expression of AChRs in differentiated myoblasts. We do not know how undifferentiated myogenic cells, in vivo or in tissue culture, differ with regard to any of these steps.(ABSTRACT TRUNCATED AT 400 WORDS)

  1. Expression of hypothalamic-pituitary-gonadal axis-related hormone receptors in low-grade serous ovarian cancer (LGSC).

    PubMed

    Feng, Zheng; Wen, Hao; Ju, Xingzhu; Bi, Rui; Chen, Xiaojun; Yang, Wentao; Wu, Xiaohua

    2017-01-25

    The aim of our study was to investigate the clinical features and expression levels of hypothalamic-pituitary-gonadal axis-related hormone receptors in low-grade serous ovarian cancer (LGSC). We retrospectively investigated the clinical features of 26 consecutive patients with LGSC who underwent primary staging or debulking surgery between April 2005 and June 2013 in our center; concomitant primary high-grade serous ovarian cancer (HGSC) patients were randomly selected at a 2:1 ratio for comparison. Tissue microarrays were constructed from the LGSC and HGSC specimens, and the expression levels of six hormone receptors in the hypothalamic pituitary-gonadal axis were analyzed by immunohistochemistry. The median (range) age of patients with LGSC was 54 (27-77) years. According to the FIGO staging system, the cases were distributed as follows: stage I, 6 (23.1%); stage II, 0 (0%); stage III, 19 (73.1%); and stage IV, 1 (3.8%). The 2-year and 5-year overall survival rates for LGSC were 91.8% and 67.5%, respectively. The expression levels of the hormone receptors were as follows: ER, 80.8%; PR, 34.6%; AR, 53.8%; FSHR, 84.0%; LHR, 65.4%; and GnRHR, 100%. Hormone receptor-positive patients had a better prognosis compared with hormone receptor-negative patients, but the difference was not significant. Our study presented a higher overall survival rate and distinctive hormone receptor expression levels of LGSC patients compared with the HGSC cohort. Patients with positive hormone receptor expression tended to have a better prognosis than the corresponding hormone receptor negative patients.

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

    SciTech Connect

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

    2011-04-15

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

  3. Receptor Expression in Rat Skeletal Muscle Cell Cultures

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.

    1996-01-01

    One on the most persistent problems with long-term space flight is atrophy of skeletal muscles. Skeletal muscle is unique as a tissue in the body in that its ability to undergo atrophy or hypertrophy is controlled exclusively by cues from the extracellular environment. The mechanism of communication between muscle cells and their environment is through a group of membrane-bound and soluble receptors, each of which carries out unique, but often interrelated, functions. The primary receptors include acetyl choline receptors, beta-adrenergic receptors, glucocorticoid receptors, insulin receptors, growth hormone (i.e., somatotropin) receptors, insulin-like growth factor receptors, and steroid receptors. This project has been initiated to develop an integrated approach toward muscle atrophy and hypertrophy that takes into account information on the populations of the entire group of receptors (and their respective hormone concentrations), and it is hypothesized that this information can form the basis for a predictive computer model for muscle atrophy and hypertrophy. The conceptual basis for this project is illustrated in the figure below. The individual receptors are shown as membrane-bound, with the exception of the glucocorticoid receptor which is a soluble intracellular receptor. Each of these receptors has an extracellular signalling component (e.g., innervation, glucocorticoids, epinephrine, etc.), and following the interaction of the extracellular component with the receptor itself, an intracellular signal is generated. Each of these intracellular signals is unique in its own way; however, they are often interrelated.

  4. Evaluation of Eph receptor and ephrin expression within the human cornea and limbus.

    PubMed

    Hogerheyde, Thomas A; Stephenson, Sally-Anne; Harkin, Damien G; Bray, Laura J; Madden, Peter W; Woolf, Mark I; Richardson, Neil A

    2013-02-01

    Eph receptor tyrosine kinases and their ligands, the ephrins, regulate the development and maintenance of multiple organs but little is known about their potential role within the cornea. The purpose of this study was to perform a thorough investigation of Eph/ephrin expression within the human cornea including the limbal stem cell niche. Initially, immunohistochemistry was performed on human donor eyes to determine the spatial distribution of Eph receptors and ephrins in the cornea and limbus. Patterns of Eph/ephrin gene expression in (1) immortalised human corneal endothelial (B4G12) or corneal epithelial (HCE-T) cell lines, and (2) primary cultures of epithelial or stromal cells established from the corneal limbus of cadaveric eye tissue were then assessed by reverse transcription (RT) PCR. Limbal epithelial or stromal cells from primary cultures were also assessed for evidence of Eph/ephrin-reactivity by immunofluorescence. Immunoreactivity for ephrinA1 and EphB4 was detected in the corneal endothelium of donor eyes. EphB4 was also consistently detected in the limbal and corneal epithelium and in cells located in the stroma of the peripheral cornea. Expression of multiple Eph/ephrin genes was detected in immortalised corneal epithelial and endothelial cell lines. Evidence of Eph/ephrin gene expression was also demonstrated in primary cultures of human limbal stromal (EphB4, B6; ephrinA5) and epithelial cells (EphA1, A2; ephrinA5, B2) using both RT-PCR and immunofluorescence. The expression of Eph receptors and ephrins within the human cornea and limbus is much wider than previously appreciated and suggests multiple potential roles for these molecules in the maintenance of normal corneal architecture. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Amino acid conjugates of lithocholic acid as antagonists of the EphA2 receptor.

    PubMed

    Incerti, Matteo; Tognolini, Massimiliano; Russo, Simonetta; Pala, Daniele; Giorgio, Carmine; Hassan-Mohamed, Iftiin; Noberini, Roberta; Pasquale, Elena B; Vicini, Paola; Piersanti, Silvia; Rivara, Silvia; Barocelli, Elisabetta; Mor, Marco; Lodola, Alessio

    2013-04-11

    The Eph receptor-ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The l-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small-molecule antagonists of the EphA2 receptor.

  6. MED12 protein expression in breast fibroepithelial lesions: correlation with mutation status and oestrogen receptor expression.

    PubMed

    Tan, Wai Jin; Chan, Jason Yongsheng; Thike, Aye Aye; Lim, Jeffrey Chun Tatt; Md Nasir, Nur Diyana; Tan, Jane Sie Yong; Koh, Valerie Cui Yun; Lim, Weng Khong; Tan, Jing; Ng, Cedric Chuan Yong; Rajasegaran, Vikneswari; Nagarajan, Sanjanaa; Bay, Boon Huat; Teh, Bin Tean; Tan, Puay Hoon

    2016-10-01

    Recent reports have identified recurrent MED12 somatic mutations in fibroadenomas and phyllodes tumours. The frequency and type of somatic mutations were noted to be similar to those of uterine leiomyomas. We aimed to investigate protein expression of MED12, correlating it to MED12 mutational status and expression of oestrogen receptors (ER). Immunohistochemistry was performed on a total of 232 fibroepithelial lesions (100 fibroadenomas, 132 phyllodes tumours) diagnosed at the Department of Pathology, Singapore General Hospital using MED12, ERα and ERβ antibodies. Expressions were evaluated in both stroma and epithelium, and correlated with MED12 mutational status. MED12 mutation was significantly associated with high MED12 protein expression (H-score >150) in the stroma (p=0.029), but not in the epithelium. It was not associated with ERα and ERβ protein expression in both stroma and epithelium. MED12 protein expression was significantly correlated with ERα in epithelial (p=0.007) and ERβ in stromal (p=0.049) components. MED12 was not significantly different between fibroadenomas and phyllodes tumours. Epithelial expression of ERα was significantly higher in fibroadenomas (p<0.001) than in phyllodes tumours. Conversely, both epithelial and stromal expression of ERβ was significantly higher in phyllodes tumours (p<0.001). Positive associations observed between MED12 and ERα, ERβ immunohistochemical expression suggest a biological interplay between the proteins. The lack of significant association of MED12 mutation with ER protein expression indicates a need to further explore the functional impact of MED12 mutations on the ER signalling pathway in breast fibroepithelial lesions. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  7. Effects of tobacco smoke condensate on estrogen receptor-alpha gene expression and activity.

    PubMed

    Martin, Mary Beth; Reiter, Ronald; Johnson, Michael; Shah, Mansi S; Iann, Mary C; Singh, Baljit; Richards, Julie Kate; Wang, Antai; Stoica, Adriana

    2007-10-01

    Metallo-estrogens are a new class of potent environmental estrogens. This study investigates whether tobacco smoke condensate (TSC), which contains metals and metalloids, elicits estrogen-like effects at environmentally relevant doses. Treatment of human breast cancer cells, MCF-7, with 40 microg/ml TSC resulted in a 2.5-fold stimulation of cell growth. TSC decreased the concentration of estrogen receptor (ER)-alpha protein and mRNA (63 and 62%, respectively), and increased the expression of the estrogen-regulated genes, progesterone receptor and pS2 (5- and 2-fold, respectively). In addition, TSC activated ER-alpha in COS-1 or CHO cells transiently transfected with wild-type ER-alpha and an ERE-CAT or an ERE-luciferase reporter gene (11- and 6-fold, respectively). TSC also activated a chimeric receptor (GAL-ER) containing the hormone binding domain of ER-alpha (3.5-fold). It blocked the binding of estradiol to the receptor without altering the affinity of estradiol (K(d) = 2.2-6.8 x 10(-10) m). Transfection assays with ER-alpha mutants identified C381, C447, H524, N532, E523, and D538 in the hormone binding domain as important for activation by TSC. In ovariectomized rats, low doses of TSC [10 or 20 mg/kg body weight (bw)] increased uterine wet weight (1.7- and 2.1-fold), and induced the expression of progesterone receptor and complement C3 in the uterus (2- and 26-fold) and mammary gland (4.4- and 15-fold). Both the in vitro and in vivo TSC effects were blocked by the antiestrogen ICI 182,780, suggesting the involvement of ER. Collectively, these results provide strong evidence that low doses of TSC, acting through the hormone binding domain, exert estrogen-like effects in cell culture and animals.

  8. In pulmonary lymphangioleiomyomatosis expression of progesterone receptor is frequently higher than that of estrogen receptor.

    PubMed

    Gao, Ling; Yue, Michael M; Davis, Jennifer; Hyjek, Elisabeth; Schuger, Lucia

    2014-04-01

    Lymphangioleiomyomatosis (LAM) of the lung is a rare low-grade malignancy affecting primarily women of childbearing age. LAM is characterized by the proliferation of SMA and HMB-45 positive spindle-shaped and epithelioid cells throughout the lung in the form of discrete lesions causing cystic destruction and ultimately respiratory insufficiency. LAM occurs sporadically or in patients with tuberous sclerosis complex (TSC) and is etiologically linked to mutations in the TSC1 and TSC2 genes. Although LAM cells are known to express estrogen and progesterone receptors (ER and PR, respectively), their respective expression level was never determined. Therefore, here we measured the immunohistochemical expression of ERs and PRs in a large series of pulmonary LAM cases using the Aperio Spectrum Analysis Platform. Our case series comprised open lung biopsy specimens from 20 LAM patients and lungs explanted during the course of lung transplant from 24 patients. All cases were positive for ER and PR. PR expression was statistically significantly higher than ER in 80 % of the biopsies while ER predominated only in one case. Specimens from explanted cases of LAM had relatively fewer PR-positive nuclei. As a result, PR expression was significantly higher than ER in 38 % of the cases, whereas ER predominated in 33 %. Overall, PR expression predominated in 57 % of cases and ER in 21 %. These data indicate that PR frequently prevails over ER in pulmonary LAM. LAM is unusual in its high PR/ER ratio; other female neoplasms show a definite prevalence of ER. Our findings therefore warrant further study of PR function in LAM.

  9. Leptin receptor expression and Gln223Arg polymorphism as prognostic markers in oral and oropharyngeal cancer.

    PubMed

    Rodrigues, P R S; Maia, L L; Santos, M; Peterle, G T; Alves, L U; Takamori, J T; Souza, R P; Barbosa, W M; Mercante, A M C; Nunes, F D; Carvalho, M B; Tajara, E H; Louro, I D; Silva-Conforti, A M A

    2015-11-25

    The leptin gene product is released into the blood stream, passes through the blood-brain barrier, and finds the leptin receptor (LEPR) in the central nervous system. This hormone regulates food intake, hematopoiesis, inflammation, immunity, differentiation, and cell proliferation. The LEPR Gln223Arg polymorphism has been reported to alter receptor function and expression, both of which have been related with prognostics in several tumor types. Furthermore, several studies have shown a relationship between the Gln223Arg polymorphism and tumor development, and its role in oral and oropharyngeal squamous cell carcinoma is now well understood. In this study, 315 DNA samples were used for LEPR Gln223Arg genotyping and 87 primary oral and oropharyngeal squamous cell carcinomas were used for immunohistochemical expression analysis, such that a relationship between these and tumor development and prognosis could be established. Homozygous LEPR Arg223 was found to be associated with a 2-fold reduction in oral and oropharyngeal cancer risk. In contrast, the presence of the Arg223 allele in tumors was associated with worse disease-free and disease-specific survival. Low LEPR expression was found to be an independent risk factor, increasing the risk for lymph node metastasis 4-fold. In conclusion, the Gln223Arg polymorphism and LEPR expression might be valuable markers for oral and oropharyngeal cancer, suggesting that LEPR might serve as a potential target for future therapies.

  10. Expression of retinoid receptors in lungs of cattle, dogs, and pigs.

    PubMed

    Channabasappa, Shankaramurthy; Ferguson, Julia; Singh, Baljit

    2014-07-01

    Retinoids play an important role in lung development and immune response. The effects of retinoids are mediated through 2 families of retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), with alpha (α), beta (β), and gamma (γ) subtypes in each family. To date, no data exist on the expression pattern of retinoid receptors in lungs of cattle, dogs, and pigs. Because of the biomedical importance of retinoid receptors in inflammation and immune responses, Western blot, immunohistology, and immunoelectron microscopy were used to determine the expression of retinoid receptors in normal lungs of cattle, dogs, and pigs (n = 2 for each species). Western blot showed expression of all 6 retinoid receptor subtypes in pig lungs. Immunohistology data indicated differential expression of retinoid receptors in airway epithelium, vascular endothelium, alveolar/septal macrophages, and alveolar septum in all 3 species. Electron microscopy showed nuclear localization of retinoid receptors in neutrophils and pulmonary intravascular macrophages. Retinoic acid receptors (RAR) α subtype were localized in cytoplasmic vacuoles of pig monocytes. These data indicate constitutive expression of retinoid receptors in the lungs of cattle, dogs, and pigs.

  11. Expression and cytokine regulation of glucocorticoid receptors in Kaposi's sarcoma.

    PubMed Central

    Guo, W. X.; Antakly, T.; Cadotte, M.; Kachra, Z.; Kunkel, L.; Masood, R.; Gill, P.

    1996-01-01

    Development of Kaposi's sarcoma (KS) after glucocorticoid therapy has been observed in a variety of clinical states including human immunodeficiency virus-1 infection and recent in vitro studies provided evidence for a direct stimulation effect of glucocorticoid hormones on KS cell proliferation. The importance of glucocorticoids in KS pathogenesis is further highlighted by the finding that glucocorticoids synergize with cytokines to promote acquired immune deficiency syndrome (AIDS)-associated KS (AIDS-KS) growth. Furthermore, cytokine effects were abrogated by the glucocorticoid antagonist RU-486. As glucocorticoid action is mediated through activation of their intracellular cognate receptors, we hypothesized that enhanced responsiveness of AIDS-KS cells to glucocorticoids may be due to elevated glucocorticoid receptor (GR) content. Indeed, high expression of GRs in AIDS-KS tumor biopsies was detected both at the level of mRNA and protein. Quantitative measurements of GRs in these specimens by a sensitive immunoassay showed that GR content was significantly elevated in the tumor tissue (4663 fmol/mg protein) compared with the uninvolved skin of the same patients (2777 fmol/mg protein), both of which were markedly above the normal skin of healthy donors (893 fmol/mg protein). Immunocytochemical analysis confirmed the presence of GRs in the cytoplasm and the nucleus of KS cells. Interestingly, four major KS cytokines, namely interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and oncostatin M, all of which are known autocrine growth factors for AIDS-KS cells, significantly increased the expression of functional GRs in cultured AIDS-KS cells. The latter result may explain, at least in part, the synergistic effect of glucocorticoid and oncostatin M on AIDS-KS cell proliferation. Thus, the high levels of GR expression in AIDS-KS and the up-regulation of GRs by KS-growth-promoting factors may confer enhanced and sustained sensitivity to the stimulatory

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

    PubMed Central

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

    2016-01-01

    The consumption of caffeine modulates working and reference memory through the antagonism of adenosine A2A receptors (A2ARs) controlling synaptic plasticity processes in hippocampal excitatory synapses. Fear memory essentially involves plastic changes in amygdala circuits. However, it is unknown if A2ARs in the amygdala regulate synaptic plasticity and fear memory. We report that A2ARs in the amygdala are enriched in synapses and located to glutamatergic synapses, where they selectively control synaptic plasticity rather than synaptic transmission at a major afferent pathway to the amygdala. Notably, the downregulation of A2ARs selectively in the basolateral complex of the amygdala, using a lentivirus with a silencing shRNA (small hairpin RNA targeting A2AR (shA2AR)), impaired fear acquisition as well as Pavlovian fear retrieval. This is probably associated with the upregulation and gain of function of A2ARs in the amygdala after fear acquisition. The importance of A2ARs to control fear memory was further confirmed by the ability of SCH58261 (0.1 mg/kg; A2AR antagonist), caffeine (5 mg/kg), but not DPCPX (0.5 mg/kg; A1R antagonist), treatment for 7 days before fear conditioning onwards, to attenuate the retrieval of context fear after 24–48 h and after 7–8 days. These results demonstrate that amygdala A2ARs control fear memory and the underlying process of synaptic plasticity in this brain region. This provides a neurophysiological basis for the association between A2AR polymorphisms and phobia or panic attacks in humans and prompts a therapeutic interest in A2ARs to manage fear-related pathologies. PMID:27312408

  13. Distribution of delta opioid receptor expressing neurons in the mouse hippocampus

    PubMed Central

    Eric, ERBS; Lauren, FAGET; Gregory, SCHERRER; Pascal, KESSLER; Didier, HENTSCH; Jean-Luc, VONESCH; Audrey, MATIFAS; Brigitte L., KIEFFER; Dominique, MASSOTTE

    2012-01-01

    Delta opioid receptors participate to the control of chronic pain and emotional responses. Recent data also identified their implication in spatial memory and drug-context associations pointing to a critical role of hippocampal delta receptors. We examined the distribution of delta receptor-expressing cells in the hippocampus using fluorescent knock-in mice that express a functional delta receptor fused at its carboxyterminus with the green fluorescent protein in place of the native receptor. Colocalization with markers for different neuronal populations was performed by immunohistochemical detection. Fine mapping in the dorsal hippocampus confirmed that delta opioid receptors are mainly present in GABAergic neurons. Indeed, they are mostly expressed in parvalbumin-immunopositive neurons both in the Ammon’s horn and dentate gyrus. These receptors, therefore, most likely participate to the dynamic regulation of hippocampal activity. PMID:22750239

  14. Expression of the aryl hydrocarbon receptor pathway and cyclooxygenase-2 in dog tumors.

    PubMed

    Giantin, M; Vascellari, M; Lopparelli, R M; Ariani, P; Vercelli, A; Morello, E M; Cristofori, P; Granato, A; Buracco, P; Mutinelli, F; Dacasto, M

    2013-02-01

    In humans, the aryl hydrocarbon receptor (AHR) gene battery constitutes a set of contaminant-responsive genes, which have been recently shown to be involved in the regulation of several patho-physiological conditions, including tumorigenesis. As the domestic dog represents a valuable animal model in comparative oncology, mRNA levels of cytochromes P450 1A1, 1A2 and 1B1 (CYP1A1, 1A2 and 1B1), AHR, AHR nuclear translocator (ARNT), AHR repressor (AHRR, whose partial sequence was here obtained) and cyclooxygenase-2 (COX2) were measured in dog control tissues (liver, skin, mammary gland and bone), in 47 mast cell tumors (MCTs), 32 mammary tumors (MTs), 5 osteosarcoma (OSA) and related surgical margins. Target genes were constitutively expressed in the dog, confirming the available human data. Furthermore, their pattern of expression in tumor biopsies was comparable to that already described in a variety of human cancers; in particular, both AHR and COX2 genes were up-regulated and positively correlated, while CYP1A1 and CYP1A2 mRNAs were generally poorly expressed. This work demonstrated for the first time that target mRNAs are expressed in neoplastic tissues of dogs, thereby increasing the knowledge about dog cancer biology and confirming this species as an useful animal model for comparative studies on human oncology. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. The EphA2 receptor drives self-renewal and tumorigenicity in stem-like tumor-propagating cells from human glioblastomas.

    PubMed

    Binda, Elena; Visioli, Alberto; Giani, Fabrizio; Lamorte, Giuseppe; Copetti, Massimiliano; Pitter, Ken L; Huse, Jason T; Cajola, Laura; Zanetti, Nadia; DiMeco, Francesco; De Filippis, Lidia; Mangiola, Annunziato; Maira, Giulio; Anile, Carmelo; De Bonis, Pasquale; Reynolds, Brent A; Pasquale, Elena B; Vescovi, Angelo L

    2012-12-11

    In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2(High) and EphA2(Low) populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. The EphA2 Receptor Drives Self-Renewal and Tumorigenicity in Stem-Like Tumor-Propagating Cells from Human Glioblastomas

    PubMed Central

    Binda, Elena; Visioli, Alberto; Giani, Fabrizio; Lamorte, Giuseppe; Copetti, Massimiliano; Pitter, Ken L.; Huse, Jason T.; Cajola, Laura; Zanetti, Nadia; DiMeco, Francesco; De Filippis, Lidia; Mangiola, Annunziato; Maira, Giulio; Anile, Carmelo; De Bonis, Pasquale; Reynolds, Brent A.; Pasquale, Elena B.; Vescovi, Angelo L.

    2014-01-01

    SUMMARY In human glioblastomas (hGBMs), tumor-propagating cells with stem-like characteristics (TPCs) represent a key therapeutic target. We found that the EphA2 receptor tyrosine kinase is overexpressed in hGBM TPCs. Cytofluorimetric sorting into EphA2High and EphA2Low populations demonstrated that EphA2 expression correlates with the size and tumor-propagating ability of the TPC pool in hGBMs. Both, ephrinA1-Fc, which caused EphA2 downregulation in TPCs, and siRNA-mediated knockdown of EPHA2 expression suppressed TPCs self-renewal ex vivo and intracranial tumorigenicity, pointing to EphA2 downregulation as a causal event in the loss of TPCs tumorigenicity. Infusion of ephrinA1-Fc into intracranial xenografts elicited strong tumor-suppressing effects, suggestive of therapeutic applications. PMID:23238013

  17. Estrogen Receptor and Progesterone Receptor Expression in Normal Terminal Duct Lobular Units Surrounding Invasive Breast Cancer

    PubMed Central

    Yang, Xiaohong R.; Figueroa, Jonine D.; Hewitt, Stephen M.; Falk, Roni T.; Pfeiffer, Ruth M.; Lissowska, Jolanta; Peplonska, Beata; Brinton, Louise A.; Garcia-Closas, Montserrat; Sherman, Mark E.

    2014-01-01

    Introduction Molecular and morphological alterations related to carcinogenesis have been found in terminal duct lobular units (TDLUs), the microscopic structures from which most breast cancer precursors and cancers develop, and therefore, analysis of these structures may reveal early changes in breast carcinogenesis and etiologic heterogeneity. Accordingly, we evaluated relationships of breast cancer risk factors and tumor pathology to estrogen receptor (ER) and progesterone receptor (PR) expression in TDLUs surrounding breast cancers. Methods We analyzed 270 breast cancer cases included in a population-based breast cancer case-control study conducted in Poland. TDLUs were mapped in relation to breast cancer: within the same block as the tumor (TDLU-T), proximal to tumor (TDLU-PT), or distant from (TDLU-DT). ER/PR was quantitated using image analysis of immunohistochemically stained TDLUs prepared as tissue microarrays. Results In surgical specimens containing ER-positive breast cancers, ER and PR levels were significantly higher in breast cancer cells than in normal TDLUs, and higher in TDLU-T than in TDLU-DT or TDLU-PT, which showed similar results. Analyses combining DT-/PT TDLUs within subjects demonstrated that ER levels were significantly lower in premenopausal women vs. postmenopausal women (odds ratio [OR]=0.38, 95% confidence interval [CI]=0.19, 0.76, P=0.0064) and among recent or current menopausal hormone therapy users compared with never users (OR=0.14, 95% CI=0.046–0.43, Ptrend=0.0006). Compared with premenopausal women, TDLUs of postmenopausal women showed lower levels of PR (OR=0.90, 95% CI=0.83–0.97, Ptrend=0.007). ER and PR expression in TDLUs was associated with epidermal growth factor receptor (EGFR) expression in invasive tumors (P=0.019 for ER and P=0.03 for PR), but not with other tumor features. Conclusions Our data suggest that TDLUs near breast cancers reflect field effects, whereas those at a distance demonstrate influences of breast

  18. Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2.

    PubMed

    Reed, J K

    1981-08-06

    The effects of phospholipase A2 treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [3H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A2 from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by beta-bungarotoxin. (2) Phospholipase A2 from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A2 treatment of [3H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.

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

    PubMed

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

    2017-01-01

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

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

    PubMed Central

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

    2017-01-01

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

  1. Expression of melatonin receptors in arteries involved in thermoregulation

    SciTech Connect

    Viswanathan, M.; Laitinen, J.T.; Saavedra, J.M.

    1990-08-01

    Melatonin binding sites were localized and characterized in the vasculature of the rat by using the melatonin analogue 2-(125I)iodomelatonin (125I-melatonin) and quantitative in vitro autoradiography. The expression of these sites was restricted to the caudal artery and to the arteries that form the circle of Willis at the base of the brain. The arterial 125I-melatonin binding was stable, saturable, and reversible. Saturation studies revealed that the binding represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 3.4 x 10(-11) M in the anterior cerebral artery and 1.05 x 10(-10) M in the caudal artery. Themore » binding capacities (Bmax) in these arteries were 19 and 15 fmol/mg of protein, respectively. The relative order of potency of indoles for inhibition of 125I-melatonin binding at these sites was typical of a melatonin receptor: 2-iodomelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine. Norepinephrine-induced contraction of the caudal artery in vitro was significantly prolonged and potentiated by melatonin in a concentration-dependent manner, suggesting that these arterial binding sites are functional melatonin receptors. Neither primary steps in smooth muscle contraction (inositol phospholipid hydrolysis) nor relaxation (adenylate cyclase activation) were affected by melatonin. Melatonin, through its action on the tone of these arteries, may cause circulatory adjustments in these arteries, which are believed to be involved in thermoregulation.« less

  2. Upregulation of IFN-γ Receptor Expression by Proinflammatory Cytokines Influences IDO Activation in Epithelial Cells

    PubMed Central

    SHIREY, KARI ANN; JUNG, JOO-YONG; MAEDER, GREGORY S.; CARLIN, JOSEPH M.

