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Sample records for activator receptor upar

  1. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm; Iversen, Frank; Liu, Zhuo; Thomsen, Karen; Pedersen, Michael; Skrydstrup, Troels; Nielsen, Niels Chr.; Ploug, Michael; Kjems, Jørgen

    2013-08-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific targeting peptide onto polyethylene glycol (PEG) coated USPIO nanoparticles by click chemistry resulted in a five times higher uptake in vitro in a uPAR positive cell line compared to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient management when combined with MRI and drug delivery.Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor patient prognosis shared by several cancers including breast, colorectal, and gastric cancers. Conjugation of a uPAR specific

  2. Targeting of peptide conjugated magnetic nanoparticles to urokinase plasminogen activator receptor (uPAR) expressing cells

    Hansen, Line; Unmack Larsen, Esben Kjær; Nielsen, Erik Holm

    2013-01-01

    Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are currently being used as a magnetic resonance imaging (MRI) contrast agent in vivo, mainly by their passive accumulation in tissues of interest. However, a higher specificity can ideally be achieved when the nanoparticles are targeted...... towards cell specific receptors and this may also facilitate specific drug delivery by an enhanced target-mediated endocytosis. We report efficient peptide-mediated targeting of magnetic nanoparticles to cells expressing the urokinase plasminogen activator receptor (uPAR), a surface biomarker for poor...... to nanoparticles carrying a non-binding control peptide. In accordance with specific receptor-mediated recognition, a low uptake was observed in the presence of an excess of ATF, a natural ligand for uPAR. The uPAR specific magnetic nanoparticles can potentially provide a useful supplement for tumor patient...

  3. Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR and concomitant activation of gelatinolytic enzymes.

    Synnøve Magnussen

    Full Text Available The urokinase plasminogen activator receptor (uPAR is associated with poor prognosis in oral squamous cell carcinoma (OSCC, and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational

  4. Structure, function and expression on blood and bone marrow cells of the urokinase-type plasminogen activator receptor, uPAR

    Plesner, T; Behrendt, N; Ploug, M

    1997-01-01

    Several important functions have been assigned to the receptor for urokinase-type plasminogen activator, uPAR. As implied by the name, uPAR was first identified as a high affinity cellular receptor for urokinase plasminogen activator (uPA). It mediates the binding of the zymogen, pro-uPA, to the ...

  5. The receptor for urokinase-plasminogen activator (uPAR) controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style.

    Margheri, Francesca; Luciani, Cristina; Taddei, Maria Letizia; Giannoni, Elisa; Laurenzana, Anna; Biagioni, Alessio; Chillà, Anastasia; Chiarugi, Paola; Fibbi, Gabriella; Del Rosso, Mario

    2014-03-30

    The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the "path generating" mesenchymal to the "path finding" amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly.

  6. The receptor for urokinase-plasminogen activator (uPAR) controls plasticity of cancer cell movement in mesenchymal and amoeboid migration style

    Taddei, Maria Letizia; Giannoni, Elisa; Laurenzana, Anna; Biagioni, Alessio; Chillà, Anastasia; Chiarugi, Paola; Fibbi, Gabriella; Rosso1, Mario Del

    2014-01-01

    The receptor for the urokinase plasminogen activator (uPAR) is up-regulated in malignant tumors. Historically the function of uPAR in cancer cell invasion is strictly related to its property to promote uPA-dependent proteolysis of extracellular matrix and to open a path to malignant cells. These features are typical of mesenchymal motility. Here we show that the full-length form of uPAR is required when prostate and melanoma cancer cells convert their migration style from the “path generating” mesenchymal to the “path finding” amoeboid one, thus conferring a plasticity to tumor cell invasiveness across three-dimensional matrices. Indeed, in response to a protease inhibitors-rich milieu, prostate and melanoma cells activated an amoeboid invasion program connoted by retraction of cell protrusions, RhoA-mediated rounding of the cell body, formation of a cortical ring of actin and a reduction of Rac-1 activation. While the mesenchymal movement was reduced upon silencing of uPAR expression, the amoeboid one was almost completely abolished, in parallel with a deregulation of small Rho-GTPases activity. In melanoma and prostate cancer cells we have shown uPAR colocalization with β1/β3 integrins and actin cytoskeleton, as well integrins-actin co-localization under both mesenchymal and amoeboid conditions. Such co-localizations were lost upon treatment of cells with a peptide that inhibits uPAR-integrin interactions. Similarly to uPAR silencing, the peptide reduced mesenchymal invasion and almost abolished the amoeboid one. These results indicate that full-length uPAR bridges the mesenchymal and amoeboid style of movement by an inward-oriented activity based on its property to promote integrin-actin interactions and the following cytoskeleton assembly. PMID:24681666