    2006-01-01

    Interferon-γ (IFN-γ) induces the enzyme indoleamine dioxygenase (IDO) in a variety of human cell types. Furthermore, tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) synergistically increase IFN-induced IDO activity. Inasmuch as cytokines can upregulate cytokine receptor expression, one mechanism of cytokine synergy may be at the level of receptor expression. To test the hypothesis that this mechanism of IDO regulation is active in epithelial cells, HeLa cells were treated with IFN-γ, TNF-β, or IL-1 α to determine optimal cytokine concentrations and time for maximal cytokine receptor expression. Flow cytometric analysis with antibodies to receptors for IFN-γ, TNF-α, or IL-1 β indicated that each cytokine upregulated expression of the other cytokine receptors by 4 h, with maximal expression observed between 16 and 20 h after cytokine treatment. Furthermore, increases in IFN-γ receptors (IFNGR) induced by IL-1 β were found to be dependent on NF-κB transactivation. To determine if increases in IFNGR expression alone contributes to synergistic IDO induction, cells were stimulated with IL-1 β to upregulate receptor expression, and the NF-κB concentration was allowed to return to basal levels. When treated with IFN-γ, enhanced Stat1 signaling and IDO induction were still observed, indicating that increased cytokine receptor expression contributes to synergistic increases in IDO activity. PMID:16426148

  3. Flumazenil decreases surface expression of α4β2δ GABAA receptors by increasing the rate of receptor internalization.

    PubMed

    Kuver, Aarti; Smith, Sheryl S

    2016-01-01

    Increases in expression of α4βδ GABAA receptors (GABARs), triggered by fluctuations in the neurosteroid THP (3α-OH-5α[β]-pregnan-20-one), are associated with changes in mood and cognition. We tested whether α4βδ trafficking and surface expression would be altered by in vitro exposure to flumazenil, a benzodiazepine ligand which reduces α4βδ expression in vivo. We first determined that flumazenil (100 nM-100 μM, IC50=∼1 μM) acted as a negative modulator, reducing GABA (10 μM)-gated current in the presence of 100 nM THP (to increase receptor efficacy), assessed with whole cell patch clamp recordings of recombinant α4β2δ expressed in HEK-293 cells. Surface expression of recombinant α4β2δ receptors was detected using a 3XFLAG reporter at the C-terminus of α4 (α4F) using confocal immunocytochemical techniques following 48 h exposure of cells to GABA (10 μM)+THP (100 nM). Flumazenil (10 μM) decreased surface expression of α4F by ∼60%, while increasing its intracellular accumulation, after 48 h. Reduced surface expression of α4β2δ after flumazenil treatment was confirmed by decreases in the current responses to 100 nM of the GABA agonist gaboxadol. Flumazenil-induced decreases in surface expression of α4β2δ were prevented by the dynamin blocker, dynasore, and by leupeptin, which blocks lysosomal enzymes, suggesting that flumazenil is acting to increase endocytosis and lysosomal degradation of the receptor. Flumazenil increased the rate of receptor removal from the cell surface by 2-fold, assessed using botulinum toxin B to block insertion of new receptors. These findings may suggest new therapeutic strategies for regulation of α4β2δ expression using flumazenil. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Irradiation affects cellular properties and Eph receptor expression in human melanoma cells

    PubMed Central

    Mosch, Birgit; Pietzsch, Doreen; Pietzsch, Jens

    2012-01-01

    X-ray irradiation influences metastatic properties of tumor cells and, moreover, metastasis and cellular motility can be modified by members of the Eph receptor/ephrin family of receptor tyrosine kinases. We hypothesized that irradiation-induced changes in cellular properties relevant for metastasis in melanoma cells could be mediated by Eph receptor/ephrin signaling. In this pilot study, we analyzed one pre-metastatic (Mel-Juso) and three metastatic human melanoma (Mel-Juso-L3, A375, and A2058) cells lines and predominantly found anti-metastatic effects of X-ray irradiation with impaired cell growth, clonal growth and motility. Additionally, we observed an irradiation-induced increase in adhesion paralleled by a decrease in migration in Mel-Juso and Mel-Juso-L3 cells and, in part, also in A375 cells. We further demonstrate a decrease of EphA2 both in expression and activity at 7 d after irradiation paralleled by an upregulation of EphA3. Analyzing downstream signaling after irradiation, we detected decreased Src kinase phosphorylation, but unchanged focal adhesion kinase (FAK) phosphorylation, indicating, in part, irradiation-induced downregulation of signaling via the EphA2-Src-FAK axis in melanoma cells. However, to which extent this finding contributes to the modification of metastasis-relevant cellular properties remains to be elucidated. PMID:22568947

  5. A2A adenosine receptor upregulation correlates with disease activity in patients with systemic lupus erythematosus.

    PubMed

    Bortoluzzi, Alessandra; Vincenzi, Fabrizio; Govoni, Marcello; Padovan, Melissa; Ravani, Annalisa; Borea, Pier Andrea; Varani, Katia

    2016-08-26

    Adenosine is a purine nucleoside implicated in the regulation of the innate and adaptive immune systems, acting through its interaction with four cell surface receptors: A1, A2A, A2B, and A3. There is intense interest in understanding how adenosine functions in health and during disease, but surprisingly little is known about the actual role of adenosine-mediated mechanisms in systemic lupus erythematosus (SLE). With this background, the aim of the present study was to test the hypothesis that dysregulation of A1, A2A, A2B, and A3 adenosine receptors (ARs) in lymphocytes of patients with SLE may be involved in the pathogenesis of the disease and to examine the correlations between the status of the ARs and the clinical parameters of SLE. ARs were analyzed by performing saturation-binding assays, as well as messenger RNA and Western blot analysis, with lymphocytes of patients with SLE in comparison with healthy subjects. We tested the effect of A2AAR agonists in the nuclear factor kB (NF-kB) pathway and on the release of interferon (IFN)-α; tumor necrosis factor (TNF)-α; and interleukin (IL)-2, IL-6, IL-1β, and IL-10. In lymphocytes obtained from 80 patients with SLE, A2AARs were upregulated compared with those of 80 age-matched healthy control subjects, while A1, A2B, and A3 ARs were unchanged. A2AAR density was inversely correlated with Systemic Lupus Erythematosus Disease Activity Index 2000 score disease activity through time evaluated according to disease course patterns, serositis, hypocomplementemia, and anti-double-stranded DNA positivity. A2AAR activation inhibited the NF-kB activation pathway and diminished inflammatory cytokines (IFN-α, TNF-α, IL-2, IL-6, IL-1β), but it potentiated the release of anti-inflammatory IL-10. These data suggest the involvement of A2AARs in the complex pathogenetic network of SLE, acting as a modulator of the inflammatory process. It could represent a compensatory pathway to better counteract disease activity. A2AAR

  6. Phenotypical characterization of the rat striatal neurons expressing the D1 dopamine receptor gene.

    PubMed Central

    Le Moine, C; Normand, E; Bloch, B

    1991-01-01

    In situ hybridization experiments were performed in rat brain sections from normal and 6-hydroxydopamine-treated rats in order to map and identify the neurons expressing the D1 receptor gene in the striatum and the substantia nigra. Procedures of combined in situ hybridization, allowing the simultaneous detection of two mRNAs in the same section or in adjacent sections, were used to characterize the phenotypes of the neurons expressing the D1 receptor gene. D1 receptor mRNA was found in neurons all over the caudate-putamen, the accumbens nucleus, and the olfactory tubercle but not in the substantia nigra. In the caudate-putamen and accumbens nucleus, most of the neurons containing D1 receptor mRNA were characterized as medium-sized substance P neurons and distinct from those containing D2 receptor mRNA. Nevertheless, 15-20% of the substance P neurons did not contain D1 receptor mRNA. The neurons containing preproenkephalin A mRNA did not contain D1 receptor mRNA but contained D2 receptor mRNA. A small number of cholinergic and somatostatinergic neurons exhibited a weak reaction for D1 receptor mRNA. These results demonstrate that dopamine acts on efferent striatal neurons through expression of distinct receptors--namely, D1 and D2 in separate cell populations (substance P and preproenkephalin A neurons, respectively)--and can also act on nonprojecting neurons through D1 receptor expression. Images PMID:1827915

  7. Expression and clinical significance of S100A2 and p63 in esophageal carcinoma

    PubMed Central

    Cao, Li-Yu; Yin, Yu; Li, Hao; Jiang, Yan; Zhang, Hong-Fu

    2009-01-01

    AIM: To investigate the expression and clinical significance of S100A2 mRNA and protein, p63 protein in esophageal squamous cell carcinoma (ESCC) and their roles in carcinogenesis and progression of esophageal carcinoma (EC). METHODS: Immunohistochemical staining (S-P method) for S100A2 and p63 protein were performed in 40 samples of ESCC and 40 samples of normal esophageal mucosa. In situ hybridization (ISH) was used to detect the expression of S100A2 mRNA. RESULTS: Expression of S100A2 mRNA in ESCC was positive in 77.5% of samples, which was lower than that in normal mucosa (100%) by ISH (P = 0.002). The expression level of S100A2 mRNA was closely related to differentiation and and node-metastasis (P = 0.012, P = 0.008). Expression of S100A2 protein was positive in 72.5% of ESCC samples and expression of p63 protein was positive in 37.5% of ESCC samples, and was lower than that in normal mucosa (100%) (P = 0.000). The expression of S100A2 protein was correlated with the differentiation and node-metastasis (P = 0.007, P = 0.001), but no relationship was observed between the expression of p63 protein and clinical pathological manifestations. S100A2 protein was positively correlated with the expression of S100A2 mRNA, and negatively associated with the expression of p63 protein (P = 0.000, P = 0.002). CONCLUSION: S100A2 and p63 protein both play important roles in the carcinogenesis of ESCC. An investigation into the combined expression of S100A2 and p63 may be helpful in early diagnosis and in evaluating the prognosis of ESCC. PMID:19725154

  8. Substance P Receptor Binding Sites are Expressed by Glia in vivo after Neuronal Injury

    NASA Astrophysics Data System (ADS)

    Mantyh, Patrick W.; Johnson, Donald J.; Boehmer, Christian G.; Catton, Mark D.; Vinters, Harry V.; Maggio, John E.; Too, Heng-Phon; Vigna, Steven R.

    1989-07-01

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

  9. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

    PubMed

    Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

    2015-06-01

    Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. Copyright © 2015. Published by Elsevier B.V.

  10. Adenosine A2A Receptor Antagonists and Parkinson’s Disease

    PubMed Central

    2011-01-01

    This Review summarizes and updates the work on adenosine A2A receptor antagonists for Parkinson’s disease from 2006 to the present. There have been numerous publications, patent applications, and press releases within this time frame that highlight new medicinal chemistry approaches to this attractive and promising target to treat Parkinson’s disease. The Review is broken down by scaffold type and will discuss the efforts to optimize particular scaffolds for activity, pharmacokinetics, and other drug discovery parameters. The majority of approaches focus on preparing selective A2A antagonists, but a few approaches to dual A2A/A1 antagonists will also be highlighted. The in vivo profiles of compounds will be highlighted and discussed to compare activities across different chemical series. A clinical report and update will be given on compounds that have entered clinical trials. PMID:22860156

  11. Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

    NASA Technical Reports Server (NTRS)

    Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

    2001-01-01

    Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

  12. Differences in adenosine A-1 and A-2 receptor density revealed by autoradiography in methylxanthine-sensitive and insensitive mice

    SciTech Connect

    Jarvis, M.F.; Williams, M.

    1988-07-01

    Two strains of inbred mice, CBA/J and SWR/J, have been identified which are, respectively, sensitive and insensitive to the behavioral and toxic effects of methylxanthines. Autoradiographic analyses of brain adenosine receptors were conducted with (/sup 3/H)CHA to label adenosine A-1 receptors and (/sup 3/H)NECA, in the presence of 50 nM CPA, to label adenosine A-2 receptors. For both mouse strains, adenosine A-1 receptors were most highly concentrated in the hippocampus and cerebellum whereas adenosine A-2 receptors were selectively localized in the striatum. CBA/J mice displayed a 30% greater density of adenosine A-1 receptors in the hippocampal CA-1 and CA-3 regionsmore » and in the cerebellum as compared to the SWR/J mice. The number of A-2 receptors (Bmax) was 40% greater in the striatum and olfactory tubercle of CBA/J as compared to SWR/J mice. No significant regional differences in A-1 or A-2 receptor affinities were observed between these inbred strains of mice. These results indicate that the differential sensitivity to methylxanthines between these mouse strains may reflect a genetically mediated difference in regional adenosine receptor densities.« less

  13. Ovary-specific depletion of the nuclear receptor Nr5a2 compromises expansion of the cumulus oophorus but not fertilization by intracytoplasmic sperm injection.

    PubMed

    Bertolin, Kalyne; Meinsohn, Marie-Charlotte; Suzuki, João; Gossen, Jan; Schoonjans, Kristina; Duggavathi, Rajesha; Murphy, Bruce D

    2017-06-01

    The orphan nuclear receptor, liver receptor homolog-1 (aka Nuclear receptor subfamily 5, Group A, Member 2 (Nr5a2)), is widely expressed in mammalian tissues, and its ovarian expression is restricted to granulosa cells of activated follicles. We employed the floxed Nr5a2 (Nr5a2f/f) mutant mouse line and two granulosa-specific Cre lines, Anti-Müllerian hormone receptor- 2 (Amhr2Cre) and transgenic cytochrome P450 family 19 subfamily A polypeptide 1 (tgCyp19Cre), to develop two tissue- and time-specific Nr5a2 depletion models: Nr5a2Amhr2-/- and Nr5a2Cyp19-/-. In the Nr5a2Cyp19-/- ovaries, Nr5a2 was depleted in mural granulosa, but not cumulus cells. We induced follicular development in mutant and wild-type (control, CON) mice with equine chorionic gonadotropin followed 44 h later treatment with human chorionic gonadotropin (hCG) to induce ovulation. Both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- cumulus-oocyte complexes underwent a reduced degree of expansion in vitro relative to wild-type mice. We found downregulation of epiregulin (Ereg), amphiregulin (Areg), betacellulin (Btc) and tumor necrosis factor stimulated gene-6 (Tnfaip6) transcripts in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- ovaries. Tnfaip6 protein abundance, by quantitative immunofluorescence, was likewise substantially reduced in the Nr5a2-depleted model. Transcript abundance for connexin 43 (Gja1) in granulosa cells was lower at 0 h and maximum at 8 h post-hCG in both Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles, while Gja1 protein was not different prior to the ovulatory signal, but elevated at 8 h in Nr5a2Amhr2-/- and Nr5a2Cyp19-/- follicles. In both mutant genotypes, oocytes can mature in vivo and resulting embryos were capable of proceeding to blastocyst stagein vitro. We conclude that Nr5a2 is essential for cumulus expansion in granulosa cells throughout follicular development. The disruption of Nr5a2 in follicular somatic cells does not affect the capacity of the oocyte to be fertilized by intracytoplasmic sperm

  14. "Moonlighting" GAPDH Protein Localizes with AMPA Receptor GluA2 and L1 Axonal Cell Adhesion Molecule at Fiber Cell Borders in the Lens.

    PubMed

    Frederikse, Peter H; Nandanoor, Anoop; Kasinathan, Chinnaswamy

    2016-01-01

    The canonical role of glyceraldehyde phosphate dehydrogenase (GAPDH) is as an enzyme in glycolysis. GAPDH is also a principal "moonlighting" protein with additional roles at diverse sites in a variety of cells. Surface GAPDH on mammalian, yeast, and bacterial cells acts as a receptor and also mediates cell contacts. In neurons, extracellular GAPDH localizes at synapses. Two GAPDH binding partners at synapses are α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor (AMPA) GluA2 subunit at dendritic spines and L1 cell adhesion molecule at pre-synaptic membranes, and both proteins are also expressed in lenses. Fiber cell membrane protrusions and dendritic spines have similar size, shape, and spacing, contain F-actin, and express clathrin/AP-2 Adaptor at their surfaces linked with Tyr-phosphatase STEP-regulated endocytosis of AMPA/GluA2 receptors. AMPA receptors work with NMDA (N-methyl-d-aspartate) and GABA (γ-aminobutyric acid) receptors, calcium calmodulin kinase II (CaMKIIα), channel proteins, STEP, and ephrin receptors, which are also expressed in lenses. In neurons, coordinate AMPA/GluA2 receptor endocytosis with GAPDH is linked with disease. GAPDH was previously characterized as a fiber cell membrane protein and shown to decrease substantially in interior fiber cells in human age-related cataract. Here, we examined GAPDH spatial expression in healthy lenses in two vertebrate species. In situ methods were used to examine GAPDH expression in lenses of healthy young adult rabbits and chickens. Immunoblots were used to detect L1 in lenses. The present study demonstrated that GAPDH is present at fiber cell borders in adult rabbit and chicken lenses with evidence of focal concentrations along the fiber cell perimeter, and overlapped with detection of p-Tyr-GluA2, L1, STEP, actin and clathrin. We observed that L1-140 kDa was the prominent form in lens. Our findings indicate investigations into GAPDH "moonlighting" activities similar to its role

  15. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant

    PubMed Central

    Croy, Carrie H.; Ruble, Cara L.; Tao, Ran; Felder, Christian C.

    2017-01-01

    Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension. PMID:29211764

  16. Identification, expression and functional characterization of M4L, a muscarinic acetylcholine M4 receptor splice variant.

    PubMed

    Schober, Douglas A; Croy, Carrie H; Ruble, Cara L; Tao, Ran; Felder, Christian C

    2017-01-01

    Rodent genomic alignment sequences support a 2-exon model for muscarinic M4 receptor. Using this model a novel N-terminal extension was discovered in the human muscarinic acetylcholine M4 receptor. An open reading frame was discovered in the human, mouse and rat with a common ATG (methionine start codon) that extended the N-terminus of the muscarinic acetylcholine M4 receptor subtype by 155 amino acids resulting in a longer variant. Transcriptional evidence for this splice variant was confirmed by RNA-Seq and RT-PCR experiments performed from human donor brain prefrontal cortices. We detected a human upstream exon indicating the translation of the mature longer M4 receptor transcript. The predicted size for the longer two-exon M4 receptor splice variant with the additional 155 amino acid N-terminal extension, designated M4L is 69.7 kDa compared to the 53 kDa canonical single exon M4 receptor (M4S). Western blot analysis from a mammalian overexpression system, and saturation radioligand binding with [3H]-NMS (N-methyl-scopolamine) demonstrated the expression of this new splice variant. Comparative pharmacological characterization between the M4L and M4S receptors revealed that both the orthosteric and allosteric binding sites for both receptors were very similar despite the addition of an N-terminal extension.

  17. Activation of Presynaptic GABAB(1a,2) Receptors Inhibits Synaptic Transmission at Mammalian Inhibitory Cholinergic Olivocochlear–Hair Cell Synapses

    PubMed Central

    Wedemeyer, Carolina; Zorrilla de San Martín, Javier; Ballestero, Jimena; Gómez-Casati, María Eugenia; Torbidoni, Ana Vanesa; Fuchs, Paul A.; Bettler, Bernhard; Elgoyhen, Ana Belén

    2013-01-01

    The synapse between olivocochlear (OC) neurons and cochlear mechanosensory hair cells is cholinergic, fast, and inhibitory. The inhibitory sign of this cholinergic synapse is accounted for by the activation of Ca2+-permeable postsynaptic α9α10 nicotinic receptors coupled to the opening of hyperpolarizing Ca2+-activated small-conductance type 2 (SK2)K+ channels. Acetylcholine (ACh) release at this synapse is supported by both P/Q- and N-type voltage-gated calcium channels (VGCCs). Although the OC synapse is cholinergic, an abundant OC GABA innervation is present along the mammalian cochlea. The role of this neurotransmitter at the OC efferent innervation, however, is for the most part unknown. We show that GABA fails to evoke fast postsynaptic inhibitory currents in apical developing inner and outer hair cells. However, electrical stimulation of OC efferent fibers activates presynaptic GABAB(1a,2) receptors [GABAB(1a,2)Rs] that downregulate the amount of ACh released at the OC–hair cell synapse, by inhibiting P/Q-type VGCCs. We confirmed the expression of GABABRs at OC terminals contacting the hair cells by coimmunostaining for GFP and synaptophysin in transgenic mice expressing GABAB1–GFP fusion proteins. Moreover, coimmunostaining with antibodies against the GABA synthetic enzyme glutamic acid decarboxylase and synaptophysin support the idea that GABA is directly synthesized at OC terminals contacting the hair cells during development. Thus, we demonstrate for the first time a physiological role for GABA in cochlear synaptic function. In addition, our data suggest that the GABAB1a isoform selectively inhibits release at efferent cholinergic synapses. PMID:24068816

  18. Titanium particles modulate expression of Toll-like receptor proteins.

    PubMed

    Pajarinen, Jukka; Mackiewicz, Zygmunt; Pöllänen, Raimo; Takagi, Michiaki; Epstein, Noah J; Ma, Ting; Goodman, Stuart B; Konttinen, Yrjö T

    2010-03-15

    The role of Toll-like receptors (TLRs) responding to microbial remnants, indolent biofilms or cellular byproducts in aseptic loosening of joint replacements is unknown. Thus, the effect of titanium (Ti) particles on TLR protein levels was evaluated. To create a model of particle-induced inflammation, an intramedullary stainless steel rod with and without Ti particles was bilaterally placed in the femora of 14 mice. The animals were sacrificed at 2 or 10 weeks postoperatively and paraffin-embedded femur sections were evaluated for TLR1, 2, 4, 5, 8, and 9 proteins using immunohistochemistry. Decrease in the number of TLR immunoreactive cells was observed between weeks 2 and 10 in both settings. Furthermore, in the presence of Ti particles, the numbers of TLR immunoreactive cells were lower than in the presence of rod only at both time points, suggesting downregulation of TLR expression by Ti-particles per se. Accordingly, in a short-term 24 h stimulation, downregulation of TLR4 mRNA (p < 0.02) was observed in vitro in RAW 264.7 cells challenged with Ti particles. Results suggest that after an initial inflammatory stage, TLRs are downregulated in response to Ti particles, possibly to inhibit excessive inflammation, although TLR downregulation might at the same time render tissues more susceptible to pathogens. (c) 2009 Wiley Periodicals, Inc.

  19. Inhibition of oxytocin receptor and estrogen receptor-alpha expression, but not relaxin receptors (LGR7), in the myometrium of late pregnant relaxin gene knockout mice.

    PubMed

    Siebel, Andrew L; Gehring, Helen M; Reytomas, Irna Grace T; Parry, Laura J

    2003-10-01

    This study used relaxin (RLX) gene knockout mice (Rlx-/-) to investigate the effects of RLX on myometrial oxytocin receptor (OTR) and estrogen receptor (ER)-alpha gene expression in late gestation. We also characterized the temporal expression of the RLX receptor (LGR7) and demonstrated gene transcripts in the myometrium of Rlx+/+ and Rlx-/- mice. There was a significant (P < 0.05) decrease in myometrial LGR7 gene expression on d 17.5 and 18.5 post coitum (pc) compared with earlier stages of gestation, but no differences between Rlx+/+ and Rlx-/- mice. Myometrial OTR mRNA levels increased at the end of gestation in Rlx+/+ but not Rlx-/- mice. ERalpha gene expression was up-regulated on d 14.5 pc in Rlx+/+ mice, with mRNA levels remaining high throughout late gestation. In contrast, ERalpha mRNA levels were significantly lower in Rlx-/- mice on d 14.5 and 18.5 pc. These data show that the increases in myometrial OTR and ERalpha expression in late pregnant Rlx+/+ mice were attenuated in Rlx-/- mice. The effects of RLX on OTRs are probably mediated via activation of ERalpha. Finally, RLX receptor expression in the myometrium of Rlx-/- mice did not differ from wild-type mice, implying that RLX does not influence expression of its receptor.

  20. Expression of mu-opioid receptors in developing rat spinal cord: an autoradiographic study.

    PubMed

    Ray, Subrata Basu; Wadhwa, Shashi

    2004-05-01

    The expression of mu-opioid receptors in the developing rat spinal cord (Postnatal days 7, 14, 30) was studied by autoradiography using [3H]DAMGO. When compared to camera lucida drawings, the receptor was noted over the entire gray matter and dorsal root ganglia at postnatal days 7 and 14. At postnatal day 30, the receptor expression decreased over the gray matter except the superficial laminae (laminae I and II). At all age groups studied, a higher expression of the receptor was noted over the superficial laminae. The study shows that micro-opioid receptors appears early in postnatal development and attains mature receptor distribution relatively late in ontogeny, suggesting a possible role in the normal development of nervous system. This is affirmed by an impairment of psychomotor development in babies born to mothers, addicted to opiates.

  1. Improving the odorant sensitivity of olfactory receptor-expressing yeast with accessory proteins.

    PubMed

    Fukutani, Yosuke; Hori, Ayaka; Tsukada, Satoshi; Sato, Ryoichi; Ishii, Jun; Kondo, Akihiko; Matsunami, Hiroaki; Yohda, Masafumi

    2015-02-15

    Olfaction depends on the selectivity and sensitivity of olfactory receptors. Previous attempts at constructing a mammalian olfactory receptor-based artificial odorant sensing system in the budding yeast Saccharomyces cerevisiae suffered from low sensitivity and activity. This result may be at least in part due to poor functional expression of olfactory receptors and/or limited solubility of some odorants in the medium. In this study, we examined the effects of two types of accessory proteins, receptor transporting protein 1 short and odorant binding proteins, in improving odor-mediated activation of olfactory receptors expressed in yeast. We found that receptor transporting protein 1 short enhanced the membrane expression and ligand-induced responses of some olfactory receptors. Coexpression of odorant binding proteins of the silkworm moth Bombyx mori enhanced the sensitivity of a mouse olfactory receptor. Our results suggest that different classes of accessory proteins can confer sensitive and robust responses of olfactory receptors expressed in yeast. Inclusion of accessory proteins may be essential in the future development of practical olfactory receptor-based odorant sensors. Copyright © 2014 Elsevier Inc. All rights reserved.

  2. Estrogen receptor α can selectively repress dioxin receptor-mediated gene expression by targeting DNA methylation

    PubMed Central

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-01-01

    Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene (CYP1A1) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene (CYP1B1) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells. PMID:23828038

  3. A role for a specific cholesterol interaction in stabilizing the Apo configuration of the human A(2A) adenosine receptor.

    PubMed

    Lyman, Edward; Higgs, Chris; Kim, Byungchan; Lupyan, Dmitry; Shelley, John C; Farid, Ramy; Voth, Gregory A

    2009-12-09

    The function of G-protein-coupled receptors is tightly modulated by the lipid environment. Long-timescale molecular dynamics simulations (totaling approximately 3 mus) of the A(2A) receptor in cholesterol-free bilayers, with and without the antagonist ZM241385 bound, demonstrate the instability of helix II in the apo receptor in cholesterol-poor membrane regions. We directly observe that the effect of cholesterol binding is to stabilize helix II against a buckling-type deformation, perhaps rationalizing the observation that the A(2A) receptor couples to G protein only in the presence of cholesterol (Zezula and Freissmuth, 2008). The results suggest a mechanism by which the A(2A) receptor may function as a coincidence detector, activating only in the presence of both cholesterol and agonist. We also observed a previously hypothesized conformation of the tryptophan "rotameric switch" on helix VI in which a phenylalanine on helix V positions the tryptophan out of the ligand binding pocket.

  4. The prognostic value of epidermal growth factor receptor mRNA expression in primary ovarian cancer.

    PubMed Central

    Bartlett, J. M.; Langdon, S. P.; Simpson, B. J.; Stewart, M.; Katsaros, D.; Sismondi, P.; Love, S.; Scott, W. N.; Williams, A. R.; Lessells, A. M.; Macleod, K. G.; Smyth, J. F.; Miller, W. R.

    1996-01-01

    The expression of mRNA for the epidermal growth factor (EGF) receptor, EGF and transforming growth factor alpha (TGF-alpha) was determined in 76 malignant, six borderline and 15 benign primary ovarian tumours using the reverse transcriptase-polymerase chain reaction and related to clinical and pathological parameters. Of the malignant tumours, 70% (53/76) expressed EGF receptor mRNA, 31% (23/75) expressed EGF mRNA and 35% (26/75) expressed TGF-alpha mRNA. For the borderline tumours, four of six (67%) expressed EGF receptor mRNA, 1/6 (17%) expressed TGF-alpha mRNA and none expressed EGF mRNA. Finally, 33% (5/15) of the benign tumours expressed EGF receptor mRNA, whereas 40% (6/15) expressed EGF mRNA and 7% (1/15) expressed TGF-alpha mRNA. The presence of the EGF receptor in malignant tumours was associated with that of TGF-alpha (P = 0.0015) but not with EGF (P = 1.00), whereas there was no relationship between the presence of EGF and TGF-alpha (P = 1.00). EGF receptor mRNA expression was significantly and positively associated with serous histology (P = 0.006) but not with stage or grade. Neither EGF nor TGF-alpha showed any link with histological subtype or stage. The survival of patients with malignant tumours possessing EGF receptor mRNA was significantly reduced compared with that of patients whose tumours were negative (P = 0.030 for all malignant tumours; P = 0.007 for malignant epithelial tumours only). In contrast, neither the expression of TGF-alpha nor EGF was related to survival. These data suggest that the presence of EGF receptor mRNA is associated with poor prognosis in primary ovarian cancer. Images Figure 1 PMID:8562334

  5. 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

  6. Regulation of LH/FSH expression by secretoglobin 3A2 in the mouse pituitary gland.

    PubMed

    Miyano, Yuki; Tahara, Shigeyuki; Sakata, Ichiro; Sakai, Takafumi; Abe, Hiroyuki; Kimura, Shioko; Kurotani, Reiko

    2014-04-01

    Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPβ, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.

  7. Dopamine D2 receptor expression in the corticotroph cells of the human normal pituitary gland.

    PubMed

    Pivonello, Rosario; Waaijers, Marlijn; Kros, Johan M; Pivonello, Claudia; de Angelis, Cristina; Cozzolino, Alessia; Colao, Annamaria; Lamberts, Steven W J; Hofland, Leo J

    2017-08-01

    The dopamine D 2 receptor is the main dopamine receptor expressed in the human normal pituitary gland. The aim of the current study was to evaluate dopamine D 2 receptor expression in the corticotroph cell populations of the anterior lobe and pars intermedia, as well as posterior lobe of the human normal pituitary gland by immunohistochemistry. Human normal pituitary gland samples obtained from routine autopsies were used for the study. In all cases, histology together with immunostaining for adrenocorticotropic hormone, melanocyte-stimulating hormone, prolactin, and neurofilaments were performed and compared to the immunostaining for D 2 receptor. D 2 receptor was heterogeneously expressed in the majority of the cell populations of the anterior and posterior lobe as well as in the area localized between the anterior and posterior lobe, and arbitrary defined as "intermediate zone". This zone, characterized by the presence of nerve fibers included the residual pars intermedia represented by the colloid-filled cysts lined by the remnant melanotroph cells strongly expressing D 2 receptors, and clusters of corticotroph cells, belonging to the anterior lobe but localized within the cysts and adjacent to the posterior lobe, variably expressing D 2 receptors. D 2 dopamine receptor is expressed in the majority of the cell populations of the human normal pituitary gland, and particularly, in the different corticotroph cell populations localized in the anterior lobe and the intermediate zone of the pituitary gland.

  8. Astrocytic Lrp4 (Low-Density Lipoprotein Receptor-Related Protein 4) Contributes to Ischemia-Induced Brain Injury by Regulating ATP Release and Adenosine-A2AR (Adenosine A2A Receptor) Signaling.

    PubMed

    Ye, Xin-Chun; Hu, Jin-Xia; Li, Lei; Li, Qiang; Tang, Fu-Lei; Lin, Sen; Sun, Dong; Sun, Xiang-Dong; Cui, Gui-Yun; Mei, Lin; Xiong, Wen-Cheng

    2018-01-01

    Lrp4 (low-density lipoprotein receptor-related protein 4) is predominantly expressed in astrocytes, where it regulates glutamatergic neurotransmission by suppressing ATP release. Here, we investigated Lrp4's function in ischemia/stroke-induced brain injury response, which includes glutamate-induced neuronal death and reactive astrogliosis. The brain-specific Lrp4 conditional knockout mice (Lrp4 GFAP-Cre ), astrocytic-specific Lrp4 conditional knockout mice (Lrp4 GFAP-creER ), and their control mice (Lrp4 f/f ) were subjected to photothrombotic ischemia and the transient middle cerebral artery occlusion. After ischemia/stroke, mice or their brain samples were subjected to behavior tests, brain histology, immunofluorescence staining, Western blot, and quantitative real-time polymerase chain reaction. In addition, primary astrocytes and neurons were cocultured with or without oxygen and glucose deprivation and in the presence or absence of the antagonist for adenosine-A 2A R (adenosine A2A receptor) or ATP-P2X7R (P2X purinoceptor 7) signaling. Gliotransmitters, such as glutamate, d-serine, ATP, and adenosine, in the condition medium of cultured astrocytes were also measured. Lrp4, largely expressed in astrocytes, was increased in response to ischemia/stroke. Both Lrp4 GFAP-Cre and Lrp4 GFAP-creER mice showed less brain injury, including reduced neuronal death, and impaired reactive astrogliosis. Mechanistically, Lrp4 conditional knockout in astrocytes increased ATP release and the production of ATP derivative, adenosine, which were further elevated by oxygen and glucose deprivation. Pharmacological inhibition of ATP-P 2 X 7 R or adenosine-A 2A R signaling diminished Lrp4 GFAP-creER 's protective effect. The astrocytic Lrp4 plays an important role in ischemic brain injury response. Lrp4 deficiency in astrocytes seems to be protective in response to ischemic brain injury, likely because of the increased ATP release and adenosine-A 2A R signaling. © 2017 American Heart

  9. Changes in the expression of SRBC receptors on human lymphocytes caused by azathioprine and puromycine.

    PubMed

    Drela, N

    1984-01-01

    The expression of human peripheral T lymphocyte receptors for sheep red blood cells in vitro was investigated. Changes in the expression of SRBC receptors were analysed in successive culture days depending on the stimulation of lymphocytes by PHA or Con A and the action of immunosuppressive agents, such as azathioprine and puromycine. The SRBC receptor variability of expression in cultures of whole peripheral blood lymphocytes was compared with that of T lymphocytes subpopulation isolated by the rosetting method. The experiments demonstrated: a) the appearance of different morphological E rosette types in cultures stimulated by plant mitogens, b) the inhibition of SRBC receptor expression by azathioprine and puromycine on lymphocytes from stimulated and non-stimulated cultures, c) higher sensitivity of SRBC receptors to immunosuppressive agents on cultured isolated T lymphocytes, d) the difference of azathioprine and puromycine action on the ability to form all types of E rosettes in stimulated and nonstimulated cultures.

  10. Beta-Adrenergic Receptor Expression in Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, K.; Vaughn, J. R.

    1999-01-01

    beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

  11. Molecular cloning and function expression of a diuretic hormone receptor from the house cricket, Acheta domesticus.

    PubMed

    Reagan, J D

    1996-01-01

    Insect diuretic hormones regulate fluid and ion secretion and the receptors with which they interact are attractive targets for new insect control agents. Recently, a diuretic hormone receptor from the moth Manduca sexta was isolated by expression cloning and found to be a member of the calcitonin/secretin/corticotropin releasing factor family of G-protein coupled receptors [Reagan J. D. (1994) J. Biol. Chem. 269, 9-12]. Degenerate oligonucleotides were designed based upon conserved regions in this receptor family and used to isolate a diuretic hormone receptor from the house cricket, Acheta domesticus. The complementary DNA isolated encodes a protein consisting of 441 amino acids with seven putative membrane spanning regions. Interestingly, unlike the M. sexta diuretic hormone receptor, the cricket diuretic hormone receptor contains a putative signal sequence. The receptor shares 53% and 38% sequence identity with the M. sexta diuretic hormone and human corticotropin releasing factor receptors respectively. When expressed in COS-7 cells, the receptor binds A. domesticus diuretic hormone with high affinity and stimulates adenylate cyclase with high potency. Four other insect diuretic hormones are considerably less effective at stimulating adenylate cyclase in COS-7 cells transfected with the receptor. This is in contrast to the M. sexta diuretic hormone receptor which is stimulated by all five insect diuretic hormones with high potency.

  12. Reduced response to the formalin test and lowered spinal NMDA glutamate receptor binding in adenosine A2A receptor knockout mice.

    PubMed

    Hussey, Martin J; Clarke, Geoffrey D; Ledent, Catherine; Hourani, Susanna M O; Kitchen, Ian

    2007-06-01

    Adenosine is a neuromodulator with complex effects on pain pathways. Mice lacking the adenosine A2A receptor are hypoalgesic, and have altered analgesic responses to receptor-selective opioid agonists. These and other findings suggest a role for the adenosine A2A receptor in sensitizing afferent fibres projecting to the spinal cord. To test this hypothesis formalin (20 microl, 5%) was injected into the paw and nociceptive responses were measured in wildtype and adenosine A2A receptor knockout mice. There was a significant reduction in nociception associated with sensory nerve activation in the knockout mice as measured by time spent biting/licking the formalin-injected paw and number of flinches seen during the first phase, but only the number of flinches was reduced during the second inflammatory phase. In addition, the selective adenosine A2A antagonist SCH58261 (3 and 10 mg/kg) also antagonised both phases of the formalin test. We also labelled NMDA glutamate and NK1 receptors in spinal cord sections as an indirect measure of nociceptive transmission from peripheral sites to the spinal cord. [3H]-Substance P binding to NK1 receptors was unaltered but there was a substantial reduction in binding of [3H]-MK801 to NMDA glutamate receptors in all regions of the spinal cord from knockout mice. The decrease in NMDA glutamate receptor binding may reflect reduced peripheral sensory input to the spinal cord during development and could relate to the hypoalgesia in this genotype. These results support a key role for the adenosine A2A receptor in peripheral nociceptive pathways.

  13. Thromboxane A2 receptor antagonist SQ29548 attenuates SH‑SY5Y neuroblastoma cell impairments induced by oxidative stress.

    PubMed

    Cai, Gaoyu; Yan, Aijuan; Fu, Ningzhen; Fu, Yi

    2018-03-27

    Thromboxane A2 receptor (TXA2R) serves a vital role in numerous neurological disorders. Our previous study indicated that SQ29548, an antagonist of TXA2R, attenuated the induced neuron damage in cerebral infarction animals; however, the underlying mechanism remains unknown. Certain studies revealed a new role of TXA2R in the regulation of oxidative stress, which is one of the basic pathological processes in neurological disorders. Thus, the present study attempted to examine whether the inhibition of TXA2R with SQ29548 helped to protect the nerve cells against oxidative stress. SQ29548 was utilized as a TXA2R antagonist, and relevant assays were performed to detect the cell viability, cellular reactive oxygen species (ROS) level, cell apoptosis, expression levels of superoxide dismutase‑2 (SOD2), catalase and caspases, and activation of mitogen‑activated protein kinase (MAPK) pathways. It was observed that hydrogen peroxide (H2O2) dose‑dependently reduced the viability of SH‑SY5Y cells. In addition, H2O2 raised the level of ROS in cells, inhibited the expression levels of SOD2 and catalase, and potentially enhanced cell apoptosis and the expression of caspases via activating the MAPK pathways. Pretreatment with SQ29548 not only rescued the viability of SH‑SY5Y cells, but also ameliorated the intracellular ROS level and the expression levels of SOD2 and catalase. Furthermore, it decreased the cell apoptosis and the expression of caspases, possibly via the inhibition of MAPK pathways. In conclusion, SQ29548, an antagonist of TXA2R, improved the antioxidant capacities of SH‑SY5Y cells and reduced the cell apoptosis through the inhibition of MAPK pathways.

  14. Sympathetic activity relates to adenosine A(2A) receptor gene variation in blood-injury phobia.

    PubMed

    Hohoff, C; Domschke, K; Schwarte, K; Spellmeyer, G; Vögele, C; Hetzel, G; Deckert, J; Gerlach, A L

    2009-06-01

    Variation in the candidate genes adenosine A(2A) receptor (A(2A)R), catechol-O-methyl-transferase (COMT), and norepinephrine transporter (NET) has been suggested to influence vulnerability to panic disorder. We therefore investigated patients with another anxiety disorder with an even higher heritability, the blood-injury phobia, for association of these variants and used sympathetic measures during venipuncture, which serve as a naturalistic trigger of anxiety and autonomic hyperarousal, as an intermediate phenotype of anxiety. Patients homozygous for the A(2A)R 1976T allele as compared to patients carrying at least one 1976C allele exhibited a significantly increased respiratory rate with a trend towards elevated measures of systolic and diastolic blood pressure and respiratory minute volume. None of the sympathetic measures were influenced by the COMT or NET polymorphisms.This study provides preliminary data suggesting an influence of the A(2A)R 1976C/T polymorphism on sympathetic psychophysiological indicators of anxiety-related arousal in blood-injury phobia and thereby further supports a role of the A(2A)R gene in the pathogenesis of anxiety disorders.

  15. Amino acid conjugates of lithocholic acid as antagonists of the EphA2 receptor

    PubMed Central

    Incerti, Matteo; Tognolini, Massimiliano; Russo, Simonetta; Pala, Daniele; Giorgio, Carmine; Hassan-Mohamed, Iftiin; Noberini, Roberta; Pasquale, Elena B.; Vicini, Paola; Piersanti, Silvia; Rivara, Silvia; Barocelli, Elisabetta; Mor, Marco; Lodola, Alessio

    2013-01-01

    The Eph receptor–ephrin system is an emerging target for the development of novel antiangiogenetic agents. We recently identified lithocholic acid (LCA) as a small molecule able to block EphA2-dependent signals in cancer cells, suggesting that its (5β)-cholan-24-oic acid scaffold can be used as a template to design a new generation of improved EphA2 antagonists. Here, we report the design and synthesis of an extended set of LCA derivatives obtained by conjugation of its carboxyl group with different α-amino acids. Structure-activity relationships indicate that the presence of a lipophilic amino acid side chain is fundamental to achieve good potencies. The L-Trp derivative (20, PCM126) was the most potent antagonist of the series disrupting EphA2-ephrinA1 interaction and blocking EphA2 phosphorylation in prostate cancer cells at low μM concentrations, thus being significantly more potent than LCA. Compound 20 is among the most potent small molecule antagonists of the EphA2 receptor. PMID:23489211

  16. Novel targeted system to deliver chemotherapeutic drugs to EphA2-expressing cancer cells.

    PubMed

    Wang, Si; Placzek, William J; Stebbins, John L; Mitra, Sayantan; Noberini, Roberta; Koolpe, Mitchell; Zhang, Ziming; Dahl, Russell; Pasquale, Elena B; Pellecchia, Maurizio

    2012-03-08

    The efficacy of anticancer drugs is often limited by their systemic toxicities and adverse side effects. We report that the EphA2 receptor is overexpressed preferentially in several human cancer cell lines compared to normal tissues and that an EphA2 targeting peptide (YSAYPDSVPMMS) can be effective in delivering anticancer agents to such tumors. Hence, we report on the synthesis and characterizations of a novel EphA2-targeting agent conjugated with the chemotherapeutic drug paclitaxel. We found that the peptide-drug conjugate is dramatically more effective than paclitaxel alone at inhibiting tumor growth in a prostate cancer xenograft model, delivering significantly higher levels of drug to the tumor site. We believe these studies open the way to the development of a new class of therapeutic compounds that exploit the EphA2 receptor for drug delivery to cancer cells.

  17. GDNF control of the glutamatergic cortico-striatal pathway requires tonic activation of adenosine A2A Receptors

    PubMed Central

    Gomes, Catarina A.R.V.; Simões, Patrícia F.; Canas, Paula M.; Quiroz, César; Sebastião, Ana M.; Ferré, Sergi; Cunha, Rodrigo A.; Ribeiro, Joaquim A.

    2009-01-01

    Glial cell line-derived neurotrophic factor (GDNF) affords neuroprotection in Parkinson’s disease in accordance with its ability to bolster nigrostriatal innervation. We previously found that GDNF facilitates dopamine release in a manner dependent on adenosine A2A receptor activation. Since motor dysfunction also involves modifications of striatal glutamatergic innervation, we now tested if GDNF and its receptor system, Ret (rearranged during transfection) and GFRα1 (GDNF family receptor alpha 1) controlled the cortico-striatal glutamatergic pathway in an A2A receptor-dependent manner. GDNF (10 ng/ml) enhanced (by ≈13%) glutamate release from rat striatal nerve endings, an effect potentiated (up to ≈ 30%) by the A2A receptor agonist CGS 21680 (10 nM) and prevented by the A2A receptor antagonist, SCH 58261 (50 nM). Triple immunocytochemical studies revealed that Ret and GFRα1 were located in 50% of rat striatal glutamatergic terminals (immunopositive for vesicular glutamate transporters-1/2), where they were found to be co-located with A2A receptors. Activation of the glutamatergic system upon in vivo electrical stimulation of the rat cortico-striatal input induced striatal Ret phosphoprylation that was prevented by pre-treatment with the A2A receptor antagonist, MSX-3 (3 mg/kg). The results provide the first functional and morphological evidence that GDNF controls cortico-striatal glutamatergic pathways in a manner largely dependent on the co-activation of adenosine A2A receptors. PMID:19141075

  18. [Peptide receptor radionuclide therapy for patients with somatostatin receptor expressing tumours. German Guideline (S1)].

    PubMed

    Poeppel, T D; Boy, C; Bockisch, A; Kotzerke, J; Buchmann, I; Ezziddin, S; Scheidhauer, K; Krause, B J; Schmidt, D; Amthauer, H; Rösch, F; Nagarajah, J; Führer, D; Lahner, H; Pöpperl, G; Hörsch, D; Walter, M A; Baum, R P

    2015-01-01

    This document describes the guideline for peptide receptor radionuclide therapy (PRRT) published by the German Society of Nuclear Medicine (DGN) and accepted by the Association of the Scientific Medical Societies in Germany (AWMF) to be included in the official AWMF Guideline Registry. These recommendations are a prerequisite for the quality management in the treatment of patients with somatostatin receptor expressing tumours using PRRT. They are aimed at guiding nuclear medicine specialists in selecting likely candidates to receive PRRT and to deliver the treatment in a safe and effective manner. The recommendations are based on an interdisciplinary consensus. The document contains background information and definitions and covers the rationale, indications and contraindications for PRRT. Essential topics are the requirements for institutions performing the therapy, e. g. presence of an expert for medical physics, intense cooperation with all colleagues involved in the treatment of a patient, and a certificate of instruction in radiochemical labelling and quality control are required. Furthermore, it is specified which patient data have to be available prior to performance of therapy and how treatment has to be carried out technically. Here, quality control and documentation of labelling are of great importance. After treatment, clinical quality control is mandatory (work-up of therapy data and follow-up of patients). Essential elements of follow-up are specified in detail. The complete treatment inclusive after-care has to be realised in close cooperation with the involved medical disciplines. Generally, the decision for PRRT should be undertaken within the framework of a multi-disciplinary tumour board.

  19. Adenosine A2A Receptor Deletion Blocks the Beneficial Effects of Lactobacillus reuteri in Regulatory T-Deficient Scurfy Mice

    PubMed Central

    He, Baokun; Hoang, Thomas K.; Tran, Dat Q.; Rhoads, Jon Marc; Liu, Yuying

    2017-01-01

    The lack of a functional Foxp3 transcription factor and regulatory T (Treg) cells causes lethal, CD4+ T cell-driven autoimmune diseases in scurfy (SF) mice and humans. Recent studies have shown that adenosine A2A receptor activation limits inflammation and tissue damage, thereby playing an anti-inflammatory role. However, the role of the adenosine A2A receptor in the development of disease in SF mice remains unclear. Using a genetic approach, we found that adenosine A2A receptor deletion in SF mice (SF⋅A2A-/-) does not affect early life events, the development of a lymphoproliferative disorder, or hyper-production of pro-inflammatory cytokines seen in the Treg-deficiency state. As shown previously, Lactobacillus reuteri DSM 17938 treatment prolonged survival and reduced multiorgan inflammation in SF mice. In marked contrast, A2A receptor deletion completely blocked these beneficial effects of L. reuteri in SF mice. Altogether, these results suggest that although absence of the adenosine A2A receptor does not affect the development of disease in SF mice, it plays a critical role in the immunomodulation by L. reuteri in Treg-deficiency disease. The adenosine A2A receptor and its activation may have a role in treating other Treg dysfunction-mediated autoimmune diseases. PMID:29270168

  20. CB1 Cannabinoid Receptor Expression in the Striatum: Association with Corticostriatal Circuits and Developmental Regulation

    PubMed Central

    Van Waes, Vincent; Beverley, Joel A.; Siman, Homayoun; Tseng, Kuei Y.; Steiner, Heinz

    2012-01-01

    Corticostriatal circuits mediate various aspects of goal-directed behavior and are critically important for basal ganglia-related disorders. Activity in these circuits is regulated by the endocannabinoid system via stimulation of CB1 cannabinoid receptors. CB1 receptors are highly expressed in projection neurons and select interneurons of the striatum, but expression levels vary considerably between different striatal regions (functional domains). We investigated CB1 receptor expression within specific corticostriatal circuits by mapping CB1 mRNA levels in striatal sectors defined by their cortical inputs in rats. We also assessed changes in CB1 expression in the striatum during development. Our results show that CB1 expression is highest in juveniles (P25) and then progressively decreases toward adolescent (P40) and adult (P70) levels. At every age, CB1 receptors are predominantly expressed in sensorimotor striatal sectors, with considerably lower expression in associative and limbic sectors. Moreover, for most corticostriatal circuits there is an inverse relationship between cortical and striatal expression levels. Thus, striatal sectors with high CB1 expression (sensorimotor sectors) tend to receive inputs from cortical areas with low expression, while striatal sectors with low expression (associative/limbic sectors) receive inputs from cortical regions with higher expression (medial prefrontal cortex). In so far as CB1 mRNA levels reflect receptor function, our findings suggest differential CB1 signaling between different developmental stages and between sensorimotor and associative/limbic circuits. The regional distribution of CB1 receptor expression in the striatum further suggests that, in sensorimotor sectors, CB1 receptors mostly regulate GABA inputs from local axon collaterals of projection neurons, whereas in associative/limbic sectors, CB1 regulation of GABA inputs from interneurons and glutamate inputs may be more important. PMID:22416230

  1. Dopamine receptor-mediated regulation of neuronal “clock” gene expression

    PubMed Central

    Imbesi, Marta; Yildiz, Sevim; Arslan, Ahmet Dirim; Sharma, Rajiv; Manev, Hari; Uz, Tolga

    2009-01-01

    Using transgenic mice model (i.e., “clock” knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulate the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e., D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2, and mBmal1 with the D1-class (i.e., D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e., rhythm shift). Collectively, our results indicate that the DA receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e., intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  2. Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage–dependent regulation of functional receptor

    PubMed Central

    Gutknecht, Eric; Hauger, Richard L.; Linden, Ile Van der; Vauquelin, Georges; Dautzenberg, Frank M.

    2011-01-01

    Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF1-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of ~100 pmoles/105 cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of ~25–30 pmoles/105 cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the ~300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 ± 1.6 nmol/L), whereas the CRF1-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 μmol/L. When the CRF1-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 ± 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 ± 0.2 μmol/L). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2(a) receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture. PMID:17976162

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

    PubMed

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

    2015-10-15

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

  4. Epigenetic regulation of olfactory receptor gene expression by the Myb-MuvB/dREAM complex.

    PubMed

    Sim, Choon Kiat; Perry, Sarah; Tharadra, Sana Khalid; Lipsick, Joseph S; Ray, Anandasankar

    2012-11-15

    In both mammals and insects, an olfactory neuron will usually select a single olfactory receptor and repress remaining members of large receptor families. Here we show that a conserved multiprotein complex, Myb-MuvB (MMB)/dREAM, plays an important role in mediating neuron-specific expression of the carbon dioxide (CO(2)) receptor genes (Gr63a/Gr21a) in Drosophila. Activity of Myb in the complex is required for expression of Gr63a/Gr21a and acts in opposition to the histone methyltransferase Su(var)3-9. Consistent with this, we observed repressive dimethylated H3K9 modifications at the receptor gene loci, suggesting a mechanism for silencing receptor gene expression. Conversely, other complex members, Mip120 (Myb-interacting protein 120) and E2F2, are required for repression of Gr63a in inappropriate neurons. Misexpression in mutants is accompanied by an increase in the H3K4me3 mark of active chromatin at the receptor gene locus. Nuclei of CO(2) receptor-expressing neurons contain reduced levels of the repressive subunit Mip120 compared with surrounding neurons and increased levels of Myb, suggesting that activity of the complex can be regulated in a cell-specific manner. Our evidence suggests a model in which olfactory receptors are regulated epigenetically and the MMB/dREAM complex plays a critical role in specifying, maintaining, and modulating the receptor-to-neuron map.