  7. The urokinase receptor (uPAR facilitates clearance of Borrelia burgdorferi.

    Joppe W R Hovius

    2009-05-01

    Full Text Available The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR; however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also, dependently or independently of ligation to uPA, directly affect leukocyte function. We here demonstrate that uPAR is upregulated on murine and human leukocytes upon exposure to B. burgdorferi both in vitro as well as in vivo. Notably, B. burgdorferi-inoculated C57BL/6 uPAR knock-out mice harbored significantly higher Borrelia numbers compared to WT controls. This was associated with impaired phagocytotic capacity of B. burgdorferi by uPAR knock-out leukocytes in vitro. B. burgdorferi numbers in vivo, and phagocytotic capacity in vitro, were unaltered in uPA, tPA (low fibrinolytic activity and PAI-1 (high fibrinolytic activity knock-out mice compared to WT controls. Strikingly, in uPAR knock-out mice partially backcrossed to a B. burgdorferi susceptible C3H/HeN background, higher B. burgdorferi numbers were associated with more severe carditis and increased local TLR2 and IL-1beta mRNA expression. In conclusion, in B. burgdorferi infection, uPAR is required for phagocytosis and adequate eradication of the spirochete from the heart by a mechanism that is independent of binding of uPAR to uPA or its role in the fibrinolytic system.

  8. Urinary-type plasminogen activator (uPA) and its receptor (uPAR) in squamous cell carcinoma of the oral cavity.

    Shi, Zonggao; Stack, M Sharon

    2007-10-15

    OSCC (oral squamous cell carcinoma) is the most common oral malignancy and is estimated to affect approx. 350000 new patients worldwide this year. OSCC is characterized by a high degree of morbidity and mortality, as most patients exhibit local, regional and distant metastasis at the time of diagnosis. Recent genome-wide screening efforts have identified the serine proteinase uPA (urinary-type plasminogen activator, also known as urokinase) as a strong biomarker for prediction of poor disease outcome and a key candidate for molecular classification of oral neoplasms using a 'gene signature' approach. The proteinase uPA binds a surface-anchored receptor designated uPAR (uPA receptor), focalizing proteolytic activity to the pericellular milieu. Furthermore, uPA-uPAR can interact with transmembrane proteins to modify multiple signal transduction pathways and influence a wide variety of cellular behaviours. Correlative clinical data show elevated uPA-uPAR in oral tumour tissues, with tumours exhibiting high levels of both uPA and uPAR as the most invasive. Combined in vitro, pre-clinical and clinical data support the need for further analysis of uPA-uPAR as a prognostic indicator as well as a potential therapeutic target in OSCC.

  9. Expression of the urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) in brain tissues of human immunodeficiency virus patients with opportunistic cerebral diseases.

    Nebuloni, Manuela; Cinque, Paola; Sidenius, Nicolai; Ferri, Angelita; Lauri, Eleonora; Omodeo-Zorini, Elisabetta; Zerbi, Pietro; Vago, Luca

    2009-01-01

    The urokinase plasminogen activator receptor (uPAR) and its ligand (uPA) play an important role in cell migration and extracellular proteolysis. We previously described uPAR/uPA overexpression in the cerebrospinal fluid (CSF) and brain tissues of patients with human immunodeficiency virus (HIV)-related cerebral diseases. In this study, we examined uPAR/uPA expression by immunohistochemistry (IHC) in brains of HIV patients with opportunistic cerebral lesions and in HIV-positive/negative controls. uPAR was found in macrophages/microglia with the highest levels in cytomegalovirus (CMV) encephalitis, toxoplasmosis, and lymphomas; in cryptococcosis and progressive multifocal leukoencephalopathy (PML) cases, only a few positive cells were found and no positivity was observed in controls. uPA expression was demonstrated only in a few macrophages/microglia and lymphocytes in all the cases and HIV-positive controls without different pattern of distribution; no uPA immunostaining was found in cryptococcosis and HIV-negative controls. The higher expression of uPAR/uPA in most of the opportunistic cerebral lesions supports their role in these diseases, suggesting their contribution to tissue injury.

  10. The urokinase receptor (uPAR) facilitates clearance of Borrelia burgdorferi

    Hovius, J.W.R.; Bijlsma, M.F.; van der Windt, G.J.W.; Wiersinga, W.J.; Boukens, B.J.D.; Coumou, J.; Oei, A.; de Beer, R.; de Vos, A.F.; van 't Veer, C.; van Dam, A.P.; Wang, P.; Fikrig, E.; Levi, M.M.; Roelofs, J.J.T.H.; van der Poll, T.

    2009-01-01

    The causative agent of Lyme borreliosis, the spirochete Borrelia burgdorferi, has been shown to induce expression of the urokinase receptor (uPAR); however, the role of uPAR in the immune response against Borrelia has never been investigated. uPAR not only acts as a proteinase receptor, but can also

  11. The urokinase receptor (uPAR) and the uPAR-associated protein (uPARAP/Endo180)

    Behrendt, Niels

    2004-01-01

    processes involve a highly organized interplay between proteases and their cellular binding sites as well as specific substrates and internalization receptors. This review article is focused on two components, the urokinase plasminogen activator receptor (uPAR) and the uPAR-associated protein (uPARAP, also...... designated Endo180), that are considered crucially engaged in matrix degradation. uPAR and uPARAP have highly diverse functions, but on certain cell types they interact with each other in a process that is still incompletely understood. uPAR is a glycosyl-phosphatidylinositol-anchored glycoprotein...

  12. Mapping the topographic epitope landscape on the urokinase plasminogen activator receptor (uPAR) by surface plasmon resonance and X-ray crystallography

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai

    2015-01-01

    as a dynamic modular protein structure composed of three homologous Ly6/uPAR domains (LU).This internally flexible protein structure of uPAR enables an allosteric regulation of the interactions with its two principal ligands: the serine protease urokinase-type plasminogen activator (uPA) and the provisional...... matrix protein vitronectin (Vn) (Mertens et al., 2012; Gårdsvoll et al., 2011; Madsen et al., 2007 [2-4]). The data presented here relates to the non-covalent trapping of one of these biologically relevant uPAR-conformations by a novel class of monoclonal antibodies (Zhao et al., 2015 [5......]) and to the general mapping of the topographic epitope landscape on uPAR. The methods required to achieve these data include: (1) recombinant expression and purification of a uPAR-hybrid protein trapped in the desired conformation [patent; WO 2013/020898 A12013]; (2) developing monoclonal antibodies with unique...

  13. Decreased expression of intercellular adhesion molecule-1 (ICAM-1) and urokinase-type plasminogen activator receptor (uPAR) is associated with tumor cell spreading in vivo.

    Donadio, Ana C; Remedi, María M; Frede, Silvia; Bonacci, Gustavo R; Chiabrando, Gustavo A; Pistoresi-Palencia, María C

    2002-01-01

    The development of an effective antitumor immune response to control tumor growth is influenced by the tumor cell itself and/or by the tumor microenvironment. Tumor invasion and tumor cell spreading require a finely tuned regulation of the formation and loosening of adhesive contacts of tumor cells with the extracellular matrix (ECM). In our laboratory, a rat tumor cell line derived from a spontaneous rat sarcoma revealed, by flow cytometry, a high frequency of intercellular adhesion molecule-1 (ICAM-1, 70.1 +/- 8.7%) and urokinase-type plaminogen activator receptor (uPAR, 51.2 +/- 5.2%) positive cells, while a weak expression of MHC class II (IA, 2.2 +/- 0.2% and IE, 17.4 +/- 3.7%) and B7 (12.1 +/- 2.2%) antigens was detected. In our tumor experimental model, after implantation of tumor cells, visible tumor masses were present at days 5-7 with a relatively fast tumor growth until day 15 (progressive phase) followed by a suppression of the tumor growth (regressive phase). Here we present data that correlates a significant decrease in the frequency of ICAM-1 and uPAR expressing tumor cells with the appearance of tumor cells in sites distant from that of the primary tumor. In addition we describe the development of a cellular immune response which controls the tumor progression and is associated with an increase in the expression of major histocompatibility complex (MHC) class II IA antigen during tumor development. The histological examination at tumor progressive and regressive time points revealed the relevant presence of polymorphonuclear neutrophils (PMNs) evidencing colliquative necrosis in tumor growth areas. Taken together, these results support the idea that the balance between adhesive interactions, proteolytic activity and tumorigenicity may lead to a tumor invasive phenotype.

  14. A flexible multidomain structure drives the function of the urokinase-type plasminogen activator receptor (uPAR)

    Mertens, Haydyn D.T.; Kjærgaard, Magnus; Mysling, Simon

    2012-01-01

    -deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted...... cell adhesion and migration as well as for translational research including targeted intervention therapy and non-invasive tumor imaging in vivo....

  15. Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

    Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla;

    2009-01-01

    : rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression......Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms...... and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay...

  16. Copenhagen uPAR prostate cancer (CuPCa) database

    Lippert, Solvej; Berg, Kasper D; Høyer-Hansen, Gunilla;

    2016-01-01

    AIM: Urokinase plasminogen activator receptor (uPAR) plays a central role during cancer invasion by facilitating pericellular proteolysis. We initiated the prospective 'Copenhagen uPAR Prostate Cancer' study to investigate the significance of uPAR levels in prostate cancer (PCa) patients. METHODS......PAR(I) levels were significantly higher in hormone-naive and castrate-resistant patients compared with patients with localized disease (both: p

  17. The intact urokinase receptor is required for efficient vitronectin binding

    Høyer-Hansen, G; Behrendt, N; Ploug, M;

    1997-01-01

    The urokinase receptor (uPAR) is a receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin. There are two forms of cell surface-bound uPAR; intact uPAR and a cleaved form, uPAR(2+3), which is formed by uPA-catalyzed cleavage of uPAR. In ligand-blotting experim...