  5. Functional pharmacology of H1 histamine receptors expressed in mouse preoptic/anterior hypothalamic neurons

    PubMed Central

    Tabarean, I V

    2013-01-01

    BACKGROUND AND PURPOSE Histamine H1 receptors are highly expressed in hypothalamic neurons and mediate histaminergic modulation of several brain-controlled physiological functions, such as sleep, feeding and thermoregulation. In spite of the fact that the mouse is used as an experimental model for studying histaminergic signalling, the pharmacological characteristics of mouse H1 receptors have not been studied. In particular, selective and potent H1 receptor agonists have not been identified. EXPERIMENTAL APPROACH Ca2+ imaging using fura-2 fluorescence signals and whole-cell patch-clamp recordings were carried out in mouse preoptic/anterior hypothalamic neurons in culture. KEY RESULTS The H1 receptor antagonists mepyramine and trans-triprolidine potently antagonized the activation by histamine of these receptors with IC50 values of 0.02 and 0.2 μM respectively. All H1 receptor agonists studied had relatively low potency at the H1 receptors expressed by these neurons. Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine had full-agonist activity with potencies similar to that of histamine. In contrast, 2-pyridylethylamine and betahistine showed only partial agonist activity and lower potency than histamine. The histamine receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide (HTMT) had no agonist activity at the H1 receptors H1 receptors expressed by mouse preoptic/anterior hypothalamic neurons but displayed antagonist activity. CONCLUSIONS AND IMPLICATIONS Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine were identified as full agonists of mouse H1 receptors. These results also indicated that histamine H1 receptors in mice exhibited a pharmacological profile in terms of agonism, significantly different from those of H1 receptors expressed in other species. PMID:23808378

  6. Functional pharmacology of H1 histamine receptors expressed in mouse preoptic/anterior hypothalamic neurons.

    PubMed

    Tabarean, I V

    2013-09-01

    Histamine H1 receptors are highly expressed in hypothalamic neurons and mediate histaminergic modulation of several brain-controlled physiological functions, such as sleep, feeding and thermoregulation. In spite of the fact that the mouse is used as an experimental model for studying histaminergic signalling, the pharmacological characteristics of mouse H1 receptors have not been studied. In particular, selective and potent H1 receptor agonists have not been identified. Ca(2+) imaging using fura-2 fluorescence signals and whole-cell patch-clamp recordings were carried out in mouse preoptic/anterior hypothalamic neurons in culture. The H1 receptor antagonists mepyramine and trans-triprolidine potently antagonized the activation by histamine of these receptors with IC50 values of 0.02 and 0.2 μM respectively. All H1 receptor agonists studied had relatively low potency at the H1 receptors expressed by these neurons. Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine had full-agonist activity with potencies similar to that of histamine. In contrast, 2-pyridylethylamine and betahistine showed only partial agonist activity and lower potency than histamine. The histamine receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide (HTMT) had no agonist activity at the H1 receptors H1 receptors expressed by mouse preoptic/anterior hypothalamic neurons but displayed antagonist activity. Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine were identified as full agonists of mouse H1 receptors. These results also indicated that histamine H1 receptors in mice exhibited a pharmacological profile in terms of agonism, significantly different from those of H1 receptors expressed in other species. © 2013 The British Pharmacological Society.

  7. The pattern of melatonin receptor expression in the brain may influence antidepressant treatment

    PubMed Central

    Hirsch-Rodriguez, Eric; Imbesi, Marta; Manev, Radmila; Uz, Tolga; Manev, Hari

    2007-01-01

    Summary The pineal hormone melatonin produces most of its biological effects via G protein-coupled receptors MT1 and MT2. In mammals, these receptors are expressed in various tissues and organs including in the brain. Recent research points to a putative role of MT1/MT2 dimerization as a mechanism that could determine the receptor-mediated biological effects of melatonin. Brain content and the ratios between MT1 and MT2 receptors are affected by illness, e.g., Alzheimer's disease, and by prolonged drug treatment, e.g., antidepressants. New drugs with antidepressant properties that bind and activate melatonin receptors have been discovered. We hypothesize that endogenous, i.e., low, levels of melatonin could contribute to antidepressant effects depending on the expression pattern of melatonin receptors in the brain. Hence, we propose that a prolonged treatment with classical antidepressant drugs alters the brain ratio of MT1/MT2 receptors to enable the endogenous melatonin, which is secreted during the night, to further improve the antidepressant effects. A corollary of this hypothesis is that antidepressants would be less effective in conditions of pathologically altered brain melatonin receptors, e.g., in Alzheimer's patients or due to genetic polymorphisms. If our hypothesis is confirmed, supplementing classical antidepressant treatment with an appropriate dose of a melatonin receptor agonist might be used to improve antidepressant effects in subjects with a susceptible pattern of brain melatonin receptor expression. PMID:17197111

  8. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    PubMed

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands. © 2015 John Wiley & Sons A/S.

  9. Embryonic expression of the transforming growth factor beta ligand and receptor genes in chicken.

    PubMed

    Cooley, James R; Yatskievych, Tatiana A; Antin, Parker B

    2014-03-01

    Transforming growth factor-beta (TGFβ) signaling regulates a myriad of biological processes during embryogenesis, in the adult, and during the manifestation of disease. TGFβ signaling is propagated through one of three TGFβ ligands interacting with Type I and Type II receptors, and Type III co-receptors. Although TGFβ signaling is regulated partly by the combinatorial expression patterns of TGFβ receptors and ligands, a comprehensive gene expression analysis has not been published. Here we report the embryonic mRNA expression patterns in chicken embryos of the canonical TGFβ ligands (TGFB1, TGFB2, and TGFB3) and receptors (TGFBR1, TGFBR2, TGFBR3), plus the Activin A receptor, type 1 (ACVR1) and co receptor Endoglin (ENG) that also transduce TGFβ signaling. TGFB ligands and receptors show dynamic and frequently overlapping expression patterns in numerous embryonic cell layers and structures. Integrating expression information identifies combinations of ligands and receptors that are involved in specific developmental processes including somitogenesis, cardiogenesis and vasculogenesis. Copyright © 2013 Wiley Periodicals, Inc.

  10. Glucagon like peptide-1 receptor expression in the human thyroid gland.

    PubMed

    Gier, Belinda; Butler, Peter C; Lai, Chi K; Kirakossian, David; DeNicola, Matthew M; Yeh, Michael W

    2012-01-01

    Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted.

  11. Histamine H1 receptors are expressed in mouse and frog semicircular canal sensory epithelia.

    PubMed

    Botta, Laura; Tritto, Simona; Perin, Paola; Laforenza, Umberto; Gastaldi, Giulia; Zampini, Valeria; Zucca, Gianpiero; Valli, Stefano; Masetto, Sergio; Valli, Paolo

    2008-03-05

    Histamine-related drugs are commonly used in the treatment of vertigo and related vestibular disorders. Their site and mechanism of action, however, are still poorly understood. To increase our knowledge of the histaminergic system in the vestibular organs, we have investigated the expression of H1 and H3 histamine receptors in the frog and mouse semicircular canal sensory epithelia. Analysis was performed by mRNA reverse transcriptase-PCR, immunoblotting and immunocytochemistry experiments. Our data show that both frog and mouse vestibular epithelia express H1 receptors. Conversely no clear evidence for H3 receptors expression was found.

  12. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ.

    PubMed

    Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik

    2015-01-01

    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

  13. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ

    PubMed Central

    Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik

    2015-01-01

    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

  14. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2.

    PubMed

    Li, Qun; Laumonnier, Yves; Syrovets, Tatiana; Simmet, Thomas

    2011-11-01

    To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×10(7) clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating protein interactions.

  15. Yeast two-hybrid screening of proteins interacting with plasmin receptor subunit: C-terminal fragment of annexin A2

    PubMed Central

    Li, Qun; Laumonnier, Yves; Syrovets, Tatiana; Simmet, Thomas

    2011-01-01

    Aim: To identify proteins that interact with the C-terminal fragment of annexin A2 (A2IC), generated by plasmin cleavage of the plasmin receptor, a heterotetramer (AA2t) containing annexin A2. Methods: The gene that encodes the A2IC fragment was obtained from PCR-amplified cDNA isolated from human monocytes, and was ligated into the pBTM116 vector using a DNA ligation kit. The resultant plasmid (pBTM116-A2IC) was sequenced with an ABI PRISM 310 Genetic Analyzer. The expression of an A2IC bait protein fused with a LexA-DNA binding domain (BD) was determined using Western blot analysis. The identification of proteins that interact with A2IC and are encoded in a human monocyte cDNA library was performed using yeast two-hybrid screening. The DNA sequences of the relevant cDNAs were determined using an ABI PRISM BigDye terminator cycle sequencing ready reaction kit. Nucleotide sequence databases were searched for homologous sequences using BLAST search analysis (http://www.ncbi.nlm.nih.gov). Confirmation of the interaction between the protein LexA-A2IC and each of cathepsin S and SNX17 was conducted using a small-scale yeast transformation and X-gal assay. Results: The yeast transformed with plasmids encoding the bait proteins were screened with a human monocyte cDNA library by reconstituting full-length transcription factors containing the GAL4-active domain (GAL4-AD) as the prey in a yeast two-hybrid approach. After screening 1×107 clones, 23 independent β-Gal-positive clones were identified. Sequence analysis and a database search revealed that 15 of these positive clones matched eight different proteins (SNX17, ProCathepsin S, RPS2, ZBTB4, OGDH, CCDC32, PAPD4, and actin which was already known to interact with annexin A2). Conclusion: A2IC A2IC interacts with various proteins to form protein complexes, which may contribute to the molecular mechanism of monocyte activation induced by plasmin. The yeast two-hybrid system is an efficient approach for investigating

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

    PubMed Central

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

    2010-01-01

    Several recent studies suggest functional and molecular interactions between striatal adenosine A2A and cannabinoid CB1 receptors. Here we demonstrate that A2A receptors selectively modulate reinforcing effects of cannabinoids. We studied effects of A2A receptor blockade on the reinforcing effects of delta-9-tetrahydrocannabinol (THC) and the endogenous CB1 receptor ligand anandamide under a fixed-ratio (FR) schedule of intravenous drug injection in squirrel monkeys. A low dose of the selective adenosine A2A 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 A2A antagonists acting preferentially at presynaptic A2A 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 A2A antagonists with more selectivity for presynaptic versus postsynaptic receptors could be potential medications for treatment of cannabis abuse. PMID:21054689

  17. Toll-like receptor 2 stimulation decreases IFN-gamma receptor expression in mouse RAW264.7 macrophages.

    PubMed

    Curry, Heather; Alvarez, Gail R; Zwilling, Bruces S; Lafuse, William P

    2004-12-01

    Interferon-gamma (IFN-gamma) is a key cytokine in the immune defense against mycobacteria. IFN-gamma activates macrophages to resist the growth of mycobacteria and induces expression of MHC class II molecules required for antigen presentation. Macrophages infected with mycobacteria or stimulated by the interaction of mycobacterial products with toll-like receptor 2 (TLR2) have reduced responses to IFN-gamma. Previous research has shown that infection of mouse macrophages with Mycobacterium avium causes decreased expression of the IFN-gamma receptor (IFNGR). In the present study, we show that TLR2 stimulation of RAW264.7 macrophages with a synthetic lipoprotein, Pam3CSK4, also causes rapid decrease in expression of IFNGR-1 protein, with little change in IFNGR-2 protein levels. The decrease in IFNGR-2 expression in TLR2-stimulated cells required receptor internalization and proteasomal degradation. The level of IFNGR-1 mRNA also decreased in TLR2-stimulated RAW264.7 cells and M. avium-infected cells. The decrease in IFNGR-1 mRNA was shown to be due to decreased transcription. In spite of the decrease in IFNGR-2 receptor expression, activation of Stat1 activation by an optimal dose of IFN-gamma was identical between control and TLR2-stimulated RAW264.7 cells. However, at low suboptimal doses of IFN-gamma, Stat1 activation was decreased in TLR2-stimulated cells.

  18. 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

  19. Effects of chemotherapy agents on Sphingosine-1-Phosphate receptors expression in MCF-7 mammary cancer cells.

    PubMed

    Ghosal, P; Sukocheva, O A; Wang, T; Mayne, G C; Watson, D I; Hussey, D J

    2016-07-01

    Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of cell proliferation and cancer progression. Increased expression of S1P receptors has been detected in advanced breast tumours with poor prognosis suggesting that S1P receptors might control tumour response to chemotherapy. However, it remains unclear how the levels of S1P receptor expression are influenced by chemotherapy agents. Western immunoblotting, PCR analysis and fluorescent microscopy techniques were used in this study to analyze expression patterns of S1P receptors 2 and 3 (S1P2/S1P3) in MCF-7 breast adenocarcinoma cells treated by Tamoxifen (TAM) and/or Medroxyprogesterone acetate (MPA). We found that TAM/MPA induce downregulation of S1P3 receptors, but stimulate expression of S1P2. According to cell viability and caspase activity analyses, as expected, TAM activated apoptosis. We also detected TAM/MPA-induced autophagy marked by formation of macroautophagosomes and increased level of Beclin 1. Combined application of TAM and MPA resulted in synergistic apoptosis- and autophagy-stimulating effects. Assessed by fluorescent microscopy with autophagosome marker LAMP-2, changes in S1P receptor expression coincided with activation of autophagy, suggestively, directing breast cancer cells towards death. Further studies are warranted to explore the utility of manipulation of S1P2 and S1P3 receptor expression as a novel treatment approach. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  20. Intratumoral Heterogeneity for Expression of Tyrosine Kinase Growth Factor Receptors in Human Colon Cancer Surgical Specimens and Orthotopic Tumors

    PubMed Central

    Kuwai, Toshio; Nakamura, Toru; Kim, Sun-Jin; Sasaki, Takamitsu; Kitadai, Yasuhiko; Langley, Robert R.; Fan, Dominic; Hamilton, Stanley R.; Fidler, Isaiah J.

    2008-01-01

    The design of targeted therapy, particularly patient-specific targeted therapy, requires knowledge of the presence and intratumoral distribution of tyrosine kinase receptors. To determine whether the expression of such receptors is constant or varies between and within individual colon cancer neoplasms, we examined the pattern of expression of the ligands, epidermal growth factor, vascular endothelial growth factor, and platelet-derived growth factor-B as well as their respective receptors in human colon cancer surgical specimens and orthotopic human colon cancers growing in the cecal wall of nude mice. The expression of the epidermal growth factor receptor and the vascular endothelial growth factor receptor on tumor cells and stromal cells, including tumor-associated endothelial cells, was heterogeneous in surgical specimens and orthotopic tumors. In some tumors, the receptor was expressed on both tumor cells and stromal cells, and in other tumors the receptor was expressed only on tumor cells or only on stromal cells. In contrast, the platelet-derived growth factor receptor was expressed only on stromal cells in both surgical specimens and orthotopic tumors. Examination of receptor expression in both individual surgical specimens and orthotopic tumors revealed that the platelet-derived growth factor receptor was expressed only on stromal cells and that the patterns of epidermal growth factor receptor and vascular endothelial growth factor receptor 2 expression differed between tumor cells. This heterogeneity in receptor expression among different tumor cells suggests that targeting a single tyrosine kinase may not yield eradication of the disease. PMID:18202197

  1. Expression and physiology of opioid receptors in the gastrointestinal tract.

    PubMed

    Mosińska, Paula; Zielińska, Marta; Fichna, Jakub

    2016-02-01

    Stimulation of opioid receptors elicits analgesic effect not only in the central nervous system, but also in the gastrointestinal tract, where a high concentration of opioid receptors can be found within the enteric nervous system as well as muscular and immune cells. Along with antinociception, opioid receptors in the stomach and intestine relay signals crucial for secretory and motor gastrointestinal function. The review focuses on the utility of opioid receptor antagonists, which is generally contributing to the management of postoperative ileus and opioid bowel dysfunction in chronic pain patients nonetheless, opioid receptor antagonists can also be useful in the treatment of irritable bowel syndrome and chronic idiopathic constipation. The study also discusses several antidiarrheal opioid agonists, as well as opioids and opioid mimetics encompassing the concept of ligand-biased agonism and truncated opioid receptor splice variants. Good understanding of the localization and the role of opioid receptors is vital for regulation of various pathophysiological processes in the gastrointestinal tract and may simultaneously provide a tempting approach in eliminating adverse effects related to centrally acting opioids.

  2. Ligand-dependent cholesterol interactions with the human A2A adenosine receptor

    PubMed Central

    Lee, Ji Young; Patel, Rohan; Lyman, Edward

    2013-01-01

    We present nearly ten microseconds of all-atom simulation data of a G-protein coupled receptor, the human A2A adenosine receptor, bound to four different ligands. Our focus is on binding of cholesterol to the “cholesterol consensus motif,” a cluster of five amino acids on the second and fourth transmembrane helices, which interact with two cholesterols in the intracellular leaflet of the bilayer. We find evidence for a ligand-specific interaction between the CCM and cholesterol, mediated by the rotameric dynamics and configuration of Trp129. Binding of the synthetic agonist UK432097 disrupts hydrogen bonding between Trp129 and Ser47, which activates the rotameric dynamics of Trp129 and disrupts the interaction with one of the two cholesterols. We also investigate the effect of four thermostabilizing mutations, three of which are located on helix two. The conformational stability of helix two has been proposed to be sensitive to interaction with cholesterol in the CCM, suggesting a mechanism for the thermostabilization. However, our data are instead suggestive of a force-field dependent “straightening” of helix two, and therefore offer no basis for rationalizing the effect of the quadruple mutant. PMID:23454349

  3. Identification of Two New Cholesterol Interaction Sites on the A2AAdenosine Receptor.

    PubMed

    Rouviere, Eric; Arnarez, Clément; Yang, Lewen; Lyman, Edward

    2017-12-05

    By mole, cholesterol is the most abundant component of animal cell plasma membranes. Many membrane proteins have been shown to be functionally dependent on cholesterol, several of which have also been shown to bind cholesterol at well-defined locations on their membrane-facing surface. In this work, a combination of coarse-grained "Martini" and all-atom simulations are used to identify two, to our knowledge, new cholesterol-binding sites on the A 2A adenosine receptor, a G-protein-coupled receptor that is a target for the treatment of Parkinson's disease. One of the sites is also observed to bind cholesterol in several recent, high-resolution crystal structures of the protein, and in the simulations, interacts with cholesterol only when bound to the inverse agonist ZM241385. Cataloguing cholesterol-binding sites is a vital step in the effort to understand cholesterol-dependent function of membrane proteins. Given that cholesterol content in plasma membranes varies with cell type and on administration of widely prescribed pharmaceuticals, such as statins, understanding cholesterol-dependent function is an important step toward exploiting membrane compositional variation for therapeutic purposes. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  4. Overexpression of Annexin A2 Receptor Inhibits Neovascularization via the Promotion of Krüppel-Like Transcription Factor 2.

    PubMed

    Guo, Ting; Song, Hongyuan; Zhao, Zichang; Qi, Zhongtian; Zhao, Shihong

    2018-04-24

    Annexin A2 receptor (AX2R) can mediate annexin A2 signalling and induce apoptosis in a variety of cells, but its role in neovascularization (NV) remains unclear. Krüppel-like transcription factor 2 (KLF2) is known to be expressed in a range of cell types and to participate in a number of processes during development and disease, such as endothelial homeostasis, vasoregulation and vascular growth/remodelling. The aim of our study was to investigate the role of AX2R in NV and the plausible molecular mechanism. We constructed a eukaryotic overexpression plasmid for AX2R (Lenti-AX2R) by using polymerase chain reaction (PCR). The full-length human AX2R gene was transfected into human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) using lentivirus vectors to overexpress AX2R. All experiments were divided into three groups: control, negative control (Lenti-EGFP), and Lenti-AX2R.Cell proliferation, cell migration, tube formation, mouse aortic ring assays and mouse matrigel plug assay were applied to analyse the effect of AX2R in NV. Furthermore, we conducted flow cytometry to evaluate whether AX2R could influence the cell cycle. A series of cell cycle-related proteins including cyclin A1, cyclin B1, cyclin D1, cyclin E1, CDK1, and p-CDC2 were detected by WB. The mRNA and protein levels of KLF2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were further quantified by RT-PCR and WB to reveal the possible mechanism. Overexpression of AX2R significantly inhibited cell proliferation, migration and tube formation in both types of endothelial cells (ECs), HRECs and HUVECs. It also suppressed vessel sprouting in the mouse aortic ring assay and NV in mouse matrigel plug assay. Furthermore, infection with Lenti-AX2R lentivirus arrested the cell cycle in S/G2 and influenced the expression of a series of cell cycle-related proteins. We also found that the overexpression of AX2R increased the

  5. Identification, molecular structure and expression of two cloned serotonin receptors from the pond snail, Helisoma trivolvis.

    PubMed

    Mapara, Sabeen; Parries, Shawn; Quarrington, Caitlin; Ahn, Kee-Chan; Gallin, Warren J; Goldberg, Jeffrey I

    2008-03-01

    Helisoma trivolvis has served as a model system to study the functions of serotonin (5-HT) from cellular, developmental, physiological and behavioural perspectives. To further explore the serotonin system at the molecular level, and to provide experimental knockout tools for future studies, in this study we identified serotonin receptor genes from the H. trivolvis genome, and characterized the molecular structure and expression profile of the serotonin receptor gene products. Degenerate oligonucleotide primers, based on conserved regions of the Lymnaea stagnalis 5-HT(1Lym) receptor, were used to amplify G protein-coupled biogenic amine receptor sequences from H. trivolvis genomic cDNA, resulting in the cloning of two putative serotonin receptors. The deduced gene products both appear to be G protein-coupled serotonin receptors, with well-conserved structure in the functional domains and high variability in the vestibule entrance of the receptor protein. Phylogenetic analysis placed these receptors in the 5-HT(1) and 5-HT(7) families of serotonin receptors. They are thus named the 5-HT(1Hel) and 5-HT(7Hel) receptors, respectively. In situ hybridization and immunofluorescence studies revealed that these genes and gene products are expressed most heavily in the ciliated pedal and mantle epithelia of H. trivolvis embryos. In adults, widespread expression occurred in all ganglia and connectives of the central nervous system. Expression of both receptor proteins was localized exclusively to neurites when examined in situ. In contrast, when isolated neurons were grown in culture, 5-HT(1Hel) and 5-HT(7Hel) immunoreactivity were located primarily in the cell body. This is the first study to reveal a 5-HT(7) receptor in a molluscan species.

  6. Leptin, its receptor and aromatase expression in deep infiltrating endometriosis.

    PubMed

    Gonçalves, Helder F; Zendron, Carolina; Cavalcante, Fernanda S; Aiceles, Verônica; Oliveira, Marco Aurélio P; Manaia, Jorge Henrique M; Babinski, Márcio A; Ramos, Cristiane F

    2015-08-05

    The aim of this study was to evaluate the leptin levels in the serum and peritoneal fluid (PF) and the protein expression in three different peritoneal ectopic implants in patients who underwent surgery for deep infiltrating endometriosis. All patients had been treated at the Department of Gynecology of the Pedro Ernesto University Hospital, Rio de Janeiro. The study group consisted of 15 patients who underwent surgery for adnexal masses and infertility, while the control group consisted of ten women who underwent surgery for tubal ligation. Peritoneal fluid and samples tissues were collected during surgery. Serum samples were obtained before anesthesia. In this study, the leptin levels in the serum and peritoneal fluid (PF) were evaluated by ELISA. The protein expression of leptin and its receptors (ObR) and aromatase enzyme were evaluated by Western blot analysis of the intestine, uterosacral ligament and vaginal septum in the ectopic implants. The t-test and one-way ANOVA with Holm-Sìdak post-test were used, and p < 0.05 was considered to be statistically significant. Compared to the controls, the serum leptin levels (control = 14.7 ng/mL ± 2.63, endometriosis = 19.2 ng/mL ± 1.84, p < 0.0001) were increased, while in PF, there was no difference (control = 6.68 ng/mL ± 0.43, endometriosis = 7.71 ng/mL ± 0.59, p = 0.18). Comparing women with and without ovarian implants, the leptin levels in both the serum and PF were significantly higher in women without ovarian implants (serum: with ovarian implant = 15.85 ± 1.99; without ovarian implant = 23.14 ± 2.60; ng/mL, p = 0.04; PF: with ovarian implant = 4.28 ± 1.30; without ovarian implant = 11.18 ± 2.98;ng/mL, p = 0.048). The leptin, ObR and aromatase protein expression levels were increased in lesions in the vaginal septum and were decreased in the intestine lesions. This study reports several interesting associations between

  7. N-methyl-D-aspartate receptors are transiently expressed in the developing spinal cord ventral horn.

    PubMed Central

    Kalb, R G; Lidow, M S; Halsted, M J; Hockfield, S

    1992-01-01

    Quantitative receptor autoradiography was used to map the distribution of N-methyl-D-aspartate (NMDA) receptors in the developing rat spinal cord. Three different specific ligands, which label partially overlapping subpopulations of NMDA receptors, were used: an agonist (L-[3H]glutamate), a noncompetitive antagonist ([3H]MK-801), and a competitive antagonist ([3H]CGP-39653). In the adult, NMDA receptors labeled with all three ligands are restricted to the substantia gelatinosa in the spinal dorsal horn. In marked distinction, at postnatal day 7 NMDA receptors labeled with L-[3H]glutamate and [3H]MK-801 are present throughout the spinal gray matter. NMDA receptors in the neonatal spinal ventral horn have a higher affinity for L-[3H]glutamate than those in the adult substantia gelatinosa. Over the second and third postnatal weeks, NMDA receptors are lost from all areas of the spinal gray matter except for the substantia gelatinosa. Neonatal NMDA receptors identified with [3H]CGP-39653 are restricted to the substantia gelatinosa. These results show that the immature ventral horn contains a subpopulation of NMDA receptors and raise the possibility that motor neurons transiently express NMDA receptors in early postnatal life. Ventral horn NMDA receptors may be a component of the mechanisms by which the mature phenotype of motor neurons is acquired through activity-dependent processes. The loss of NMDA receptors over the course of development may play a role in limiting the period of motor neuron plasticity. Images PMID:1356265

  8. Design, synthesis and bioevaluation of an EphA2 receptor-based targeted delivery system.

    PubMed

    Barile, Elisa; Wang, Si; Das, Swadesh K; Noberini, Roberta; Dahl, Russell; Stebbins, John L; Pasquale, Elena B; Fisher, Paul B; Pellecchia, Maurizio

    2014-07-01

    Because of its overexpression in a range of solid tumors, the EphA2 receptor is a validated target for cancer therapeutics. We recently described a new targeted delivery system based on specific EphA2-targeting peptides conjugated with the chemotherapeutic agent paclitaxel. Here, we investigate the chemical determinants responsible for the stability and degradation of these agents in plasma. Introducing modifications in both the peptide and the linker between the peptide and paclitaxel resulted in drug conjugates that are both long-lived in rat plasma and that markedly decrease tumor size in a prostate cancer xenograft model compared with paclitaxel alone treatment. These studies identify critical rate-limiting degradation sites on the peptide-drug conjugates, enabling the design of agents with increased stability and efficacy. These results provide support for our central hypothesis that peptide-drug conjugates targeting EphA2 represent an innovative and potentially effective strategy to selectively deliver cytotoxic drugs to cancer cells. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Expression of muscarinic acetylcholine and dopamine receptor mRNAs in rat basal ganglia

    SciTech Connect

    Weiner, D.M.; Levey, A.I.; Brann, M.R.

    1990-09-01

    Within the basal ganglia, acetylcholine and dopamine play a central role in the extrapyramidal control of motor function. The physiologic effects of these neurotransmitters are mediated by a diversity of receptor subtypes, several of which have now been cloned. Muscarinic acetylcholine receptors are encoded by five genes (m1-m5), and of the two known dopamine receptor subtypes (D1 and D2) the D2 receptor gene has been characterized. To gain insight into the physiological roles of each of these receptor subtypes, the authors prepared oligodeoxynucleotide probes to localize receptor subtype mRNAs within the rat striatum and substantia nigra by in situ hybridizationmore » histochemistry. Within the striatum, three muscarinic (m1, m2, m4) receptor mRNAs and the D2 receptor mRNA were detected. The m1 mRNA was expressed in most neurons; the m2 mRNA, in neurons which were both very large and rare; and the m4 and D2 mRNAs, in 40-50% of the neurons, one-third of which express both mRNAs. Within the substantia nigra, pars compacta, only the m5 and D2 mRNAs were detected, and most neurons expressed both mRNAs. These data provide anatomical evidence for the identity of the receptor subtypes which mediate the diverse effects of muscarinic and dopaminergic drugs on basal ganglia function.« less

  10. Comparative genomics reveals tissue-specific regulation of prolactin receptor gene expression

    USDA-ARS?s Scientific Manuscript database

    Prolactin (PRL), acting via the prolactin receptor, fulfills a diversity of biological functions including the maintenance of solute balance and mineral homeostasis via tissues such as the heart, kidneys and intestine. Expression and activity of the prolactin receptor (PRLR) is regulated by various ...

  11. The atypical dopamine D1 receptor agonist SKF 83959 induces striatal Fos expression in rats.

    PubMed

    Wirtshafter, David; Osborn, Catherine V

    2005-12-28

    The effects of dopamine D1 receptor agonists are often presumed to result from an activation of adenylyl cyclase, but dopamine D1 receptors may also be linked to other signal transduction cascades and the relative importance of these various pathways is currently unclear. SKF 83959 is an agonist at dopamine D1 receptors linked to phospholipase C, but has been reported to be an antagonist at receptors linked to adenylyl cyclase. The current report demonstrates that SKF 83959 induces pronounced, nonpatchy, expression of the immediate-early gene product Fos in the striatum of intact rats which can be converted to a patchy pattern by pretreatment with the dopamine D2-like receptor agonist quinpirole. In rats with unilateral 6-hydroxydopamine lesions SKF 83959 induces strong behavioral rotation and a greatly potentiated Fos response. All of the responses to SKF 83959, in both intact and dopamine-depleted animals, can be blocked by pretreatment with the dopamine D1 receptor antagonist SCH-23390. In intact subjects, SKF 83959 induced Fos expression less potently than the standard dopamine D1 receptor agonist SKF 82958, but the two drugs were approximately equipotent in deinnervated animals. These results demonstrate for the first time that possession of full efficacy at dopamine D1 receptors linked to adenylyl cyclase is not a necessary requirement for the induction of striatal Fos expression in intact animals and suggest that alternative signal transduction pathways may play a role in dopamine agonist induced Fos expression, especially in dopamine-depleted subjects.

  12. Histamine H1Receptor Gene Expression and Drug Action of Antihistamines.

    PubMed

    Fukui, Hiroyuki; Mizuguchi, Hiroyuki; Nemoto, Hisao; Kitamura, Yoshiaki; Kashiwada, Yoshiki; Takeda, Noriaki

    2017-01-01

    The upregulation mechanism of histamine H 1 receptor through the activation of protein kinase C-δ (PKCδ) and the receptor gene expression was discovered. Levels of histamine H 1 receptor mRNA and IL-4 mRNA in nasal mucosa were elevated by the provocation of nasal hypersensitivity model rats. Pretreatment with antihistamines suppressed the elevation of mRNA levels. Scores of nasal symptoms were correlatively alleviated to the suppression level of mRNAs above. A correlation between scores of nasal symptoms and levels of histamine H 1 receptor mRNA in the nasal mucosa was observed in patients with pollinosis. Both scores of nasal symptoms and the level of histamine H 1 receptor mRNA were improved by prophylactic treatment of antihistamines. Similar to the antihistamines, pretreatment with antiallergic natural medicines showed alleviation of nasal symptoms with correlative suppression of gene expression in nasal hypersensitivity model rats through the suppression of PKCδ. Similar effects of antihistamines and antiallergic natural medicines support that histamine H 1 receptor-mediated activation of histamine H 1 receptor gene expression is an important signaling pathway for the symptoms of allergic diseases. Antihistamines with inverse agonist activity showed the suppression of constitutive histamine H 1 receptor gene expression, suggesting the advantage of therapeutic effect.

  13. Larvae of small white butterfly, Pieris rapae, express a novel serotonin receptor

    USDA-ARS?s Scientific Manuscript database

    The biogenic amine serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter in vertebrates and invertebrates. It acts in regulation and modulation of many physiological and behavioral processes through G protein-coupled receptors. Insects express five 5-HT receptor subtypes that share high simila...

  14. Pregnane X Receptor Expression in Human Pancreatic Adenocarcinoma: Associations With Clinicopathologic Parameters, Tumor Proliferative Capacity, Patients' Survival, and Retinoid X Receptor Expression.

    PubMed

    Koutsounas, Ioannis; Giaginis, Constantinos; Alexandrou, Paraskevi; Zizi-Serbetzoglou, Adamantia; Patsouris, Efstratios; Kouraklis, Gregorios; Theocharis, Stamatios

    2015-10-01

    Pregnane X receptor (PXR) has been involved in human malignancy, either by directly affecting carcinogenesis or by inducing drug-drug interactions and chemotherapy resistance. The present study aimed to assess the clinical significance of PXR in pancreatic adenocarcinoma. Pregnane X receptor and its heterodimers' PXR/retinoid X receptor α (RXR-α), RXR-β, and RXR-γ expression were assessed immunohistochemically on tumoral samples from 55 pancreatic adenocarcinoma patients and were associated with clinicopathologic parameters, tumor proliferative capacity, and patients' survival. Enhanced PXR expression was noted in 24 (43.6%) of 55 pancreatic adenocarcinoma cases. Pancreatic adenocarcinoma patients presenting increased histological grade of tumor differentiation showed a significant increased incidence of elevated PXR expression (P = 0.023). Enhanced PXR/RXR-β expression was significantly associated with smaller tumor size and earlier clinical stage (P = 0.005 and P = 0.003, respectively). Elevated PXR/RXR-γ expression was significantly associated with smaller tumor size and earlier clinical stage (P = 0.012 and P = 0.014, respectively) and borderline with the absence of lymph node metastases (P = 0.056). In addition, pancreatic adenocarcinoma patients presenting enhanced PXR/RXR-γ expression showed marginally longer survival times compared with those with decreased expression (log-rank test, P = 0.053). This study supported evidence that PXR and its copartners' overexpression may be associated with favorable clinicopathologic parameters and better outcome in pancreatic adenocarcinoma.

  15. Expression of the human ABCC6 gene is induced by retinoids through the retinoid X receptor

    SciTech Connect

    Ratajewski, Marcin; Department of Molecular Biophysics, University of Lodz, Lodz; Bartosz, Grzegorz

    2006-12-01

    Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although Physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. Thismore » constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.« less

  16. Crystal structure of the adenosine A2A receptor bound to an antagonist reveals a potential allosteric pocket

    PubMed Central

    Sun, Bingfa; Bachhawat, Priti; Chu, Matthew Ling-Hon; Wood, Martyn; Ceska, Tom; Sands, Zara A.; Mercier, Joel; Lebon, Florence; Kobilka, Tong Sun; Kobilka, Brian K.

    2017-01-01

    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-phase 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. PMID:28167788

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

    SciTech Connect

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

    2017-02-06

    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

  18. The dopamine D1 receptor is expressed and facilitates relaxation in airway smooth muscle

    PubMed Central

    2013-01-01

    Background Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gi-coupled “D2-like” receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. Methods The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Results Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The

  19. Identification and Expression Analysis of Putative Chemosensory Receptor Genes in Microplitis mediator by Antennal Transcriptome Screening

    PubMed Central

    Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Gu, Shao-Hua; Li, Rui-Jun; Zhou, Jing-Jiang; Zhang, Yong-Jun; Guo, Yu-Yuan

    2015-01-01

    Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor perception through sensory organs such as antennae. Antennal chemoreception is extremely important for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level. PMID:26078716

  20. Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors

    PubMed Central

    Houari, Sophia; Loiodice, Sophia; Jedeon, Katia; Berdal, Ariane; Babajko, Sylvie

    2016-01-01

    Endocrine disrupting chemicals (EDCs) play a part in the modern burst of diseases and interfere with the steroid hormone axis. Bisphenol A (BPA), one of the most active and widely used EDCs, affects ameloblast functions, leading to an enamel hypomineralization pattern similar to that of Molar Incisor Hypomineralization (MIH). In order to explore the molecular pathways stimulated by BPA during amelogenesis, we thoroughly investigated the receptors known to directly or indirectly mediate the effects of BPA. The expression patterns of high affinity BPA receptors (ERRγ, GPR30), of ketosteroid receptors (ERs, AR, PGR, GR, MR), of the retinoid receptor RXRα, and PPARγ were established using RT-qPCR analysis of RNAs extracted from microdissected enamel organ of adult rats. Their expression was dependent on the stage of ameloblast differentiation, except that of ERβ and PPARγ which remained undetectable. An additional large scale microarray analysis revealed three main groups of receptors according to their level of expression in maturation-stage ameloblasts. The expression level of RXRα was the highest, similar to the vitamin D receptor (VDR), whereas the others were 13 to 612-fold lower, with AR and GR being intermediate. Immunofluorescent analysis of VDR, ERα and AR confirmed their presence mainly in maturation- stage ameloblasts. These data provide further evidence that ameloblasts express a specific combination of hormonal receptors depending on their developmental stage. This study represents the first step toward understanding dental endocrinology as well as some of the effects of EDCs on the pathophysiology of amelogenesis. PMID:27853434

  1. Prolactin receptor, growth hormone receptor, and putative somatolactin receptor in Mozambique tilapia: tissue specific expression and differential regulation by salinity and fasting.

    PubMed

    Pierce, A L; Fox, B K; Davis, L K; Visitacion, N; Kitahashi, T; Hirano, T; Grau, E G

    2007-01-01

    In fish, pituitary growth hormone family peptide hormones (growth hormone, GH; prolactin, PRL; somatolactin, SL) regulate essential physiological functions including osmoregulation, growth, and metabolism. Teleost GH family hormones have both differential and overlapping effects, which are mediated by plasma membrane receptors. A PRL receptor (PRLR) and two putative GH receptors (GHR1 and GHR2) have been identified in several teleost species. Recent phylogenetic analyses and binding studies suggest that GHR1 is a receptor for SL. However, no studies have compared the tissue distribution and physiological regulation of all three receptors. We sequenced GHR2 from the liver of the Mozambique tilapia (Oreochromis mossambicus), developed quantitative real-time PCR assays for the three receptors, and assessed their tissue distribution and regulation by salinity and fasting. PRLR was highly expressed in the gill, kidney, and intestine, consistent with the osmoregulatory functions of PRL. PRLR expression was very low in the liver. GHR2 was most highly expressed in the muscle, followed by heart, testis, and liver, consistent with this being a GH receptor with functions in growth and metabolism. GHR1 was most highly expressed in fat, liver, and muscle, suggesting a metabolic function. GHR1 expression was also high in skin, consistent with a function of SL in chromatophore regulation. These findings support the hypothesis that GHR1 is a receptor for SL. In a comparison of freshwater (FW)- and seawater (SW)-adapted tilapia, plasma PRL was strongly elevated in FW, whereas plasma GH was slightly elevated in SW. PRLR expression was reduced in the gill in SW, consistent with PRL's function in freshwater adaptation. GHR2 was elevated in the kidney in FW, and correlated negatively with plasma GH, whereas GHR1 was elevated in the gill in SW. Plasma IGF-I, but not GH, was reduced by 4 weeks of fasting. Transcript levels of GHR1 and GHR2 were elevated by fasting in the muscle. However

  2. Flavonoids inhibit the platelet TxA2 signalling pathway and antagonize TxA2 receptors (TP) in platelets and smooth muscle cells

    PubMed Central

    Guerrero, José A; Navarro-Nuñez, Leyre; Lozano, María L; Martínez, Constantino; Vicente, Vicente; Gibbins, Jonathan M; Rivera, José

    2007-01-01

    What is already known about this subject Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid–TP interaction inhibits signalling downstream TP has not been shown. What this study adds This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Aims Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA2 receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Methods Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPα- and TPβ-transfected HEK 293T cells was explored using binding assays and the TP antagonist 3H-SQ29548. Results Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium

  3. The calcium-sensing receptor regulates parathyroid hormone gene expression in transfected HEK293 cells

    PubMed Central

    Galitzer, Hillel; Lavi-Moshayoff, Vardit; Nechama, Morris; Meir, Tomer; Silver, Justin; Naveh-Many, Tally

    2009-01-01

    Background The parathyroid calcium receptor determines parathyroid hormone secretion and the response of parathyroid hormone gene expression to serum Ca2+ in the parathyroid gland. Serum Ca2+ regulates parathyroid hormone gene expression in vivo post-transcriptionally affecting parathyroid hormone mRNA stability through the interaction of trans-acting proteins to a defined cis element in the parathyroid hormone mRNA 3'-untranslated region. These parathyroid hormone mRNA binding proteins include AUF1 which stabilizes and KSRP which destabilizes the parathyroid hormone mRNA. There is no parathyroid cell line; therefore, we developed a parathyroid engineered cell using expression vectors for the full-length human parathyroid hormone gene and the human calcium receptor. Results Co-transfection of the human calcium receptor and the human parathyroid hormone plasmid into HEK293 cells decreased parathyroid hormone mRNA levels and secreted parathyroid hormone compared with cells that do not express the calcium receptor. The decreased parathyroid hormone mRNA correlated with decreased parathyroid hormone mRNA stability in vitro, which was dependent upon the 3'-UTR cis element. Moreover, parathyroid hormone gene expression was regulated by Ca2+ and the calcimimetic R568, in cells co-transfected with the calcium receptor but not in cells without the calcium receptor. RNA immunoprecipitation analysis in calcium receptor-transfected cells showed increased KSRP-parathyroid hormone mRNA binding and decreased binding to AUF1. The calcium receptor led to post-translational modifications in AUF1 as occurs in the parathyroid in vivo after activation of the calcium receptor. Conclusion The expression of the calcium receptor is sufficient to confer the regulation of parathyroid hormone gene expression to these heterologous cells. The calcium receptor decreases parathyroid hormone gene expression in these engineered cells through the parathyroid hormone mRNA 3'-UTR cis element and the

  4. G Protein-Coupled Estrogen Receptor (GPER) Expression in Normal and Abnormal Endometrium

    PubMed Central

    Lessey, Bruce A.; Taylor, Robert N.; Wang, Wei; Bagchi, Milan K.; Yuan, Lingwen; Scotchie, Jessica; Fritz, Marc A.; Young, Steven L.

    2012-01-01

    Rapid estrogen effects are mediated by membrane receptors, and evidence suggests a role for both a membrane-associated form of estrogen receptor alpha (ESR1; ERα) and G-protein coupled receptor 30 (GPER; GPR30). Considering estrogen’s importance in endometrial physiology and endometriosis pathophysiology, we hypothesized that GPER could be involved in both cyclic changes in endometrial estrogen action and that aberrant expression might be seen in the eutopic endometrium of women with endometriosis. Using real-time reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemical analysis of normal endometrium, endometrial samples demonstrated cycle-regulated expression of GPER, with maximal expression in the proliferative phase. Eutopic and ectopic endometrium from women with endometriosis overexpressed GPER as compared to eutopic endometrium of normal participants. Ishikawa cells, an adenocarcinoma cell line, expressed GPER, with increased expression upon treatment with estrogen or an ESR1 agonist, but not with a GPER-specific agonist. Decreased expression was seen in Ishikawa cells stably transfected with progesterone receptor A. Together, these data suggest that normal endometrial GPER expression is cyclic and regulated by nuclear estrogen and progesterone receptors, while expression is dysregulated in endometriosis. PMID:22378861

  5. Selenoprotein W controls epidermal growth factor receptor surface expression, activation and degradation via receptor ubiquitination

    USDA-ARS?s Scientific Manuscript database

    Epidermal growth factor (EGF) receptor (EGFR) is the founding member of the ErbB family of growth factor receptors that modulate a complex network of intracellular signaling pathways controlling growth, proliferation and differentiation. Selenoprotein W (SEPW1) is a diet-regulated, highly conserved...

  6. Progesterone receptor expression during prostate cancer progression suggests a role of this receptor in stromal cell differentiation.

    PubMed

    Yu, Yue; Yang, Ou; Fazli, Ladan; Rennie, Paul S; Gleave, Martin E; Dong, Xuesen

    2015-07-01

    The progesterone receptor, like the androgen receptor, belongs to the steroid receptor superfamily. Our previous studies have reported that the PR is expressed specifically in prostate stroma. PR inhibits proliferation of, and regulates cytokine secretion by stromal cells. However, PR protein expression in cancer-associated stroma during prostate cancer progression has not been profiled. Since the phenotypes of prostate stromal cells change dynamically as tumors progress, whether the PR plays a role in regulating stromal cell differentiation needs to be investigated. Immunohistochemistry assays measured PR protein levels on human prostate tissue microarrays containing 367 tissue cores from benign prostate, prostate tumors with different Gleason scores, tumors under various durations of castration therapy, and tumors at the castration-resistant stage. Immunoblotting assays determined whether PR regulated the expression of alpha smooth muscle actin (α-SMA), vimentin, and fibroblast specific protein (FSP) in human prostate stromal cells. PR protein levels decreased in cancer-associated stroma when compared with that in benign prostate stroma. This reduction in PR expression was not correlated with Gleason scores. PR protein levels were elevated by castration therapy, but reduced to pre-castration levels when tumors progressed to the castration-resistant stage. Enhanced PR expression in human prostate stromal cells increased α-SMA, but decreased vimentin and FSP protein levels ligand-independently. These results suggest that PR plays an active role in regulating stromal cell phenotypes during prostate cancer progression. © 2015 Wiley Periodicals, Inc.

  7. Expression of IGF-1, IL-27 and IL-35 Receptors in Adjuvant Induced Rheumatoid Arthritis Model.

    PubMed

    Abdi, Elham; Najafipour, Hamid; Joukar, Siyavash; Dabiri, Shahriar; Esmaeli-Mahani, Saeed; Abbasloo, Elham; Houshmandi, Nasrin; Afsharipour, Abbas

    2018-03-01

    IGF-1 and certain other cytokines have been shown to exert inflammatory/anti-inflammatory roles in chronic joint diseases. To assess the effect of IGF-1, IL-27 and IL-35, their interaction and their receptor expression in a rheumatoid arthritis model. Freund's adjuvant-induced chronic joint inflammation was operated on 160 male rats. Animals were divided into histopathology and receptor expression groups, each composed of 10 subgroups including; control, vehicle, IGF-1, IL-27, IL-35, their antagonists, IGF-1+IL-27 antagonist and IGF-1+IL-35 antagonist. After two weeks, vehicle or agonist/antagonists were injected into the joint space every other day until day 28 where joint histopathology was performed. The expression of IGF-1, IL-27 and IL-35 receptors were assessed by western blot analysis. IGF-1 did not show pro- or anti- inflammatory functions; endogenous IL-27 and IL-35, on the other hand, exerted inflammatory effects. IL-27 and IL-35 antagonists exerted the highest anti-inflammatory effects. The total inflammation scores were 0.55 ± 0.06, 4.63 ± 0.40, 3.63 ± 0.60, 2.50 ± 0.38 and 1.63 ± 0.40 regarding control, vehicle, IGF-1 Ant., IL-27 Ant. and IL-35 Ant., respectively. IGF-1 receptor expression was reduced in chronic joint inflammation and all three antagonists augmented the IGF-1 receptor expression. IL-27 and IL-35 receptors were up-regulated by chronic joint inflammation. Overall, the results demonstrated the pro-inflammatory role of endogenous IL-27 and IL-35 along with the over expression of their receptors in chronic joint inflammation. IL-27 and IL-35 antagonists exerted the most anti-inflammatory effects and increased IGF-1 receptor expression. These two antagonists may be potential agents for new treatment strategies in chronic joint inflammatory diseases.

  8. Homer1 gene products orchestrate Ca2+-permeable AMPA receptor distribution and LTP expression

    PubMed Central

    Rozov, Andrei; Zivkovic, Aleksandar R.; Schwarz, Martin K.

    2012-01-01

    We studied the role of Homer1 gene products on the presence of synaptic Ca2+-permeable AMPA receptors (AMPARs) and long-term potentiation (LTP) generation in hippocampal CA1 pyramidal neurons, using mice either lacking all Homer1 isoforms (Homer1 KO) or overexpressing the immediate early gene (IEG) product Homer1a (H1aTG). We found that Homer1 KO caused a significant redistribution of the AMPAR subunit GluA2 from the dendritic compartment to the soma. Furthermore, deletion of Homer1 enhanced the AMPAR-mediated component of glutamatergic currents at Schaffer collateral synapses as demonstrated by increased AMPA/NMDA current ratios. Meanwhile, LTP generation appeared to be unaffected. Conversely, sustained overexpression of Homer1a strongly reduced AMPA/NMDA current ratios and polyamine sensitivity of synaptic AMPAR, indicating that the proportion of synaptic GluA2-containing AMPAR increased relative to WT. LTP maintenance was abolished in H1aTG. Notably, overexpression of Homer1a in Homer1 KO or GluA2 KO mice did not affect LTP expression, suggesting activity-dependent interaction between Homer1a and long Homer1 isoforms with GluA2-containing AMPAR. Thus, Homer1a is essential for the activity-dependent regulation of excitatory synaptic transmission. PMID:23133416

  9. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    SciTech Connect

    Nie,Y.; Hobbs, J.; Vigues, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain amore » large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.« less

  10. Expression of group III metabotropic glutamate receptors in the reproductive system of male mice.

    PubMed

    Marciniak, Marcin; Chruścicka, Barbara; Lech, Tomasz; Burnat, Grzegorz; Pilc, Andrzej

    2016-03-01

    Although the presence of metabotropic glutamate (mGlu) receptors in the central nervous system is well documented, they have recently been found in peripheral and non-neuronal tissues. In the present study we investigated the expression of group III mGlu receptors in the reproductive system of male mice. Reverse transcription-polymerase chain reaction analysis revealed the presence of mGlu6, mGlu7 and mGlu8 (but not mGlu4) receptor transcripts in testes and epididymides from adult mice. In addition, expression of mGlu6 (Grm6) and mGlu8 receptor (Grm8) mRNA was detected in spermatozoa isolated from the vas deferens. The vas deferens was found to contain only mGlu7 receptor (Grm7) mRNA, which was particularly intense in 21-day-old male mice. In penile homogenates, only the mGlu7 receptor signal was detected. Genetic ablation of the mGlu7 receptor in males led to fertility disorders manifested by decreased insemination capability as well as deterioration of sperm parameters, particularly sperm motility, vitality, sperm membrane integrity and morphology, with a simultaneous increase in sperm concentration. These results indicate that constitutively expressed mGlu receptors in the male reproductive system may play an important role in ejaculation and/or erection processes, as well as in the formation and maturation of spermatozoa.

  11. Toll-Like Receptor 3 Signalling Up-Regulates Expression of the HIV Co-Receptor G-Protein Coupled Receptor 15 on Human CD4+ T Cells

    PubMed Central

    Kiene, Miriam; Rethi, Bence; Jansson, Marianne; Dillon, Stephanie; Lee, Eric; Lantto, Rebecka; Wilson, Cara; Pöhlmann, Stefan; Chiodi, Francesca

    2014-01-01

    Background Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. Results Here, we show that GPR15 expression in CD4+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF) and was more prominent on gut-homing compared to lymph node-homing CD4+ T cells. Conclusion These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4+ T cells to HIV infection and target cell availability in the gut in some infected individuals. PMID:24558379

  12. Neuroblastomas and medulloblastomas exhibit more Coxsackie adenovirus receptor expression than gliomas and other brain tumors.