  18. uPAR as anti-cancer target

    Lund, Ida K; Illemann, Martin; Thurison, Tine

    2011-01-01

    , and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to u......Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted...... up-regulated during cancer progression and is primarily confined to the tumor-associated stromal compartment. Furthermore, both uPAR and uPA have proven to be prognostic markers in several types of cancer; high levels indicating poor survival. The cleaved forms of uPAR are also prognostic markers...

  19. Identification of a new epitope in uPAR as a target for the cancer therapeutic monoclonal antibody ATN-658, a structural homolog of the uPAR binding integrin CD11b (αM.

    Xiang Xu

    Full Text Available The urokinase plasminogen activator receptor (uPAR plays a role in tumor progression and has been proposed as a target for the treatment of cancer. We recently described the development of a novel humanized monoclonal antibody that targets uPAR and has anti-tumor activity in multiple xenograft animal tumor models. This antibody, ATN-658, does not inhibit ligand binding (i.e. uPA and vitronectin to uPAR and its mechanism of action remains unclear. As a first step in understanding the anti-tumor activity of ATN-658, we set out to identify the epitope on uPAR to which ATN-658 binds. Guided by comparisons between primate and human uPAR, epitope mapping studies were performed using several orthogonal techniques. Systematic site directed and alanine scanning mutagenesis identified the region of aa 268-275 of uPAR as the epitope for ATN-658. No known function has previously been attributed to this epitope Structural insights into epitope recognition were obtained from structural studies of the Fab fragment of ATN-658 bound to uPAR. The structure shows that the ATN-658 binds to the DIII domain of uPAR, close to the C-terminus of the receptor, corroborating the epitope mapping results. Intriguingly, when bound to uPAR, the complementarity determining region (CDR regions of ATN-658 closely mimic the binding regions of the integrin CD11b (αM, a previously identified uPAR ligand thought to be involved in leukocyte rolling, migration and complement fixation with no known role in tumor progression of solid tumors. These studies reveal a new functional epitope on uPAR involved in tumor progression and demonstrate a previously unrecognized strategy for the therapeutic targeting of uPAR.

  20. A deficiency of uPAR alters endothelial angiogenic function and cell morphology

    Balsara Rashna D

    2011-05-01

    Full Text Available Abstract The angiogenic potential of a cell requires dynamic reorganization of the cytoskeletal architecture that involves the interaction of urokinase-type plasminogen activator receptor (uPAR with the extracellular matrix. This study focuses on the effect of uPAR deficiency (uPAR-/- on angiogenic function and associated cytoskeletal organization. Utilizing murine endothelial cells, it was observed that adhesion, migration, proliferation, and capillary tube formation were altered in uPAR-/- cells compared to wild-type (WT cells. On a vitronectin (Vn matrix, uPAR-/- cells acquired a "fried egg" morphology characterized by circular actin organization and lack of lamellipodia formation. The up-regulation of β1 integrin, FAK(P-Tyr925, and paxillin (P-Tyr118, and decreased Rac1 activation, suggested increased focal adhesions, but delayed focal adhesion turnover in uPAR-/- cells. This accounted for the enhanced adhesion, but attenuated migration, on Vn. VEGF-enriched Matrigel implants from uPAR-/- mice demonstrated a lack of mature vessel formation compared to WT mice. Collectively, these results indicate that a uPAR deficiency leads to decreased angiogenic functions of endothelial cells.

  1. Endothelial progenitor cell-dependent angiogenesis requires localization of the full-length form of uPAR in caveolae.

    Margheri, Francesca; Chillà, Anastasia; Laurenzana, Anna; Serratì, Simona; Mazzanti, Benedetta; Saccardi, Riccardo; Santosuosso, Michela; Danza, Giovanna; Sturli, Niccolò; Rosati, Fabiana; Magnelli, Lucia; Papucci, Laura; Calorini, Lido; Bianchini, Francesca; Del Rosso, Mario; Fibbi, Gabriella

    2011-09-29

    Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

  2. Circulating intact and cleaved forms of the urokinase-type plasminogen activator receptor

    Sørensen, Tine Thurison; Christensen, Ib J; Lund, Ida K;

    2015-01-01

    BACKGROUND: High levels of circulating forms of the urokinase-type plasminogen activator receptor (uPAR) are significantly associated to poor prognosis in cancer patients. Our aim was to determine biological variations and reference intervals of the uPAR forms in blood, and in addition, to test t...