    PubMed

    Persson, Annette; Fan, Xiaolong; Salford, Leif G; Widegren, Bengt; Englund, Elisabet

    2007-06-01

    Adenoviral vector-mediated treatment is a potential therapy for tumors of the central nervous system. To obtain a significant therapeutic effect by adenoviral vectors, a sufficient infection is required, the power of which depends predominantly on the level of Coxsackie adenovirus receptors. We stained surgical biopsies of central nervous system tumors and neuroblastomas for Coxsackie adenovirus receptors. For gliomas, the level of the receptor was low and markedly variable among individual tumors. By contrast, neuroblastomas and medulloblastomas exhibited a higher degree of Coxsackie adenovirus receptor expression than gliomas and other brain tumors. We conclude that neuroblastomas and medulloblastomas could be suitable for adenovirus-mediated gene therapy. Adverse effects of the treatment, however, must be considered because neurons and reactive astrocytes also express a significant amount of the receptor.

  13. Expression cloning of a high-affinity melatonin receptor from Xenopus dermal melanophores.

    PubMed Central

    Ebisawa, T; Karne, S; Lerner, M R; Reppert, S M

    1994-01-01

    Using an expression cloning strategy, a high-affinity melatonin receptor cDNA has been isolated from Xenopus laevis dermal melanophores. Transient expression of the cDNA in COS-7 cells resulted in high-affinity 2-[125I]-iodomelatonin binding (Kd = 6.3 +/- 0.3 x 10(-11) M). In addition, six ligands exhibited a rank order of inhibition of specific 2-[125I]iodomelatonin binding that was identical to that reported for endogenous high-affinity receptors. Functional studies of CHO cells stably expressing the receptor cDNA showed that melatonin acting through the cloned receptor inhibited forskolin-stimulated cAMP accumulation in a dose-dependent manner. Northern blot analysis showed that melatonin receptor transcripts are moderately expressed in Xenopus dermal melanophores. The cDNA encodes a protein of 420 amino acids, which contains seven hydrophobic segments. Structural analysis revealed that the receptor protein is a newly discovered member of the guanine nucleotide binding protein-coupled receptor family. Images PMID:7517042

  14. Expression and cellular localization of the Mas receptor in the adult and developing mouse retina.

    PubMed

    Prasad, Tuhina; Verma, Amrisha; Li, Qiuhong

    2014-01-01

    Recent studies have provided evidence that a local renin-angiotensin system (RAS) exists in the retina and plays an important role in retinal neurovascular function. We have recently shown that increased expression of ACE2 and angiotensin (1-7) [Ang (1-7)], two components of the protective axis of the RAS, in the retina via adeno-associated virus (AAV)-mediated gene delivery, conferred protection against diabetes-induced retinopathy. We hypothesized that the protective molecular and cellular mechanisms of Ang (1-7) are mediated by its receptor, Mas, and the expression level and cellular localization dictate the response to Ang (1-7) and activation of subsequent protective signaling pathways. We tested this hypothesis by examining the expression and cellular localization of the Mas receptor in adult and developing mouse retinas. The cellular localization of the Mas receptor protein was determined with immunofluorescence of the eyes of adult and postnatal day 1 (P1), P5, P7, P15, and P21 mice using the Mas receptor-specific antibody, and mRNA was detected with in situ hybridization of paraffin-embedded sections. Western blotting and real-time reverse-transcription (RT)-PCR analysis were performed to determine the relative levels of the Mas protein and mRNA in adult and developing retinas, as well as in cultured retinal Müller glial and RPE cells. In the adult eye, the Mas receptor protein was abundantly present in retinal ganglion cells (RGCs) and photoreceptor cells; a lower level of expression was observed in endothelial cells, Müller glial cells, and other neurons in the inner nuclear layer of the retina. In the developing retina, Mas receptor mRNA and protein expression was detected in the inner retina at P1, and the expression levels increased with age to reach the adult level and pattern by P15. In the adult mouse retina, Mas receptor mRNA was expressed at a much higher level when compared to angiotensin II (Ang II) type I (AT1R) and type II (AT2R) receptor m

  15. Tissue expression of steroid hormone receptors is associated with differential immune responsiveness

    PubMed Central

    Butts, Cherié L.; Jones, Yava L.; Lim, Jean K.; Salter, Caroline E.; Belyavskaya, Elena; Sternberg, Esther M.

    2010-01-01

    Glucocorticoids and other steroid hormones have been used as treatments against a number of diseases, especially inflammatory conditions in which the immune system is overactive. These treatments have varying degrees of responsiveness among individuals and in different tissues (including brain); therefore, it is important to determine what could account for these differences. In this study, we evaluated expression of steroid hormone receptors in immune cells from lymphoid and non-lymphoid tissues as a possible explanation for tissue-specific differences. We analyzed leukocytes (CD45+) in kidney, liver, spleen, and thymus tissues from healthy mice for expression of the receptor for stress hormone (glucocorticoid - GR) as well as other steroid hormones (androgen - AR, progesterone - PR) and found that all tissues expressed these steroid hormone receptors but with varying expression patterns. To determine whether tissue-specific differences were related to immune cell composition, we examined steroid hormone receptor expression in T lymphocytes from each of these tissues and found similar patterns of expression in these cells regardless of tissue source. Because glucocorticoids can also impact brain function, we further examined expression of the stress hormone receptor in brain tissue and found GR expressed in immune cells at this site. In order to investigate the potential impact in an area of neuropathology, we utilized a mouse model of West Nile Virus (WNV). We observed pathological changes in brains of WNV-infected animals and T lymphocytes in the areas of inflammation; however, these cells did not express GR. These data indicate that tissue-specific differences in steroid hormone receptor expression by immune cells could determine responsiveness with steroid hormone treatment. PMID:21074604

  16. Architecture of fully occupied GluA2 AMPA receptor – TARP complex elucidated by cryo-EM

    PubMed Central

    Zhao, Yan; Chen, Shanshuang; Yoshioka, Craig; Baconguis, Isabelle; Gouaux, Eric

    2016-01-01

    Fast excitatory neurotransmission in the mammalian central nervous system is largely carried out by AMPA-sensitive ionotropic glutamate receptors. Localized within the postsynaptic density of glutamatergic spines, AMPA receptors are composed of heterotetrameric receptor assemblies associated with auxiliary subunits, the most common of which are transmembrane AMPA-receptor regulatory proteins (TARPs). The association of TARPs with AMPA receptors modulates the kinetics of receptor gating and pharmacology, as well as trafficking. Here we report the cryo-EM structure of the homomeric GluA2 AMPA receptor saturated with TARP γ2 subunits, showing how the TARPs are arranged with four-fold symmetry around the ion channel domain, making extensive interactions with the M1, M2 and M4 TM helices. Poised like partially opened ‘hands’ underneath the two-fold symmetric ligand binding domain (LBD) ‘clamshells’, one pair of TARPs are juxtaposed near the LBD dimer interface, while the other pair is near the LBD dimer-dimer interface. The extracellular ‘domains’ of TARP are positioned to not only modulate LBD ‘clamshell’ closure, but also to affect conformational rearrangements of the LBD layer associated with receptor activation and desensitization, while the TARP transmembrane (TM) domains buttress the ion channel pore. PMID:27368053

  17. [Expression and function of receptors for advanced glycation end products in bovine corneal endothelial cells].

    PubMed

    Kaji, Yuichi

    2005-11-01

    Corneal endothelial cell loss is a change that occurs with age, but its mechanism is still unclear. We postulated that interaction between advanced glycation end product(AGE) and its receptors is implicated in the corneal endothelial cell loss with age. We investigated the expression of AGE receptors: receptors for AGE(RAGE) and galectin-3 in bovine corneal endothelial cells by reverse transcription-polymerase chain reaction(RT-PCR) and immunohistochemistry. In addition, we investigated the effect of AGE on the cultured corneal endothelial cells. Expression of RAGE and galectin-3 was detected in bovine corneal endothelial cells. Galectin-3 was important in the internalization of AGE. In contrast, RAGE was important in the generation of reactive oxygen species and induction of apoptosis. Based on these data, the interaction of AGE in aqueous humor and AGE receptors expressed on the corneal endothelial cells was speculated to have a role in the corneal endothelial cell loss with age.

  18. Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta

    PubMed Central

    Lager, Susanne; Ramirez, Vanessa I.; Gaccioli, Francesca; Jansson, Thomas; Powell, Theresa L.

    2014-01-01

    Fatty acids can function as signaling molecules, acting through receptors in the cytosol or on the cell surface. G-Protein Receptor (GPR)120 is a membrane-bound receptor mediating anti-inflammatory and insulin-sensitizing effects of the omega-3 fatty acid docohexaenoic acid (DHA). GPR120 dysfunction is associated with obesity in humans. Cellular localization of GPR120 and the influence of maternal obesity on GPR120 protein expression in the placenta are unknown. Herein we demonstrate that GPR120 is predominantly expressed in the microvillous membrane (MVM) of human placenta and that the expression level of this receptor in MVM is not altered by maternal body mass index (BMI). PMID:24844436

  19. 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.

  20. Oleocanthal Modulates Estradiol-Induced Gene Expression Involving Estrogen Receptor α.

    PubMed

    Keiler, Annekathrin Martina; Djiogue, Sefirin; Ehrhardt, Tino; Zierau, Oliver; Skaltsounis, Leandros; Halabalaki, Maria; Vollmer, Günter

    2015-09-01

    Oleocanthal is a bioactive compound from olive oil. It has attracted considerable attention as it is anti-inflammatory, antiproliferative, and has been shown to possess neuroprotective properties in vitro and in vivo. Delineated from its polyphenolic structure, the aim of this study was to characterize oleocanthal towards estrogenic properties. This might contribute to partly explain the beneficial effects described for the Mediterranean diet. Estrogenic properties of oleocanthal were assessed by different methods: a) stimulation of reporter gene activity in MVLN or RNDA cells either expressing estrogen receptor α or β, b) stimulation of luciferase reporter gene activity in U2OS osteosarcoma cells expressing estrogen receptor α or β, and c) elucidation of the impact on estradiol-induced gene expression in U2OS cells transduced with both estrogen receptors. Depending on the cell line origin, oleocanthal inhibited luciferase activity (MVLN, U2OS-estrogen receptor β) or weakly induced reporter gene activity at 10 µM in U2OS-estrogen receptor α cells. However, oleocanthal inhibited stimulation of luciferase activity by estradiol from both estrogen receptors. Oleocanthal, if given alone, did not stimulate gene expression in U2OS cells, but it significantly modulated the response of estradiol. Oleocanthal enhanced the effect of estradiol on the regulation of those genes, which are believed to be regulated through heterodimeric estrogen receptors. As the estrogenic response pattern of oleocanthal is rather unique, we compared the results obtained with oleacein. Oleocanthal binds to both estrogen receptors inducing estradiol-agonistic or antiagonistic effects depending on the cell line. Regarding regulation of gene expression in U2OS-estrogen receptor α/β cells, oleocanthal and oleacein enhanced estradiol-mediated regulation of heterodimer-regulated genes. Georg Thieme Verlag KG Stuttgart · New York.

  1. Expression of growth factor receptors and targeting of EGFR in cholangiocarcinoma cell lines.

    PubMed

    Xu, Ling; Hausmann, Martin; Dietmaier, Wolfgang; Kellermeier, Silvia; Pesch, Theresa; Stieber-Gunckel, Manuela; Lippert, Elisabeth; Klebl, Frank; Rogler, Gerhard

    2010-06-18

    Cholangiocarcinoma (CC) is a malignant neoplasm of the bile ducts or the gallbladder. Targeting of growth factor receptors showed therapeutic potential in palliative settings for many solid tumors. The aim of this study was to determine the expression of seven growth factor receptors in CC cell lines and to assess the effect of blocking the EGFR receptor in vitro. Expression of EGFR (epithelial growth factor receptor), HGFR (hepatocyte growth factor receptor) IGF1R (insulin-like growth factor 1 receptor), IGF2R (insulin-like growth factor 2 receptor) and VEGFR1-3 (vascular endothelial growth factor receptor 1-3) were examined in four human CC cell lines (EGI-1, HuH28, OZ and TFK-1). The effect of the anti-EGFR-antibody cetuximab on cell growth and apoptosis was studied and cell lines were examined for KRAS mutations. EGFR, HGFR and IGFR1 were present in all four cell lines tested. IGFR2 expression was confirmed in EGI-1 and TFK-1. No growth-inhibitory effect was found in EGI-1 cells after incubation with cetuximab. Cetuximab dose-dependently inhibited growth in TFK-1. Increased apoptosis was only seen in TFK-1 cells at the highest cetuximab dose tested (1 mg/ml), with no dose-response-relationship at lower concentrations. In EGI-1 a heterozygous KRAS mutation was found in codon 12 (c.35G>A; p.G12D). HuH28, OZ and TFK-1 lacked KRAS mutation. CC cell lines express a pattern of different growth receptors in vitro. Growth factor inhibitor treatment could be affected from the KRAS genotype in CC. The expression of EGFR itself does not allow prognoses on growth inhibition by cetuximab.

  2. [Role of the expression of c-Met receptor in the progression of gastric cancer].

    PubMed

    Amemiya, Hideki; Menolascino, Francisco; Peña, Alix

    2010-09-01

    The product of the proto-oncogene C-MET (the c-Met receptor) and its ligand, hepatocyte growth factor (HGF), have been implicated in the progression of gastric cancer. The aim of this study was to analyze the expression of c-Met receptor, HGF and proliferating cell nuclear antigen (PCNA) by the immunohistochemistry method of labeled streptavidin-biotin, as well as survival, and they were correlated with anatomopathological factors in stomach specimens of 40 patients, who underwent gastrectomy for gastric cancer in the Department of General Surgery, Hospital Central Universitario "Antonio María Pineda" in Barquisimeto, Venezuela, in 2001-2004. High expression of c-Met receptor and PCNA was observed in patients with advanced stages of gastric cancer (III and IV) compared with early stages (I and II) (p<0.01). There was also overexpression of the c-Met receptor in histologic variables with low degree of differentiation, deeper tumor invasion into the submucosa, liver metastases and it is reported a lower survival rate in patients with increased receptor expression (+++ and ++++) when compared with patients with the lowest expression (+ and ++) (p<0.01). The expression of HGF was constant in both, advanced and early groups. The c-Met receptor is associated with proliferation and cell migration in Venezuelan patients with gastric cancer and could be used as a prognostic factor in this pathology.

  3. Avian TVB (DR5-like) death receptor expression in hen ovarian follicles.

    PubMed

    Bridgham, Jamie T; Johnson, Alan L

    2002-02-22

    TVB is an avian death domain-containing receptor belonging to the TNF receptor family and is proposed to be the ortholog to mammalian DR5. Although TVB receptor activation has been demonstrated to mediate apoptosis in chick embryo fibroblasts, there is essentially no information regarding TVB expression or regulation in the mature hen ovary, and in particular within the follicle granulosa layer where apoptosis is known to promote atresia. Significantly, the TVB receptor represents the fourth death domain-containing receptor (also including Fas, TNF-R1, and DR6) found to be expressed within hen granulosa cells. Levels of TVB expression are higher in prehierarchal follicles actively undergoing atresia compared to healthy follicles. However, increased TVB expression does not precede follicle death induced in vitro. Furthermore, TVB expression within granulosa cells is highest during the final stages of follicle development when follicles are not normally susceptible to undergoing atresia. These results provide evidence that TVB receptor signaling in the ovary may function in a capacity other than solely to mediate granulosa cell death and follicle atresia. ©2002 Elsevier Science (USA).

  4. Ghrelin receptors are expressed by distal tubules of the mouse kidney.

    PubMed

    Venables, Gene; Hunne, Billie; Bron, Romke; Cho, Hyun-Jung; Brock, James A; Furness, John B

    2011-10-01

    Ghrelin, a peptide hormone from the stomach, has been recently discovered to reduce sodium excretion from the kidney. Although the effects on the kidney suggest actions in the distal nephron, the sites of expression of ghrelin receptors have not been localised. In the present work we have used a mouse that expresses green fluorescent protein under the control of the ghrelin receptor promoter to locate sites of receptor expression in the kidney. Receptor expression was confined to the straight parts of the distal tubules and the thin limbs of the loops of Henle. No expression was detected in other structures, including the glomeruli, proximal tubules and collecting ducts. Ghrelin receptors were not found in extra-renal or intra-renal arteries, despite observations that ghrelin is a vasodilator. The distribution revealed by in situ hybridisation histochemistry was the same as that revealed by the reporter. In conclusion, ghrelin receptors have a restricted distribution in the kidney. The location in the straight parts of the distal tubules accords with observations that ghrelin promotes sodium retention.

  5. Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer.

    PubMed

    Chang, Q; Jorgensen, C; Pawson, T; Hedley, D W

    2008-10-07

    Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src.

  6. Effects of dasatinib on EphA2 receptor tyrosine kinase activity and downstream signalling in pancreatic cancer

    PubMed Central

    Chang, Q; Jorgensen, C; Pawson, T; Hedley, D W

    2008-01-01

    Eph receptors constitute the largest family of receptor tyrosine kinases in the human genome. EphA2 is one prominent member that is overexpressed and functionally altered in many invasive cancers, including pancreatic cancer. Dasatinib, which is a multi-targeted kinase inhibitor mainly developed for Bcr-Abl and Src family kinases, has recently been shown to have significant activity against EphA2. As selective small molecule EphA2 inhibitors are not currently available, we investigated the therapeutic potential to target EphA2 by dasatinib in pancreatic cancer cell lines. Using in vitro kinase assays, we found that EphA2 receptor tyrosine kinase was inhibited directly by dasatinib in a dose-dependent manner. Stimulation with ephrinA1 produced rapid increases of EphA2 phosphorylation that were inhibited by dasatinib, although the effects on activation of downstream signalling differed among the pancreatic cancer cell lines. Dasatinib also inhibited ligand-induced binding of EphA2 to the ubiquitin ligase Cbl, and the internalisation and degradation of EphA2, suggesting that these processes are dependent on kinase activity. Treatment with dasatinib decreased EphA2 phosphorylation in BxPC-3 xenografts, suggesting that dasatinib might have activity in pancreatic cancer due to EphA2 inhibition, besides its effects on Src. PMID:18797457

  7. Control of mu opioid receptor expression by modification of cDNA 5'- and 3'-noncoding regions.

    PubMed

    Zöllner, C; Johnson, P S; Bei Wang, J; Roy, A J; Layton, K M; Min Wu, J; Surratt, C K

    2000-06-23

    Removal of a 712 base pair (bp) sequence following the coding region of a human micro opioid receptor (hmuOR) cDNA unexpectedly increased expression of the receptor protein. A series of 3'-noncoding region deletion mutants revealed that at least three discrete regions following the stop codon influenced receptor expression levels. Deletion of the 205-bp 5'-noncoding region immediately preceding the Kozak sequence doubled receptor expression relative to wild type, and simultaneous deletion of 5'- and 3'-noncoding regions increased expression several fold. The hmuOR noncoding regions may participate in a regulatory mechanism that controls the number of cell surface receptors.

  8. Heterologous expression of G protein-coupled receptors in U-2 OS osteosarcoma cells.

    PubMed

    Ames, Robert; Nuthulaganti, Parvathi; Fornwald, Jim; Shabon, Usman; van-der-Keyl, Harjeet; Elshourbagy, Nabil

    2004-01-01

    Recombinant baculoviruses, in which the insect cell-specific polyhedrin promoter has been replaced with a mammalian cell-active expression cassette (BacMam viruses), are efficient gene delivery vehicles for many mammalian cell types. BacMam viruses have been generated for expression of G protein-coupled receptors (GPCRs) and used to establish Ca2+mobilization assays in HEK-293 human embryonic kidney cells and U-2 OS human osteosarcoma cells. U-2 OS cells are highly susceptible to BacMam-based gene delivery and lack many of the endogenous receptors present on HEK-293 and other mammalian cell lines typically used for heterologous expression of GPCRs. U-2 OS cells were found to have a null background for muscarine, ADP, ATP, UTP, UDP, and lysophosphatidic acid (LPA). Consequently, U-2 OS cells transduced with BacMam constructs encoding the muscarinic acetylcholine receptors (M1, M2, M3, M4, and M5subtypes), the P2Y receptors (P2Y1, P2Y2), or the LPA receptors (EDG-2, EDG-7) were used for the establishment of whole-cell Ca2+mobilization assays, assays that cannot readily be established in HEK-293 cells. U-2 OS cells were susceptible to simultaneous expression of multiple genes delivered by BacMam vectors. In U-2 OS cells the functional expression of the Gi-coupled M2and M4receptors was dependent on co-expression of the receptor and a G protein chimera, both of which were delivered to the cells via BacMam viruses. The use of U-2 OS cells and BacMam-based gene delivery has facilitated development of whole-cell-based GPCR functional assays, especially for P2Y, muscarininc acetylcholine, and LPA receptors.

  9. TRA-418, a thromboxane A2 receptor antagonist and prostacyclin receptor agonist, inhibits platelet-leukocyte interaction in human whole blood.

    PubMed

    Miyamoto, Mitsuko; Ohno, Michihiro; Yamada, Naohiro; Ohtake, Atsushi; Matsushita, Teruo

    2010-10-01

    TRA-418, a compound with both thromboxane A2 receptor (TP receptor) antagonistic and prostacyclin receptor (IP receptor) agonistic activities, was synthesised in our laboratory as a new antithrombotic agent. In this study, we examined the effects of TRA-418 on platelet-leukocyte interactions in human whole blood. Platelet-leukocyte interactions were induced by U-46619 in the presence of epinephrine (U-46619 + epinephrine) or with thrombin receptor agonist peptide 1-6 (TRAP). Platelet-leukocyte interactions were assessed by flow cytometry, with examination of both platelet-neutrophil and platelet-monocyte complexes. In a control experiment, the TP receptor antagonist SQ-29548 significantly inhibited the induction of platelet-leukocyte complexes by the combination of U-46619 and epinephrine, but not TRAP-induced formation of platelet-leukocyte complexes. Conversely, the IP receptor agonist beraprost sodium inhibited platelet-leukocyte complex formation induced by both methods, although the IC50 values of beraprost sodium for U-46619 + epinephrine were at least 10-fold greater than for TRAP. Under such conditions, TRA-418 inhibited both U-46619 + epinephrine-induced and TRAP-induced platelet-leukocyte complex formation in a concentration-dependent manner, in a similar range. These results suggest that TRA-418 exerts its inhibitory effects on platelet-leukocyte interactions by acting as a TP receptor antagonist as well as an IP receptor agonist in an additive or synergistic manner. These inhibitory effects of TRA-418 on formation of platelet-leukocyte complexes suggest the compound is beneficial effects as an antithrombotic agent.

  10. Estrogen receptor β agonists affect growth and gene expression of human breast cancer cell lines.

    PubMed

    Lattrich, Claus; Stegerer, Anette; Häring, Julia; Schüler, Susanne; Ortmann, Olaf; Treeck, Oliver

    2013-02-01

    Expression of estrogen receptor β (ERβ) has been described to reduce growth of cancer cell lines derived from hormone-dependent tumors, like breast cancer. In this study we tested to what extent two ERβ agonists, androgen derivative 3β-Adiol and flavonoid Liquiritigenin, would affect growth and gene expression of different ERβ-positive human breast cancer cell lines. Under standard cell culture conditions, we observed 3β-Adiol to inhibit growth of MCF-7 cells in a dose-dependent manner, whereas growth of BT-474 and MCF-10A cells was suppressed by the maximum concentration (100 nM) only. When treated in serum-free medium, all cell lines except of MDA-MB-231 were responsive to 1 nM 3β-Adiol, and ZR75-1 cells exhibited a dose-dependent antiproliferative response. Providing putative mechanisms underlying the observed growth-inhibitory effect, expression of Ki-67 or cyclins A2 and B1 was downregulated after 3β-Adiol treatment in all responsive lines. In contrast, treatment with lower doses of Liquiritigenin did not affect growth. In MCF-7 cells, the highest dose of this flavonoid exerted proliferative effects accompanied by increased expression of cyclin B1, PR and PS2, indicating unspecific activation of ERα. In conclusion, the ERβ agonists tested exerted distinct concentration-dependent and cell line-specific effects on growth and gene expression. The observed inhibitory effects of 3β-Adiol on breast cancer cell growth encourage further studies on the potential of this and other ERβ agonists as targeted drugs for breast cancer therapy. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. 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.

  12. Molecular Cloning, Characterization, and Expression Analysis of an Estrogen Receptor-Related Receptor Homologue in the Cricket, Teleogryllus emma

    PubMed Central

    He, Hui; Xi, Gengsi; Lu, Xiao

    2010-01-01

    The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood. A homologue of human ERR was isolated from the cricket Teleogryllus emma (Ohmachi and Matsumura) (Orthoptera: Gryllidae). The full-length cDNA of T. emma ERR, termed TeERR, has 1618 base pair (bp) and contains a 5′?-untranslated region of 140 bp and a 3′?-untranslated region of 272 bp. The open reading frame of TeERR encodes a deduced 401 amino acid peptide with a predicted molecular mass of 45.75 kilodaltons. The results of sequence alignments indicate that the TeERR protein shares an overall identity of 65%–82% with other known ERR homologues, and is most closely related to that of Nasonia vitripennis (Hymenoptera: Pteromalidae) and Apis mellifera (Apidae). Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare the TeERR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeERR mRNA is differentially expressed during T. emma development, with the highest expression level in embryos and the lowest in the body of late-instar larvae. The levels of TeERR transcripts also varied throughout gonad development; interestingly testicles had higher higher expression levels than ovaries at every development stage. These results suggest that TeERR has potential significance in the regulation of development in T. emma, due to its expression during different developmental periods. PMID:21265615

  13. Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain

    PubMed Central

    de Oliveira-Carlos, Vanessa; Ganz, Julia; Hans, Stefan; Kaslin, Jan; Brand, Michael

    2013-01-01

    The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches. PMID:24039926

  14. Oestrogen and progesterone receptor expression in subtypes of canine mammary tumours in intact and ovariectomised dogs.

    PubMed

    Mainenti, M; Rasotto, R; Carnier, P; Zappulli, V

    2014-10-01

    The objective of this study was to investigate as a potential prognostic indicator the relationship between histological subtype of canine mammary tumours (CMTs) and oestrogen-α (ORα) and progesterone (PR) receptor expression. Using immunohistochemistry, receptor expression in neoplastic epithelial cells was assessed in 12 different subtypes in 113 CMTs (34 benign, 79 malignant) and 101 surrounding normal tissues. Sixty-eight and 45 CMTs were from intact and ovariectomised bitches, respectively. Histological subtype strongly influenced ORα/PR expression: simple and complex adenomas as well as simple tubular carcinomas exhibited the greatest expression, whereas immunohistochemical labelling for these receptors was weakest in carcinoma and malignant myoepitheliomas, as well as in solid/anaplastic carcinomas and comedocarcinomas. Receptor expression was generally higher in benign relative to malignant neoplasms, and in the latter it was significantly lower in ovariectomised vs. intact bitches. Lymphatic invasion, mitotic index, nodule diameter, and tumour grade were significantly associated with ORα/PR expression. Although not found to be an independent prognostic indicator, tumours from dogs with <10% cells with ORα/PR expression had a poorer prognosis. Lymphatic invasion, the state of the margins of excision, and mitotic index were found to be independent prognostic indicators. Overall, the results suggest that differences in histological subtype and whether or not a bitch has been ovariectomised should be considered when evaluating the significance of ORα and PR expression in CMTs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Developmental expression of a tyramine receptor gene in the brain of the honey bee, Apis mellifera.