  3. Quantitation of the receptor for urokinase plasminogen activator by enzyme-linked immunosorbent assay

    Rønne, E; Behrendt, N; Ploug, M;

    1994-01-01

    variant of uPAR, suPAR, has been constructed by recombinant technique and the protein content of a purified suPAR standard preparation was determined by amino acid composition analysis. The sensitivity of the assay (0.6 ng uPAR/ml) is strong enough to measure uPAR in extracts of cultured cells and cancer......Binding of the urokinase plasminogen activator (uPA) to a specific cell surface receptor (uPAR) plays a crucial role in proteolysis during tissue remodelling and cancer invasion. An immunosorbent assay for the quantitation of uPAR has now been developed. This assay is based on two monoclonal...... tissue. Recent studies have shown that a high uPA level in tumor extracts is in some cancers associated with poor prognosis. The present assay will now allow similar prognostic studies of uPAR levels....

  4. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis.

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.

  5. Differential uPAR recruitment in caveolar-lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

    Margheri, Francesca; Papucci, Laura; Schiavone, Nicola; D'Agostino, Riccardo; Trigari, Silvana; Serratì, Simona; Laurenzana, Anna; Biagioni, Alessio; Luciani, Cristina; Chillà, Anastasia; Andreucci, Elena; Del Rosso, Tommaso; Margheri, Giancarlo; Del Rosso, Mario; Fibbi, Gabriella

    2015-01-01

    Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid-rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR-GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar-raft partitioning of uPAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis. PMID:25313007

  6. uPAR as anti-cancer target: evaluation of biomarker potential, histological localization, and antibody-based therapy

    Lund, Ida K; Illemann, Martin; Sørensen, Tine Thurison

    2011-01-01

    , and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to u......Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted...... up-regulated during cancer progression and is primarily confined to the tumor-associated stromal compartment. Furthermore, both uPAR and uPA have proven to be prognostic markers in several types of cancer; high levels indicating poor survival. The cleaved forms of uPAR are also prognostic markers...

  7. Urokinase, urokinase receptor, and plasminogen activator inhibitor-1 expression on podocytes in immunoglobulin A glomerulonephritis

    Lee, Ji-Hye; Oh, Mee-Hye; Park, Jae-seok; Na, Gyoung-Jae; Gil, Hye-Wook; Yang, Jong-Oh; Lee, Eun-Young; Hong, Sae-Yong

    2014-01-01

    Background/Aims The purpose of this study was to investigate the expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor (PAI)-1 on podocytes in immunoglobulin A (IgA) glomerulonephritis (GN). Methods Renal biopsy specimens from 52 IgA GN patients were deparaffinized and subjected to immunohistochemical staining for uPA, PAI-1, and uPAR. The biopsies were classified into three groups according to the expression of uPA and uPAR on podo...

  8. A conserved TATA-less proximal promoter drives basal transcription from the urokinase-type plasminogen activator receptor gene

    Soravia, E; Grebe, A; De Luca, P;

    1995-01-01

    The urokinase-type plasminogen activator receptor (uPAR) focuses at the cell surface the activation of pro-uPA and, hence, the formation of plasmin, thus enhancing directional extracellular proteolysis. To characterize the transcriptional regulatory mechanisms that control receptor expression, we...... have cloned an uPAR DNA segment containing upstream regulatory sequences from both the human and murine genomes. We report that a proximal promoter, contained within 180 bp from the major transcription start sites of the human uPAR gene, drives basal transcription. This region lacks TATA and CAAT boxes...

  9. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express u...... with other osteoblasts markers. On the resorptive side, the number of osteoclasts formed in vitro from uPAR KO monocytes was decreased. Podosome imaging in uPAR KO osteoclasts revealed a defect in actin ring formation. CONCLUSIONS: The defective proliferation and differentiation of bone cells, coincident...

  10. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1 Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    Ekaterina N Lyukmanova

    Full Text Available SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1 differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM of human oral keratinocytes (Het-1A cells. Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM. It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1 did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the

  11. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor.

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis; Bychkov, Maxim L; Kulbatskii, Dmitrii S; Kasheverov, Igor E; Astapova, Maria V; Feofanov, Alexey V; Thomsen, Morten S; Mikkelsen, Jens D; Shenkarev, Zakhar O; Tsetlin, Victor I; Dolgikh, Dmitry A; Kirpichnikov, Mikhail P

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation of keratinocytes and controls inflammation and malignant cell transformation. The majority of previous studies of SLURP-1 have been made using fusion constructs containing, in addition to the native protein, extra polypeptide sequences. Here we describe the activity and pharmacological profile of a recombinant analogue of human SLURP-1 (rSLURP-1) differing from the native protein only by one additional N-terminal Met residue. rSLURP-1 significantly inhibited proliferation (up to ~ 40%, EC50 ~ 4 nM) of human oral keratinocytes (Het-1A cells). Application of mecamylamine and atropine,--non-selective inhibitors of nicotinic acetylcholine receptors (nAChRs) and muscarinic acetylcholine receptors, respectively, and anti-α7-nAChRs antibodies revealed α7 type nAChRs as an rSLURP-1 target in keratinocytes. Using affinity purification from human cortical extracts, we confirmed that rSLURP-1 binds selectively to the α7-nAChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding to the receptor. These findings imply an allosteric antagonist-like mode of SLURP-1 interaction with α7-nAChRs outside the classical ligand-binding site. Contrary to rSLURP-1, other inhibitors of α7-nAChRs (mecamylamine, α-bungarotoxin and Lynx1) did not suppress the proliferation of keratinocytes. Moreover, the co-application of α-bungarotoxin with rSLURP-1 did not influence antiproliferative activity of the latter. This supports the hypothesis that

  12. uPAR Expression Pattern in Patients with Urothelial Carcinoma of the Bladder

    Dohn, Line Hammer; Pappot, Helle; Iversen, Benedikte Richter;

    2015-01-01

    The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling...... or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated...... investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system....

  13. Interferon-alpha (Intron A) upregulates urokinase-type plasminogen activator receptor gene expression.

    Wu, Shanshan; Murrell, George A C; Wang, Yao

    2002-07-01

    The regulation of urokinase plasminogen activator receptor (uPAR) gene expression by interferon-alpha (IFN-alpha, or Intron A) and interferon-gamma (IFN-gamma) was studied in a HCT116 colon cancer cell line. uPAR mRNA levels were increased in a dose- and time-dependent manner in cells stimulated with IFN-alpha or IFN-gamma. uPAR protein levels reflected IFN-alpha and IFN-gamma induction of uPAR mRNA production. Cycloheximide, a protein synthesis inhibitor, also induced uPAR mRNA accumulation either alone or in combination with IFN-alpha or IFN-gamma, suggesting that the effect on uPAR mRNA levels activated by IFN-alpha or IFN-gamma does not require de novo protein synthesis. Both sodium butyrate and amiloride inhibited the uPAR mRNA levels induced by IFN-alpha or IFN-gamma. These results may provide useful information for the treatment of patients receiving IFN-alpha or IFN-gamma.

  14. The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis

    Engelholm, L H; Nielsen, B S; Netzel-Arnett, S;

    2001-01-01

    The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and ......, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V....

  15. Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

    Katia Cortese

    Full Text Available BACKGROUND: The urokinase receptor (uPAR/CD87 is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments. CONCLUSIONS/SIGNIFICANCE: These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

  16. Urokinase plasminogen activator receptor on invasive cancer cells: A prognostic factor in distal gastric adenocarcinoma

    Alpizar, Warner Enrique Alpizar; Christensen, Ib Jarle; Santoni-Rugiu, Eric

    2012-01-01

    Gastric cancer is the second cancer causing death worldwide. The five-year survival for this malignancy is below 25% and few parameters have shown an impact on the prognosis of the disease. The receptor for urokinase plasminogen activator (uPAR) is involved in extracellular matrix degradation...... by mediating cell surface associated plasminogen activation, and its presence on gastric cancer cells is linked to micrometastasis and poor prognosis. Using immunohistochemistry, the prognostic significance of uPAR was evaluated in tissue samples from a retrospective series of 95 gastric cancer patients. u...... association between the expression of uPAR on tumor cells in the peripheral invasion zone and overall survival of gastric cancer patients (HR = 2.16; 95% CI: 1.13-4.14; p = 0.02). Multivariate analysis showed that uPAR immunoreactivity in cancer cells at the invasive front is an independent prognostic factor...

  17. Urokinase plasminogen activator receptor affects bone homeostasis by regulating osteoblast and osteoclast function

    Furlan, Federico; Galbiati, Clara; Jørgensen, Niklas R;

    2007-01-01

    reorganization in mature osteoclasts. INTRODUCTION: Urokinase receptor (uPAR) is actively involved in the regulation of important cell functions, such as proliferation, adhesion, and migration. It was previously shown that the major players in bone remodeling, osteoblasts and osteoclasts, express u...... to mechanical tests. UPAR KO calvaria osteoblasts were characterized by proliferation assays, RT-PCR for important proteins secreted during differentiation, and immunoblot for activator protein 1 (AP-1) family members. In vitro osteoclast formation was tested with uPAR KO bone marrow monocytes in the presence...... a proliferative advantage with no difference in apoptosis, higher matrix mineralization, and earlier appearance of alkaline phosphatase (ALP). Surface RANKL expression at different stages of differentiation was not altered. AP-1 components, such as JunB and Fra-1, were upregulated in uPAR KO osteoblasts, along...