    PubMed

    Mustard, Julie A; Kurshan, Peri T; Hamilton, Ingrid S; Blenau, Wolfgang; Mercer, Alison R

    2005-02-28

    This study reveals that the tyramine receptor gene, Amtyr1, is expressed in the developing brain, as well as in the brain of the adult worker honey bee. Changes in levels of Amtyr1 expression were examined using Northern analysis. Age-related increases in Amtyr1 transcript levels were observed not only during metamorphic adult development, but also in the brain of the adult worker bee. RNA in situ hybridization revealed the pattern of Amtyr1 expression. Cell bodies staining intensely for tyramine receptor-gene transcript were observed throughout the somata rind, with well-defined clusters of cells associated with developing mushroom bodies, optic lobes, and antennal lobes of the brain. Staining for Amtyr1 transcript was particularly intense within the three major divisions of mushroom body intrinsic neurons (outer compact, noncompact, and inner compact cells), suggesting that Amtyr1 is highly expressed in these structures. Activation of AmTYR1 receptors heterologously expressed in insect (Spodoptera frugiperda) cells led to a reduction in intracellular levels of cAMP similar to that reported for AmTYR1 receptors expressed in mammalian (HEK 293) cells (Blenau et al. [2000] J Neurochem 74:900-908). Taken together, these results suggest that AmTYR1 receptors may play a role in the developing brain as well as in the brain of the adult worker bee. The actions of tyramine are likely to be mediated, at least in part, via the cAMP-signaling pathway. (c) 2005 Wiley-Liss, Inc.

  16. 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

  17. Expression of VEGF Receptors on Endothelial Cells in Mouse Skeletal Muscle

    PubMed Central

    Imoukhuede, Princess I.; Popel, Aleksander S.

    2012-01-01

    VEGFR surface localization plays a critical role in converting extracellular VEGF signaling towards angiogenic outcomes, and the quantitative characterization of these parameters is critical for advancing computational models; however the levels of these receptors on blood vessels is currently unknown. Therefore our aim is to quantitatively determine the VEGFR localization on endothelial cells from mouse hindlimb skeletal muscles. We contextualize this VEGFR quantification through comparison to VEGFR-levels on cells in vitro. Using quantitative fluorescence we measure and compare the levels of VEGFR1 and VEGFR2 on endothelial cells isolated from C57BL/6 and BALB/c gastrocnemius and tibialis anterior hindlimb muscles. Fluorescence measurements are calibrated using beads with known numbers of phycoerythrin molecules. The data show a 2-fold higher VEGFR1 surface localization relative to VEGFR2 with 2,000–3,700 VEGFR1/endothelial cell and 1,300–2,000 VEGFR2/endothelial cell. We determine that endothelial cells from the highly glycolytic muscle, tibialis anterior, contain 30% higher number of VEGFR1 surface receptors than gastrocnemius; BALB/c mice display ∼17% higher number of VEGFR1 than C57BL/6. When we compare these results to mouse fibroblasts in vitro, we observe high levels of VEGFR1 (35,800/cell) and very low levels of VEGFR2 (700/cell), while in human endothelial cells in vitro, we observe that the balance of VEGFRs is inverted, with higher levels VEGFR2 (5,800/cell) and lower levels of VEGFR1 (1,800/cell). Our studies also reveal significant cell-to-cell heterogeneity in receptor expression, and the quantification of these dissimilarities ex vivo for the first time provides insight into the balance of anti-angiogenic or modulatory (VEGFR1) and pro-angiogenic (VEGFR2) signaling. PMID:22984559

  18. Enhanced neuronal differentiation of NTera-2 cells expressing neuronally restricted beta2 adrenergic receptor.

    PubMed

    Fennell, M; Khawaja, X Z; Cockett, M I; Wood, A

    1998-07-20

    NTera-2/D1 (NT2) is a human teratocarcinoma cell line which can be cultured as a dividing population of precursor cells that can be manipulated with retinoic acid (RA) to yield post-mitotic neurons. Precursor cells were transfected with the human beta2 adrenergic receptor, controlled by the neuronal cell specific rat synapsin-1 promoter. Transfected precursor cells did not display elevated expression of the beta2 adrenergic receptor. Upon differentiation to a neuronal phenotype with RA, betaSyn2 and betaSyn4 (beta2Syn-NT2; ATCC CRL-12356) displayed elevated beta2 adrenergic receptor levels, and an elevated coupling to cAMP production. It was also observed that the elevated expression of the beta2 adrenergic receptor in the neuronal NT2 cells resulted in an enhanced level of neuronal differentiation compared to the wild type cells. These results demonstrate that the neuronally restricted expression of the human beta2 adrenergic receptor in NT2 neurons results in increased receptor levels and receptor stimulated generation of cAMP, this may result in the observed improvement in neuronal differentiation. Copyright 1998 Elsevier Science B.V. All rights reserved.

  19. Olfactory Plasticity: Variation in the Expression of Chemosensory Receptors in Bactrocera dorsalis in Different Physiological States.

    PubMed

    Jin, Sha; Zhou, Xiaofan; Gu, Feng; Zhong, Guohua; Yi, Xin

    2017-01-01

    Changes in physiological conditions could influence the perception of external odors, which is important for the reproduction and survival of insect. With the alteration of physiological conditions, such as, age, feeding state, circadian rhythm, and mating status, insect can modulate their olfactory systems accordingly. Ionotropic, gustatory, and odorant receptors (IR, GR, and ORs) are important elements of the insect chemosensory system, which enable insects to detect various external stimuli. In this study, we investigated the changes in these receptors at the mRNA level in Bactrocera dorsalis in different physiological states. We performed transcriptome analysis to identify chemosensory receptors: 21 IRs, 12 GRs, and 43 ORs were identified from B. dorsalis antennae, including almost all previously known chemoreceptors in B. dorsalis and a few more. Quantitative real-time polymerase chain reaction analysis revealed the effects of feeding state, mating status and time of day on the expression of IR, GR, and OR genes. The results showed that expression of chemosensory receptors changed in response to different physiological states, and these changes were completely different for different types of receptors and between male and female flies. Our study suggests that the expressions of chemosensory receptors change to adapt to different physiological states, which may indicate the significant role of these receptors in such physiological processes.

  20. Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1.

    PubMed

    Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard

    2015-01-01

    G protein-coupled receptors (GPCRs) are associated with a wide array of diseases and are targets of most of the medicines sold worldwide. Despite their clinical importance, only 25 unique GPCR structures have been determined as of April 2014. The first step for structural studies is to establish the expression of correctly folded, functional receptors in recombinant host cells at quantities to allow subsequent purification and crystallization trials. Here we describe the T-REx™-inducible expression system to construct and select a stable HEK293 cell line for high-level expression of functional neurotensin receptor type I (NTSR1). We also present the protocols used for the adaptation of the cells into suspension culture, as well as the optimization of the induction parameters for NTSR1 expression, which led to 1 mg of purified NTSR1 per liter suspension culture in bioreactors.

  1. Airborne fine particulate matter alters the expression of endothelin receptors in rat coronary arteries.

    PubMed

    Xiao, Xue; Cao, Lei; Wang, Rong; Shen, Zhen-Xing; Cao, Yong-Xiao

    2016-11-01

    Exposure to airborne fine particulate matter (PM 2.5 ) is associated with cardiovascular diseases. However, a comprehensive understanding of the underlying mechanisms by which PM 2.5 induces or aggravates these diseases is still insufficiently clear. The present study investigated whether PM 2.5 alters the expression of the endothelin subtype B (ET B ) and endothelin subtype A (ET A ) receptors in the coronary artery and examined the underlying mechanisms. Rat coronary artery segments were cultured with PM 2.5 in the presence or absence of MEK/ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was measured by myography. ET B and ET A receptor expression was evaluated using RT-PCR, western blot and immunohistochemistry. Compared with fresh arteries, the cultured coronary arteries showed a significantly enhanced contraction mediated by the ET B receptor and an unaltered contraction mediated by the ET A receptor. Culture with PM 2.5 significantly enhanced the contraction and the mRNA and protein expression levels of the ET B and ET A receptors in the coronary arteries, suggesting that PM 2.5 induces an upregulation of ET A and ET B receptors. In addition, the PM 2.5 -induced increases in ET B - and ET A -mediated vasoconstriction and receptor expressions could be notably decreased by MEK1/2 inhibitor, U0126 and Raf inhibitor, SB386023, suggesting that the upregulation of ET B and ET A receptors is related with MEK/ERK1/2 pathway. In conclusion, PM 2.5 induces the ET B and ET A receptor upregulation in rat coronary arteries, and the MEK/ERK1/2 pathway may be involved in this process. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Finasteride Treatment Alters Tissue Specific Androgen Receptor Expression in Prostate Tissues

    PubMed Central

    Bauman, Tyler M.; Sehgal, Priyanka D.; Johnson, Karen A.; Pier, Thomas; Bruskewitz, Reginald C.; Ricke, William A.; Huang, Wei

    2014-01-01

    BACKGROUND Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (DHT) from testosterone by 5α-reductase. Finasteride is a selective inhibitor of 5α-reductase 2, one isozyme of 5α-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens. METHODS Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients. RESULTS Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy. CONCLUSIONS In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment. PMID:24789081

  3. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    PubMed

    Ziemba, Paul M; Schreiner, Benjamin S P; Flegel, Caroline; Herbrechter, Robin; Stark, Timo D; Hofmann, Thomas; Hatt, Hanns; Werner, Markus; Gisselmann, Günter

    2015-11-27

    Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants. Copyright © 2015. Published by Elsevier Inc.

  4. Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

    SciTech Connect

    Bahouth, S.W.; Malbon, C.C.

    1987-05-01

    Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

  5. Identification and Expression Patterns of Anoplophora chinensis (Forster) Chemosensory Receptor Genes from the Antennal Transcriptome

    PubMed Central

    Sun, Long; Zhang, Ya-Nan; Qian, Jia-Li; Kang, Ke; Zhang, Xiao-Qing; Deng, Jun-Dan; Tang, Yan-Ping; Chen, Cheng; Hansen, Laura; Xu, Tian; Zhang, Qing-He; Zhang, Long-Wa

    2018-01-01

    The citrus long-horned beetle (CLB), Anoplophora chinensis (Forster) is a destructive native pest in China. Chemosensory receptors including odorant receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs) function to interface the insect with its chemical environment. In the current study, we assembled the antennal transcriptome of A. chinensis by next-generation sequencing. We assembled 44,938 unigenes from 64,787,784 clean reads and annotated their putative gene functions based on gene ontology (GO) and Clusters of Orthologous Groups of proteins (COG). Overall, 74 putative receptor genes from chemosensory receptor gene families, including 53 ORs, 17 GRs, and 4 IRs were identified. Expression patterns of these receptors on the antennae, maxillary and labial palps, and remaining body segments of both male and female A. chinensis were performed using quantitative real time-PCR (RT-qPCR). The results revealed that 23 ORs, 6 GRs, and 1 IR showed male-biased expression profiles, suggesting that they may play a significant role in sensing female-produced sex pheromones; whereas 8 ORs, 5 GRs, and 1 IR showed female-biased expression profiles, indicating that these receptors may be involved in some female-specific behaviors such as oviposition site seeking. These results lay a solid foundation for deeply understanding CLB olfactory processing mechanisms. Moreover, by comparing our results with those from chemosensory receptor studies in other cerambycid species, several highly probable pheromone receptor candidates were highlighted, which may facilitate the identification of additional pheromone and/or host attractants in CLB. PMID:29497384

  6. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 11; Express/T-160E Project Express A2 and A3 Data Agreement Document

    NASA Technical Reports Server (NTRS)

    Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.; Dunning, John (Technical Monitor)

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80deg E. and 11deg W., respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3-99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  7. Hall Effect Thruster Interactions Data From the Russian Express-A2 and Express-A3 Satellites. Part 12; Express/T-160 Project Express A2 and A3 Sensors Operations Procedures Document

    NASA Technical Reports Server (NTRS)

    Dunning, John (Technical Monitor); Sitnikova, N.; Volkov, D.; Maximov, I.; Petrusevich, V.; Allen, D.

    2003-01-01

    This 12-part report documents the data obtained from various sensor measurements taken aboard the Russian Express-A2 and Express-A3 spacecraft in Geosynchronous Earth Orbit (GEO). These GEO communications satellites, which were designed and built by NPO Prikladnoy Mekhaniki (NPO PM) of Zheleznogorsk, Russia, utilize Hall thruster propulsion systems for north-south and east-west stationkeeping and as of June 2002, were still operating at 80 deg. E. and 11 deg. W respectively. Express-A2 was launched on March 12, 2000, while Express-A3 was launched on June 24, 2000. The diagnostic equipment from which these data were taken includes electric field strength sensors, ion current and energy sensors, and pressure sensors. The diagnostics and the Hall thruster propulsion systems are described in detail along with lists of tabular data from those diagnostics and propulsion system and other satellite systems. Space Power, Inc., now part of Pratt & Whitney's Chemical Systems Division, under contract NAS3 99151 to the NASA Glenn Research Center, obtained these data over several periods from March 12, 2000, through September 30, 2001. Each of the 12 individual reports describe, in detail, the propulsion systems as well as the diagnostic sensors utilized. Finally, parts 11 and 12 include the requirements to which NPO PM prepared and delivered these data.

  8. Receptor EphA2 activation with ephrinA1 suppresses growth of malignant mesothelioma (MM).

    PubMed

    Nasreen, Najmunnisa; Mohammed, Kamal A; Lai, Yimu; Antony, Veena B

    2007-12-18

    The objective of this study was to understand the possible mechanisms of activation of receptor EphA2 by its ligand ephrinA1 in malignant mesothelioma cell (MMC) growth. Activation of receptor EphA2 by its ligand ephrinA1 triggered the phosphorylation of EphA2. Ligand activation of EphA2 also induced phosphorylation of ERK1/2 and significantly decreased MMC proliferation. Ligand activated and ephrinA1 vector (pcDNA/EFNA1) transfected MMC demonstrated decreased clonal growth in 3-D matrigels when compared to resting MMC. These studies suggest that EphA2 activation by its ligand ephrinA1 transmits intracellular signals from cell membrane to nucleus via ERK1/2 signaling cascade and inhibits MM growth.

  9. Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas

    PubMed Central

    2012-01-01

    Background Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. Results The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. Conclusions Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding. PMID:22647582

  10. Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas.

    PubMed

    Michel, Erika; Feldmann, Stefanie K; Kowalewski, Mariusz P; Bley, Carla Rohrer; Boos, Alois; Guscetti, Franco; Reichler, Iris M

    2012-05-30

    Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding.

  11. Association of Hormone Receptor Expression with Survival in Ovarian Endometrioid Carcinoma: Biological Validation and Clinical Implications.

    PubMed

    Rambau, Peter; Kelemen, Linda E; Steed, Helen; Quan, May Lynn; Ghatage, Prafull; Köbel, Martin

    2017-02-27

    This paper aims to validate whether hormone receptor expression is associated with longer survival among women diagnosed with ovarian endometrioid carcinoma (EC), and whether it identifies patients with stage IC/II tumors with excellent outcome that could be spared from toxic chemotherapy. Expression of estrogen receptor (ER) and progesterone receptor (PR) was assessed on 182 EC samples represented on tissue microarrays using the Alberta Ovarian Tumor Type (AOVT) cohort. Statistical analyses were performed to test for associations with ovarian cancer specific survival. ER or PR expression was present in 87.3% and 86.7% of cases, respectively, with co-expression present in 83.0%. Expression of each of the hormonal receptors was significantly higher in low-grade tumors and tumors with squamous differentiation. Expression of ER (Hazard Ratio (HR) = 0.18, 95% confidence interval 0.08-0.42, p = 0.0002) and of PR (HR = 0.22, 95% confidence interval 0.10-0.53, p = 0.0011) were significantly associated with longer ovarian cancer specific survival adjusted for age, grade, treatment center, stage, and residual disease. However, the five-year ovarian cancer specific survival among women with ER positive stage IC/II EC was 89.0% (standard error 3.3%) and for PR positive tumors 89.9% (standard error 3.2%), robustly below the 95% threshold where adjuvant therapy could be avoided. We validated the association of hormone receptor expression with ovarian cancer specific survival independent of standard predictors in an independent sample set of EC. The high ER/PR co-expression frequency and the survival difference support further testing of the efficacy of hormonal therapy in hormone receptor-positive ovarian EC. The clinical utility to identify a group of women diagnosed with EC at stage IC/II that could be spared from adjuvant therapy is limited.

  12. Cloning and pharmacologic characterization of a thromboxane A2 receptor from K562 (human chronic myelogenous leukemia) cells.

    PubMed

    D'Angelo, D D; Davis, M G; Ali, S; Dorn, G W

    1994-11-01

    Pharmacologic and molecular evidence conflicts in regard to the existence of tissue-specific subtypes of thromboxane A2 receptors (TXR). The full length TXR complementary DNA (cDNA) was cloned from a platelet-like cell line. It was expressed and its pharmacology was characterized. Northern analysis of TXR transcripts in multiple tissues showed strong hybridization to K562 chronic myelogenous leukemia messenger RNA. Therefore, a K562 cDNA library was screened and a full-length TXR cDNA (K562TXR) was isolated. K562TXR encodes a protein identical to the previously characterized placenta TXR cDNA, except for a single amino acid substitution (Glu21-->Lys). Similar to thromboxane receptors on K562 cells, K562TXR transiently expressed in HEK 293 cells (K562TXR/293) bound the thromboxane agonist 125I-labeled [1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3S),4 alpha]-7-[3-(3-hydroxy-4-(p- iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptane-2-yl]-5- heptenoic acid ([125I]BOP) with a Kd of 5.5 +/- 1.1 nM, a Bmax of 289,056 +/- 60,220 sites/cell and a Hill coefficient of -0.94 +/- 0.01 (n = 6). K562TXR/293 cells also demonstrated concentration-dependent increases in intracellular calcium in response to the thromboxane agonist (15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid. In contrast to the single [125I]BOP binding site observed in K562TXR/293, [125I]BOP binding to placental membranes resulted in a Hill coefficient significantly less than unity with a statistically superior two-site model for binding [KdH 0.63 +/- 0.18 nM and KdL of 12.5 +/- 5.0 nM, with Bmaxs of 29 +/- 9 and 212 +/- 41 fmol/mg of protein, respectively (n = 7)].(ABSTRACT TRUNCATED AT 250 WORDS)

  13. Vitamin D receptor and progesterone receptor protein and gene expression in papillary thyroid carcinomas: associations with histological features.

    PubMed

    Yavropoulou, M P; Panagiotou, G; Topouridou, K; Karayannopoulou, G; Koletsa, T; Zarampoukas, T; Goropoulos, A; Chatzaki, E; Yovos, J G; Pazaitou-Panayiotou, K

    2017-12-01

    Vitamin D receptor (VDR) and progesterone receptor (PR) expression has been described in papillary thyroid carcinoma (PTC) but data regarding association with tumor histological characteristics and localization of the protein expression are scarce. Formalin-fixed, paraffin-embedded specimens from 45 patients with PTC (cases) were retrieved and tumor histological data were recorded. We analyzed gene and protein expression of VDR and PR and gene expression of vitamin D-inactivating 24-hyroxylase (CYP24A1) and the activating 1-alpha-hydroxylase (CYP27B1) enzymes in follicular cancer cells and the adjacent non-neoplastic thyroid tissue (NNTT). VDR mRNA and protein expression was higher in PTC compared with NNTT (p < 0.05). The protein was globally localized in the cytoplasm and cell membranes of the neoplastic cells in all cases, with differences in intensity. Cytoplasmic positivity was stronger in the majority of cases. Membranous positivity was also evident in cases, whereas in NNTT was generally weak and in a low percentage of the cells. Expression of CYP 24A1, but not CYP27B1, was increased in approximately all PTC specimens and was associated with lymph node metastasis and extrathyroidal extension. PR mRNA was increased in 34% and protein expression was present in 57% of cases, and none of NNTT. PR, but not VDR, mRNA expression was significantly associated with the tumor size (r = 0.645, p = 0.007). We provide evidence for the expression pattern of VDR, PR and CYP24A1 in the progression of PTC. Rapid anti-tumor responses of vitamin D in PTC may be blocked due to inactivation of local vitamin D metabolism.

  14. β2-Adrenergic receptor-dependent chemokine receptor 2 expression regulates leukocyte recruitment to the heart following acute injury

    PubMed Central

    Grisanti, Laurel A.; Traynham, Christopher J.; Repas, Ashley A.; Gao, Erhe; Koch, Walter J.; Tilley, Douglas G.

    2016-01-01

    Following cardiac injury, early immune cell responses are essential for initiating cardiac remodeling and tissue repair. We previously demonstrated the importance of β2-adrenergic receptors (β2ARs) in the regulation of immune cell localization following acute cardiac injury, with deficient leukocyte infiltration into the damaged heart. The purpose of this study was to investigate the mechanism by which immune cell-expressed β2ARs regulate leukocyte recruitment to the heart following acute cardiac injury. Chemokine receptor 2 (CCR2) expression and responsiveness to C-C motif chemokine ligand 2 (CCL2)-mediated migration were abolished in β2AR knockout (KO) bone marrow (BM), both of which were rescued by β2AR reexpression. Chimeric mice lacking immune cell-specific CCR2 expression, as well as wild-type mice administered a CCR2 antagonist, recapitulated the loss of monocyte/macrophage and neutrophil recruitment to the heart following myocardial infarction (MI) observed in mice with immune cell-specific β2AR deletion. Converse to β2AR ablation, β2AR stimulation increased CCR2 expression and migratory responsiveness to CCL2 in BM. Mechanistically, G protein-dependent β2AR signaling was dispensable for these effects, whereas β-arrestin2–biased β2AR signaling was required for the regulation of CCR2 expression. Additionally, activator protein 1 (AP-1) was shown to be essential in mediating CCR2 expression in response to β2AR stimulation in both murine BM and human monocytes. Finally, reconstitution of β2ARKO BM with rescued expression of a β-arrestin–biased β2AR in vivo restored BM CCR2 expression as well as cardiac leukocyte infiltration following MI. These results demonstrate the critical role of β-arrestin2/AP-1–dependent β2AR signaling in the regulation of CCR2 expression and recruitment of leukocytes to the heart following injury. PMID:27956622

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

    PubMed

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

    2006-09-05

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

  16. Cloning and olfactory expression of progestin receptors in the Chinese black sleeper Bostrichthys sinensis

    PubMed Central

    Zhang, Yu Ting; Liu, Dong Teng; Zhu, Yong; Chen, Shi Xi; Hong, Wan Shu

    2017-01-01

    Our previous studies suggested that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), an oocyte maturation inducing progestin, also acts as a sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. The electroolfactogram response to DHP increased during the breeding season. In the present study, we cloned the cDNAs of the nine progestin receptors (pgr, paqr5, 6, 7(a, b), 8, 9, pgrmc1, 2) from B. sinensis, analyzed their tissue distribution, and determined the expression in the olfactory rosette during the reproductive cycle in female and male fish. The deduced amino acid sequences of the nine progestin receptors share high sequence identities with those of other fish species and relatively lower homology with their mammalian counterparts, and phylogenetic analyses classified the nine B. sinensis progestin receptors into their respective progestin receptor groups. Tissue distribution of B. sinensis progestin receptors showed differential expression patterns, but all these nine genes were expressed in the olfactory rosette. Interestingly, paqr5 mRNA was found in the intermediate and basal parts of the olfactory epithelium but not in the central core using in situ hybridization, and its expression level was the highest in the olfactory rosette among the tissues examined. These results suggested Paqr5 may have an important role for transmitting progestin signaling in the olfactory system. The expression levels of paqr7a and paqr7b, pgr and pgrmc2 mRNA peaked around the mid meiotic stage, and that of paqr8 peaked at late meiotic stage in the olfactory rosette in males, while the olfactory expression of paqr5 decreased gradually as spermatogenesis progressed. In contrast, the expression of the progestin receptors did not change significantly during the development of the ovary in the olfactory rosette in females, except that of pgr. Interestingly, the changes of paqr8 expression in

  17. Steroid receptors in the adult zebra finch syrinx: a sex difference in androgen receptor mRNA, minimal expression of estrogen receptor alpha and aromatase.

    PubMed

    Veney, Sean L; Wade, Juli

    2004-04-01

    The zebra finch syrinx (sound production organ) is a sexually dimorphic component of the song system. Only male zebra finches sing, and in parallel, the overall mass and size of fibers in the two largest syrinx muscles are greater in males than females. Despite these obvious sexual dimorphisms, little is known about the role of steroid hormones in the maintenance of the structure and/or function of the syrinx. In this report, we used in situ hybridization to assess the expression of androgen receptor (AR), estrogen receptor alpha (ERalpha), and aromatase (AROM) mRNAs in the syrinx of adult male and female zebra finches. Increased AR mRNA expression was noted in males compared to females in two regions, over the ventralis muscle and in a band of connective tissue neighboring cartilage (perichondria). In contrast, we did not detect specific ERalpha or AROM mRNA expression within the syrinx. However, substantial ERalpha mRNA was present in oviduct, and aromatase mRNA was expressed at high levels in ovary. In parallel, an assay for AROM detected activity in ovary, but not in syrinx tissue from males or females. Taken together, these data suggest that the adult syrinx is sensitive to androgens; that sex differences in function and morphology of the syrinx may in part be due to increased expression of AR in males compared to females. In contrast, estrogen receptor alpha and AROM appear to have limited roles.

  18. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    SciTech Connect

    Takemura, Kenichi; Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto; Sakashita, Naomi

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased.more » An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.« less

  19. Vitamin D receptor expression in canine mammary gland and relationship with clinicopathological parameters and progesterone/oestrogen receptors.