  18. Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    Sayers Ian

    2009-07-01

    Full Text Available Abstract Background Expression of the urokinase plasminogen activator receptor (UPAR has been shown to have clinical relevance in various cancers. We have recently identified UPAR as an asthma susceptibility gene and there is evidence to suggest that uPAR may be upregulated in lung diseases such as COPD and asthma. uPAR is a key receptor involved in the formation of the serine protease plasmin by interacting with uPA and has been implicated in many physiological processes including proliferation and migration. The current aim was to determine key regulatory regions and splice variants of UPAR and quantify its expression in primary human tissues and cells (including lung, bronchial epithelium (HBEC, airway smooth muscle (HASM and peripheral cells. Results Using Rapid Amplification of cDNA Ends (RACE a conserved transcription start site (-42 to -77 relative to ATG was identified and multiple transcription factor binding sites predicted. Seven major splice variants were identified (>5% total expression, including multiple exon deletions and an alternative exon 7b (encoding a truncated, soluble, 229aa protein. Variants were differentially expressed, with a high proportion of E7b usage in lung tissue and structural cells (55–87% of transcripts, whereas classical exon 7 (encoding the GPI-linked protein was preferentially expressed in peripheral cells (~80% of transcripts, often with exon 6 or 5+6 deletions. Real-time PCR confirmed expression of uPAR mRNA in lung, as well as airway and peripheral cell types with ~50–100 fold greater expression in peripheral cells versus airway cells and confirmed RACE data. Protein analysis confirmed expression of multiple different forms of uPAR in the same cells as well as expression of soluble uPAR in cell supernatants. The pattern of expression did not directly reflect that seen at the mRNA level, indicating that post-translational mechanisms of regulation may also play an important role. Conclusion We have

  19. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  20. Quantitative PET of human urokinase-type plasminogen activator receptor with 64Cu-DOTA-AE105

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expressi...... levels of uPAR across different human cancer xenograft models in mice and to illustrate the clinical potential of uPAR PET in future settings for individualized therapy.......Expression levels of the urokinase-type plasminogen activator receptor (uPAR) represent an established biomarker for poor prognosis in a variety of human cancers. The objective of the present study was to explore whether noninvasive PET can be used to perform a quantitative assessment of expression...

  1. Stabilizing a flexible interdomain hinge region harboring the SMB binding site drives uPAR into its closed conformation

    Zhao, Baoyu; Gandhi, Sonu; Yuan, Cai;

    2015-01-01

    The urokinase-type plasminogen activator receptor (uPAR) is a multidomain glycolipid-anchored membrane protein, which facilitates extracellular matrix remodeling by focalizing plasminogen activation to cell surfaces via its high-affinity interaction with uPA. The modular assembly of its three LU ...

  2. uPAR EXPRESSION IN CANINE NORMAL PROSTATE AND WITH PROLIFERATIVE DISORDERS

    Mariana Rodrigues Faleiro

    2013-06-01

    Full Text Available Prostatic lesions such as prostatic intraepithelial neoplasia (PIN and proliferative inflammatory atrophy (PIA are studied in human and canine species due to their malignance potential. The plasminogen activator (PA system has been suggested to play a central role in cell adhesion, angiogenesis, inflammation, and tumor invasion. The urokinase-type plasminogen activator receptor (uPAR is a component of the PA, with a range of expression in tumor and stromal cells. In this study, uPAR expression in both canine normal prostates and with proliferative disorders (benign prostatic hyperplasia-BPH, proliferative inflammatory atrophy-PIA, prostatic intraepithelial neoplasia-PIN, and carcinoma-PC was evaluated by immunohistochemistry in a tissue microarray (TMA slide to establish the role of this enzyme in extracellular matrix (ECM remodeling and in the processes of tissue invasion. A total of 298 cores and 355 diagnoses were obtained, with 36 (10.1% normal prostates, 46 (13.0% with BPH, 128 (36.1% with PIA, 74 (20.8% with PIN and 71 (20.0% with PC. There is variation in the expression of uPAR in canine prostate according to the lesion, with lower expression in normal tissue and with BPH, and higher expression in tissue with PIA, PIN and PC. The high expression of uPAR in inflammatory and neoplastic microenvironment indicates increased proteolytic activity in canine prostates with PIA, PIN, and PC.

  3. Urokinase receptor forms in serum from non-small cell lung cancer patients

    Almasi, Charlotte Elberling; Christensen, Ib Jarle; Høyer-Hansen, Gunilla;

    2011-01-01

    To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients.......To study the prognostic impact of the different forms of the receptor for urokinase plasminogen activator (uPAR) in serum from 171 non-small cell lung cancer (NSCLC) patients....

  4. Cadmium induces urokinase-type plasminogen activator receptor expression and the cell invasiveness of human gastric cancer cells via the ERK-1/2, NF-κB, and AP-1 signaling pathways.