    PubMed

    Sánchez-Céspedes, R; Fernández-Martínez, M D; Raya, A; Pineda, C; López, I; Millán, Y

    2018-03-01

    The vitamin D receptor (VDR) belongs to the nuclear class II receptor family. VDR is a ligand transcription factor and mediates the actions of calcitriol, the active product of vitamin D synthesis. Nowadays, it is known that the biological actions of calcitriol include the capacity to modulate cancer features, such as proliferation and differentiation, apoptosis, angiogenesis, invasion and metastasis. VDR expression has been demonstrated in human breast cancer and vitamin D has emerged as a promising targeted therapy. We analyse the VDR expression in normal and neoplastic canine mammary tissue samples and its relationship with clinicopathological parameters and progesterone/oestrogens receptors (PR/ER). Expression of VDR, Ki67 (to evaluate the proliferation index, PI), PR and ER was assessed in 50 mammary gland tissue samples from 41 female dogs by immunohistochemistry. VDR-positive staining was found in the nuclei of both myoepithelial and luminal epithelial cell layers. VDR expression was higher in normal mammary tissue (37/37 cases, 100%) then followed by benign tumours (6/15 cases, 40%) and malignant tumours (9/34 cases, 26.5%) (P = .001). Female dogs aged ≥10 years had lower VDR expression compared with dogs younger (P = .017). Relationship between VDR and breed, number of tumours, tumour size, histologic subtype, histologic grade of malignancy, PI and PR and ER expression was not observed. Studies with more samples are necessary to further evaluate the possible role of VDR in the biological behaviour of canine mammary tumours, and to corroborate the possibility to use the dog as model for human breast cancer. © 2017 John Wiley & Sons Ltd.

  20. The DAF-7 TGF-β signaling pathway regulates chemosensory receptor gene expression in C. elegans

    PubMed Central

    Nolan, Katherine M.; Sarafi-Reinach, Trina R.; Horne, Jennifer G.; Saffer, Adam M.; Sengupta, Piali

    2002-01-01

    Regulation of chemoreceptor gene expression in response to environmental or developmental cues provides a mechanism by which animals can alter their sensory responses. Here we demonstrate a role for the daf-7 TGF-β pathway in the regulation of expression of a subset of chemoreceptor genes in Caenorhabditis elegans. We describe a novel role of this pathway in maintaining receptor gene expression in the adult and show that the DAF-4 type II TGF-β receptor functions cell-autonomously to modulate chemoreceptor expression. We also find that the alteration of receptor gene expression in the ASI chemosensory neurons by environmental signals, such as levels of a constitutively produced pheromone, may be mediated via a DAF-7-independent pathway. Receptor gene expression in the ASI and ASH sensory neurons appears to be regulated via distinct mechanisms. Our results suggest that the expression of individual chemoreceptor genes in C. elegans is subject to multiple modes of regulation, thereby ensuring that animals exhibit the responses most appropriate for their developmental stage and environmental conditions. PMID:12464635

  1. Inhibition of microglial P2X4 receptors attenuates morphine tolerance, Iba1, GFAP and μ opioid receptor protein expression while enhancing perivascular microglial ED2

    PubMed Central

    Horvath, Ryan J.; Romero-Sandoval, Edgar A.; De Leo, Joyce A.

    2010-01-01

    Antinociceptive tolerance to opioids is a well-described phenomenon, which severely limits the clinical efficacy of opioids for the treatment of chronic pain syndromes. The mechanisms that drive antinociceptive tolerance, however, are less well understood. We have previously shown that glia have a central role in the development of morphine tolerance and that administration of a glial modulating agent attenuated tolerance formation. Recently, we have demonstrated that morphine enhances microglial Iba1 expression and P2X4 receptor-mediated microglial migration via direct μ opioid receptor signaling in in vitro microglial cultures. We hypothesize that P2X4 receptors drive morphine tolerance and modulate morphine-induced spinal glial reactivity. Additionally, we hypothesize that perivascular microglia play a role in morphine tolerance and that P2X4 receptor expression regulates perivascular microglia ED2 expression. To test these hypotheses, rats were implanted with osmotic minipumps releasing morphine or saline subcutaneously for seven days. Beginning three days prior to morphine treatment, P2X4 receptor antisense oligonucleotide (asODN) was injected intrathecally daily, to selectively inhibit P2X4 receptor expression. P2X4 receptor asODN treatment inhibited morphine-induced P2X4 receptor expression and blocked antinociceptive tolerance to systemically administered morphine. P2X4 receptor asODN treatment also attenuated the morphine-dependent increase of spinal ionized calcium binding protein (Iba1), glial fibrillary acidic protein (GFAP) and μ opioid receptor protein expression. Chronic morphine also decreased perivascular microglial ED2 expression, which was reversed by P2X4 receptor asODN. Together, these data suggest that modulation of P2X4 receptor expression on microglia and perivascular microglia may prove an attractive target for adjuvant therapy to attenuate opioid-induced antinociceptive tolerance. PMID:20573450

  2. Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

    PubMed

    Vulfius, Catherine A; Kasheverov, Igor E; Starkov, Vladislav G; Osipov, Alexey V; Andreeva, Tatyana V; Filkin, Sergey Yu; Gorbacheva, Elena V; Astashev, Maxim E; Tsetlin, Victor I; Utkin, Yuri N

    2014-01-01

    Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

  3. The expression and function of the NKRP1 receptor family in C57BL/6 mice.

    PubMed

    Aust, Jonathan G; Gays, Frances; Mickiewicz, Katarzyna M; Buchanan, Ella; Brooks, Colin G

    2009-07-01

    NKRP1 receptors were discovered more than 20 years ago, but due to a lack of appropriate reagents, our understanding of them has remained limited. Using a novel panel of mAbs that specifically recognize mouse NKRP1A, D, and F molecules, we report here that NKRP1D expression is limited to a subpopulation of NK cells, but in contrast to Ly49 receptors appears to be expressed in a normal codominant manner. NKRP1D(-) and NKRP1D(+) NK cells are functionally distinct, NKRP1D(+) cells showing reduced expression of various Ly49 receptors, elevated expression of CD94/NKG2 receptors, and higher IFN-gamma secretion and cytotoxicity than NKRP1D(-) cells. Furthermore, NKRP1D(+) NK cells were unable to kill transfected cells expressing high levels of Clr-b molecules, but readily killed MHC class-I-deficient blast cells that express only low levels of Clr-b. NKRP1A and NKRP1F were expressed at low levels on all splenic and bone marrow NK cells, but mAb-induced cross-linking of NKRP1A and NKRP1F caused no significant enhancement or inhibition of NK cell cytotoxicity and no detectable production of IFN-gamma. NKRP1A, D, and F expression could not be detected on NKT cells, all of which express NKRP1C, and although some activated T cells expressed NKRP1C and perhaps low levels of NKRP1A, no significant expression of NKRP1D or F could be detected. NKRP1 molecules expressed on NK cells or transfectants were down-regulated by cross-linking with mAbs or cell surface ligands, and using this phenomenon as a functional assay for NKRP1-ligand interaction revealed that NKRP1F can recognize CLR-x.

  4. Profile of BAFF and its receptors' expression in lupus nephritis is associated with pathological classes.

    PubMed

    Suso, J P; Posso-Osorio, I; Jiménez, C A; Naranjo-Escobar, J; Ospina, F E; Sánchez, A; Cañas, C A; Tobón, G J

    2018-04-01

    Background/Objective B-cell activating factor (BAFF) plays an important role in the pathogenesis of systemic lupus erythematosus. However, the role of BAFF in lupus nephritis (LN) is not understood. Our aim was to evaluate the expression of BAFF and its three receptors in renal biopsy samples from patients with LN and investigate a relationship with pathological class. Methods We conducted a prospective descriptive study (2011-2014) on 52 kidney biopsy samples from patients with LN. Immunohistochemistry for BAFF, its receptors (transmembrane activator and calcium modulator and cyclophilin ligand interaction (TACI), protein maturation of B cells (BCMA), and BAFF-receptor (BAFF-R)), and CD20 expression was performed. Samples were scored according to the percentage of cells with positive expression. Results In class II LN, BAFF-R and TACI were not expressed, whereas BCMA and BAFF were lowly expressed in the interstitial inflammatory infiltrates. Proliferative class III/IV had elevated BAFF expression in the glomeruli, and TACI was expressed in interstitial inflammatory infiltrates and the glomeruli. Interestingly, the class IV cases with vasculopathy ( n = 4) had endothelial BAFF expression, which was not visible in thrombotic microangiopathy ( n = 4). Class V was characterized by low BAFF expression in interstitial inflammatory infiltrates and by BAFF, TACI, and BCMA expression in the glomeruli. BAFF expression was associated with inflammatory scores and CD20 positive infiltrates, mainly in class IV. Conclusions Expression patterns of BAFF and its receptors differ according to LN class. Our study provides evidence that BAFF could be used as a routine marker in LN biopsies and to determine which patients will benefit from anti-BAFF therapy.

  5. A Role for a Specific Cholesterol Interaction in Stabilizing the Apo Configuration of the Human A2A Adenosine Receptor

    PubMed Central

    Lyman, Edward; Higgs, Chris; Kim, Byungchan; Lupyan, Dmitry; Shelley, John C.; Farid, Ramy; Voth, Gregory A.

    2009-01-01

    SUMMARY The function of G-protein coupled receptors is tightly modulated by the lipid environment. Long timescale molecular dynamics simulations (totaling ~3 microsec) of the A2A receptor in cholesterol-free bilayers, with and without the antagonist ZM241385 bound, demonstrate an instability of helix II in the apo receptor in cholesterol-poor membrane regions. We directly observe that the effect of cholesterol binding is to stabilize helix II against a buckling type deformation, perhaps rationalizing the observation that the A2A receptor couples to G-protein only in the presence of cholesterol (Zezula and Freissmuth, 2008). The results suggest a mechanism by which the A2A receptor may function as a coincidence detector, activating only in the presence of both cholesterol and agonist. We also observed a previously hypothesized conformation of the tryptophan “rotameric switch” on helix VI in which a phenylalanine on helix V positions the tryptophan out of the ligand binding pocket. PMID:20004169

  6. Heterogeneous expression of cholecystokinin and gastrin receptor in stomach and pancreatic cancer: An immunohistochemical study.

    PubMed

    Rai, Rajani; Kim, Jong Joo; Tewari, Mallika; Shukla, Hari Shankar

    2016-01-01

    Cholecystokinin (CCK) and gastrin (Gs) are a well known trophic factor for the gastrointestinal tract and their trophic effects are shown mainly toward pancreas and stomach, respectively. Though, the exact characterization of CCK and Gs receptors subtype (cholecystokinin type A receptor [CCKAR] and cholecystokinin type B receptor/gastrin receptor [CCKBR/GR]) in stomach cancer (SC) and pancreatic cancer (PC) is still controversial and necessities further validation. CCKAR and CCKBR/GR expression was analyzed by immunohistochemistry in 55 SC, 25 benign gastric diseases (BGDs), 38 PC (including periampullary carcinoma), and 10 normal pancreatic tissue. The results were statistically correlated with the patient's clinical history to observe the prognostic significance if any. CCKAR expression was detected in 18.2% of SC, 20% of BGD, 65.8% of PC, and 30.0% of normal pancreas tissue samples. The CCKBR/GR expression was detected in 58.2% of SC, 48.0% of BGD, 18.4% of PC, and 60.0% of normal pancreas tissue samples. CCKBR/GR expression was significantly high in well and moderately differentiated SC samples as compared to poorly differentiated samples. Our study showed significantly higher expression of CCKAR and down regulation of CCKBR in PC as compared to control while CCKBR/GR was detected in majority of SC samples. Thus, our study suggests that CCK and Gs receptors may have diagnostic and therapeutic implications. However, study need to be validated in significantly bigger sample size and need to be replicated in different cohorts.

  7. Temporal and spatial expression patterns of Hedgehog receptors in the developing inner and middle ear.

    PubMed

    Shin, Jeong-Oh; Ankamreddy, Harinarayana; Jakka, Naga Mahesh; Lee, Seokwon; Kim, Un-Kyung; Bok, Jinwoong

    2017-01-01

    The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development.

  8. Effects of phytoestrogen genistein on genioglossus function and oestrogen receptors expression in ovariectomized rats.

    PubMed

    Li, Wen; Liu, Yue-hua

    2009-11-01

    This study was designed to investigate the effects of genistein on genioglossal muscle function and the expression of oestrogen receptors (ERs) in the ovariectomized rats. Fifty female Sprague-Dawley rats were randomly divided into 5 groups: the control group (SHAM), the ovariectomized group (OVX), the ovariectomized rats receiving low genistein dosage (OVX+L), the moderate genistein dosage group (OVX+M) and the high genistein dosage group (OVX+H). Oestradiol level was detected by radioimmunity. The isometric twitch tension (P(t)) and tetanic tension (P(0)) of the GG muscle were measured in response to electrical field stimulation. The expression of ERs on the mRNA and protein levels was measured by real-time PCR and western blot respectively. Ovariectomy decreased muscle fatigue resistance and the expression of different ERs significantly. Genistein treatment resulted in a dose-dependent protective effect on muscle fatigability and a parallel dose-responsive increase in the expression of oestrogen receptors mRNA and protein levels in genioglossus, with larger effects on oestrogen receptor beta vs. alpha. In contrast to the improvements in fatigability, there was no treatment effect on isometric twitch or tetanic tensions. The results indicated that genistein increased muscle fatigue resistance in addition to effects on receptors, and the up-regulation of receptors expression may be a possible mechanism by which genistein improved fatigue.

  9. Recombinant expression of extracellular domain of mutant Epidermal Growth Factor Receptor in prokaryotic and baculovirus expression systems.

    PubMed

    Vettath, Sunitha Kodengil; Shivashankar, Gaganashree; Menon, Krishnakumar N; Vijayachandran, Lakshmi S

    2018-04-15

    Epidermal Growth Factor Receptor variant III (EGFRvIII) is a tumor specific antigen detected in various tumors including gliomas, breast cancer, lung cancer, head and neck squamous cell carcinoma (HNSCC). Screening of EGFRvIII targeting drug molecules can be accelerated by developing drug screening platforms using recombinantly expressed protein. Choice of expression system is one of the major factors deciding the success of recombinant expression of a protein. In our study, we have tried to express and purify the extracellular domain (ECD) of this highly unstable protein using bacterial and baculovirus expression systems to select the expression system suited for our purpose. Even though the protein was successfully expressed in prokaryotic system, purification could be done only under denaturing conditions. But in the baculovirus expression system, the protein was expressed in soluble form and could be purified under native conditions, with single step of purification. Based on our results, we conclude that insect cells are better choice over E. coli cells for expressing EGFRvIII ECD in soluble form. This study provides insights for other researchers involved in expression of similar unstable membrane proteins, on selecting the best expression system and challenges involved. Copyright © 2018 Elsevier B.V. All rights reserved.

  10. Metformin sensitizes triple-negative breast cancer to proapoptotic TRAIL receptor agonists by suppressing XIAP expression.

    PubMed

    Strekalova, Elena; Malin, Dmitry; Rajanala, Harisha; Cryns, Vincent L

    2017-06-01

    Despite robust antitumor activity in diverse preclinical models, TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists have not demonstrated efficacy in clinical trials, underscoring the need to identify agents that enhance their activity. We postulated that the metabolic stress induced by the diabetes drug metformin would sensitize breast cancer cells to TRAIL receptor agonists. Human triple (estrogen receptor, progesterone receptor, and HER2)-negative breast cancer (TNBC) cell lines were treated with TRAIL receptor agonists (monoclonal antibodies or TRAIL peptide), metformin, or the combination. The effects on cell survival, caspase activation, and expression of TRAIL receptors and the antiapoptotic protein XIAP were determined. In addition, XIAP was silenced by RNAi in TNBC cells and the effects on sensitivity to TRAIL were determined. The antitumor effects of metformin, TRAIL, or the combination were evaluated in an orthotopic model of metastatic TNBC. Metformin sensitized diverse TNBC cells to TRAIL receptor agonists. Metformin selectively enhanced the sensitivity of transformed breast epithelial cells to TRAIL receptor agonist-induced caspase activation and apoptosis with little effect on untransformed breast epithelial cells. These effects of metformin were accompanied by robust reductions in the protein levels of XIAP, a negative regulator of TRAIL-induced apoptosis. Silencing XIAP in TNBC cells mimicked the TRAIL-sensitizing effects of metformin. Metformin also enhanced the antitumor effects of TRAIL in a metastatic murine TNBC model. Our findings indicate that metformin enhances the activity of TRAIL receptor agonists, thereby supporting the rationale for additional translational studies combining these agents.

  11. 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

  12. Regulation of Expression of Citrate Synthase by the Retinoic Acid Receptor-Related Orphan Receptor α (RORα)

    PubMed Central

    Crumbley, Christine; Wang, Yongjun; Banerjee, Subhashis; Burris, Thomas P.

    2012-01-01

    The retinoic acid receptor-related orphan receptor α (RORα) is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS) is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE) in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression. PMID:22485150

  13. Expression of chemokines and chemokine receptors during human renal transplant rejection.

    PubMed

    Segerer, S; Cui, Y; Eitner, F; Goodpaster, T; Hudkins, K L; Mack, M; Cartron, J P; Colin, Y; Schlondorff, D; Alpers, C E

    2001-03-01

    Infiltration of renal allografts by leukocytes is a hallmark of acute transplant rejection. Chemokines attract leukocytes bearing specific chemokine receptors, and the specific leukocyte chemokine receptor phenotype is associated with types of immune responses, ie, T helper subtype 1 (Th1; CXC chemokine receptor 3 [CXCR3], CC chemokine receptor 5 [CCR5]) versus Th2 (CCR3, CCR4, CCR8). We studied the expression of the chemokine monocyte chemoattractant protein-1 and the chemokine receptors CCR2B and CXCR4 messenger RNA (mRNA) by in situ hybridization, as well as the chemokine receptors Duffy antigen receptor for chemokines (DARC) and CCR5 protein by immunohistochemistry in renal biopsy specimens with acute cellular rejection (n = 12) and acute vascular rejection (n = 8), transplant nephrectomy specimens (n = 6), and normal areas of tumor nephrectomy specimens (n = 5). CC chemokines and CC chemokine receptor mRNA expression were evaluated by ribonuclease protection assay in specimens from four transplant nephrectomies and one tumor nephrectomy. Upregulation of mRNAs for the chemokines, interferon-inducible protein-10 (IP-10); regulated on activation normal T-cell expressed and secreted; macrophage inflammatory protein-1alpha (MIP-1alpha); MIP-1beta; and lymphotactin, as well as the chemokine receptors, CCR2 and CCR5, were documented during allograft rejection. CCR1 mRNA was detectable in both allografts and controls, but CCR3 and CCR8 were absent. The number of CXCR4, CCR5, and CCR2B mRNAs expressing leukocytes and DARC-positive vessels increased during rejection episodes. CXCR4 mRNA was the most widely expressed. Leukocytes in diffuse interstitial infiltrates were mainly CCR5 positive, but in areas in which leukocytes formed nodular aggregates of infiltrating cells, the number of CCR5-positive cells was low. Instead, leukocytes in these nodular aggregates mainly expressed CXCR4. DARC was expressed on peritubular capillaries, where it was upregulated in areas of

  14. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  15. Brain-penetrant tetrahydronaphthalene thromboxane A2-prostanoid (TP) receptor antagonists as prototype therapeutics for Alzheimer's disease.

    PubMed

    Soper, James H; Sugiyama, Shimpei; Herbst-Robinson, Katie; James, Michael J; Wang, Xiaozhao; Trojanowski, John Q; Smith, Amos B; Lee, Virginia M-Y; Ballatore, Carlo; Brunden, Kurt R

    2012-11-21

    A hallmark pathological feature of the Alzheimer's disease (AD) brain is the presence of senile plaques, which comprise amyloid β (Aβ) peptides that are derived from the amyloid precursor protein (APP). The plaque-containing AD brain is thought to be under oxidative stress, as evidenced by increased lipid oxidation products that include isoprostane-F2αIII (iPF2αIII). IPF2αIII can bind to and activate the thromboxane A2-prostanoid (TP) receptor, and TP receptor activation causes increased Aβ production through enhancement of APP mRNA stability. Moreover, TP receptor antagonists have been shown to block iPF2αIII-induced increases of Aβ secretion. Thus, the TP receptor may be a potential drug target for AD therapy. However, here we show that existing TP receptor antagonists have poor blood-brain barrier (BBB) permeability, likely due to the presence of a carboxylic acid moiety that is believed to be important for receptor interaction, but which may hamper passive diffusion across the BBB. We now report selected analogues of a known tetrahydronaphthalene TP receptor antagonist, wherein the carboxylic acid moiety has been replaced by heterocyclic bioisosteres. These heterocyclic analogues retained relatively high affinity for the mouse and human TP receptors, and, unlike the parent carboxylic acid compound, several examples freely diffused across the BBB into the brain upon administration to mice. These results reveal that brain-penetrant tetrahydronaphthalene TP receptor antagonists can be developed by substituting the carboxylic acid moiety with a suitable nonacidic bioisostere. Compounds of this type hold promise as potential lead structures to develop drug candidates for the treatment of AD.

  16. Sequence, genomic organization and expression of two channel catfish, Ictalurus punctatus, ghrelin receptors.

    PubMed

    Small, Brian C; Quiniou, Sylvie M A; Kaiya, Hiroyuki

    2009-12-01

    Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration on target tissue mRNA expression. Analysis of sequence similarities indicated two genes putatively encoding GHS-R1 and GHS-R2, respectively, which have been known to be present in zebrafish. Organization and tissue expression of the GHS-R1 gene was similar to that reported for other species, and likewise yielded two detectable mRNA products as a result of alternative splicing. Expression of both full-length, GHS-R1a, and splice variant, GHS-R1b, mRNA was highest in the pituitary. Gene organization of GHS-R2 was similar to GHS-R1, but no splice variant was identified. Expression of GHS-R2a mRNA was highest in the Brockmann bodies. GHS-R1a mRNA was detected in unfertilized eggs and throughout embryogenesis, whereas GHR-R2a mRNA was not expressed in unfertilized eggs or early developing embryos and was the highest at the time of hatching. Catfish intraperitoneally injected with catfish ghrelin-Gly had greater mRNA expression of GHS-R1a in pituitaries at 2 h and Brockmann bodies at 4 h, and of GHS-R2a in Brockmann bodies at 6 h post injection. Amidated catfish ghrelin (ghrelin-amide) had no observable effect on expression of either pituitary receptor; however, GHS-R1a and GHS-R2a mRNA expression levels were increased 4 h post injection of ghrelin-amide in Brockmann bodies. This is the first characterization of GHS-R2a and suggests regulatory and functional differences between the two catfish receptors.

  17. CHARACTERIZATION OF THE EXPRESSION PATTERN OF ADRENERGIC RECEPTORS IN RAT TASTE BUDS

    PubMed Central

    ZHANG, Y.; KOLLI, T.; HIVLEY, R.; JABER, L.; ZHAO, F-l.; YAN, J.; HERNESS, S.

    2010-01-01

    Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of α1A, α1B, α1D, α2A, α2B, α2C, β1, and the β2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine β-hydroxylase (DβH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell RT-PCR. These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play

  18. Expression of Toll-like Receptor 2 and Toll-like Receptor 4 in Tuberculous Pleural Effusion.

    PubMed

    Lin, Yingzhong; Feng, Tingmei; Lan, Jiao; Chen, Chunxia; Qin, Zhiqiang; Wu, Yanbin; Shi, Huanzhong; Ye, Jianbo; Wei, Caizhou; Wang, Wu; Huang, Luying

    2017-01-01

    Toll-like receptor-2 (TLR2) and Toll-like receptor-4 (TLR4) have been reported to play a crucial role in tuberculosis, however, little is known about their expression in tuberculous pleuritis. The goal of this work is to explore the expressions of TLR2 and TLR4 in tuberculous pleuritis and their predominant expressions on cells. Levels of soluble TLR2 and TLR4 by enzyme linked immunosorbent assay (ELISA) in 58 patients with tuberculous pleural effusion (PE) and 43 patients with malignant PE were determined. The related genes were analyzed by RT-PCR and the membrane expressions of TLR2 and TLR4 on CD3+, CD14+, and CD19+ monocytes were assessed by using flow cytometry in 20 of 58 patients with tuberculous pleuritis. Our results showed that the levels of ADA, IL-27 and IFN-γ in tuberculous PE were obviously higher than in malignant PE. Moreover, the concentrations of soluble TLR2 and soluble TLR4 in PE were significantly higher than those in peripheral blood of the same patients, as well as the levels of soluble TLR2 in tuberculous PE were significantly higher than those in malignant effusions. Furthermore, the levels of TLR2, TLR4 and IFN-γ mRNA expression were marked increased in the tuberculous PE when compared with the correspondent serum. Importantly, we found that the predominant expressions of TLR2 in monocyte were on CD19 B cells, and the predominant expressions of TLR4 were on CD14 monocytes/macrophages. Our findings provided the evidence of a role for TLRs expression in tuberculous PE. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  19. Arx together with FoxA2, regulates Shh floor plate expression.

    PubMed

    Cho, Ginam; Lim, Youngshin; Cho, Il-Taeg; Simonet, Jacqueline C; Golden, Jeffrey A

    2014-09-01

    Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Arx together with FoxA2, regulates Shh floor plate expression

    PubMed Central

    Cho, Ginam; Lim, Youngshin; Cho, Il-Taeg; Simonet, Jacqueline C.; Golden, Jeffrey A.

    2014-01-01

    Mutations in the Aristaless related homeodomain transcription factor (ARX) are associated with a diverse set of X-linked mental retardation and epilepsy syndromes in humans. Although most studies have been focused on its function in the forebrain, ARX is also expressed in other regions of the developing nervous system including the floor plate (FP) of the spinal cord where its function is incompletely understood. To investigate the role of Arx in the FP, we performed gain-of-function studies in the chick using in ovo electroporation, and loss-of-function studies in Arx-deficient mice. We have found that Arx, in conjunction with FoxA2, directly induces Sonic hedgehog (Shh) expression through binding to a Shh floor plate enhancer (SFPE2). We also observed that FoxA2 induces Arx through its transcriptional activation domain whereas Nkx2.2, induced by Shh, abolishes this induction. Our data support a feedback loop model for Arx function; through interactions with FoxA2, Arx positively regulates Shh expression in the FP, and Shh signaling in turn activates Nkx2.2, which suppresses Arx expression. Furthermore, our data are evidence that Arx plays a role as a context dependent transcriptional activator, rather than a primary inducer of Shh expression, potentially explaining how mutations in ARX are associated with diverse, and often subtle, defects. PMID:24968361