    Khoi, Pham Ngoc; Xia, Yong; Lian, Sen; Kim, Ho Dong; Kim, Do Hyun; Joo, Young Eun; Chay, Kee-Oh; Kim, Kyung Keun; Jung, Young Do

    2014-10-01

    Cadmium exposure has been linked to human cancers, including stomach cancer. In this study, the effects of cadmium on urokinase-type plasminogen activator receptor (uPAR) expression in human gastric cancer cells and the underlying signal transduction pathways were investigated. Cadmium induced uPAR expression in a time- and concentration-dependent manner. Cadmium also induced uPAR promoter activity. Additionally, cadmium induced the activation of extracellular signal regulated kinase-1/2 (ERK-1/2), p38 mitogen-activated protein kinase (MAPK), and the activation of c-Jun amino terminal kinase (JNK). A specific inhibitor of MEK-1 (PD98059) inhibited cadmium-induced uPAR expression, while JNK and p38 MAPK inhibitors did not. Expression vectors encoding dominant-negative MEK-1 (pMCL-K97M) also prevented cadmium-induced uPAR promoter activity. Site-directed mutagenesis and electrophoretic mobility shift studies showed that sites for the transcription factors nuclear factor (NF)-κB and activator protein-1 (AP-1) were involved in cadmium-induced uPAR transcription. Suppression of the cadmium-induced uPAR promoter activity by a mutated-type NF-κB-inducing kinase and I-κB and an AP-1 decoy oligonucleotide confirmed that the activation of NF-κB and AP-1 are essential for cadmium-induced uPAR upregulation. Cells pretreated with cadmium showed markedly enhanced invasiveness and this effect was partially abrogated by uPAR-neutralizing antibodies and by inhibitors of ERK-1/2, NF-κB, and AP-1. These results suggest that cadmium induces uPAR expression via ERK-1/2, NF-κB, and AP-1 signaling pathways and, in turn, stimulates cell invasiveness in human gastric cancer AGS cells.

  5. Cellular receptors for plasminogen activators recent advances.

    Ellis, V

    1997-10-01

    The generation of the broad-specificity protease plasmin by the plasminogen activators urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) is implicated in a variety of pathophysiological processes, including vascular fibrin dissolution, extracellular matrix degradation and remodeling, and cell migration. A mechanism for the regulation of plasmin generation is through binding of the plasminogen activators to specific cellular receptors: uPA to the glycolipid-anchored membrane protein urokinase-type plasminogen activator receptor (uPAR) and tPA to a number of putative binding sites. The uPA-uPAR complex can interact with a variety of ligands, including plasminogen, vitronectin, and integrins, indicating a multifunctional role for uPAR, regulating not only efficient and spatially restricted plasmin generation but also having the potential to modulate cell adhesion and signal transduction. The cellular binding of tPA, although less well characterized, also has the capacity to regulate plasmin generation and to play a significant role in vessel-wall biology. (Trends Cardiovasc Med 1997;7:227-234). © 1997, Elsevier Science Inc.

  6. Reversibility of epithelial-mesenchymal transition (EMT) induced in breast cancer cells by activation of urokinase receptor-dependent cell signaling.

    Jo, Minji; Lester, Robin D; Montel, Valerie; Eastman, Boryana; Takimoto, Shinako; Gonias, Steven L

    2009-08-21

    Hypoxia induces expression of the urokinase receptor (uPAR) and activates uPAR-dependent cell signaling in cancer cells. This process promotes epithelial-mesenchymal transition (EMT). uPAR overexpression in cancer cells also promotes EMT. In this study, we tested whether uPAR may be targeted to reverse cancer cell EMT. When MDA-MB 468 breast cancer cells were cultured in 1% O(2), uPAR expression increased, as anticipated. Cell-cell junctions were disrupted, vimentin expression increased, and E-cadherin was lost from cell surfaces, indicating EMT. Transferring these cells back to 21% O(2) decreased uPAR expression and reversed the signs of EMT. In uPAR-overexpressing MDA-MB 468 cells, EMT was reversed by silencing expression of endogenously produced urokinase-type plasminogen activator (uPA), which is necessary for uPAR-dependent cell signaling, or by targeting uPAR-activated cell signaling factors, including phosphatidylinositol 3-kinase, Src family kinases, and extracellular signal-regulated kinase. MDA-MB 231 breast cancer cells express high levels of uPA and uPAR and demonstrate mesenchymal cell morphology under normoxic culture conditions (21% O(2)). Silencing uPA expression in MDA-MB-231 cells decreased expression of vimentin and Snail, and induced changes in morphology characteristic of epithelial cells. These results demonstrate that uPAR-initiated cell signaling may be targeted to reverse EMT in cancer.

  7. Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor by Hypoxia-Inducible Factor 1 Is Crucial for Invasion of Pancreatic and Liver Cancer

    Peter Büchler

    2009-02-01

    Full Text Available Angioinvasion is critical for metastasis with urokinase-type plasminogen activator receptor (uPAR and tumor hypoxia-activated hypoxia-inducible factor 1 (HIF-1 as key players. Transcriptional control of uPAR expression by HIF has never been reported. The aim of the present study, therefore, was to test whether tumor hypoxia-induced HIF expression may be linked to transcriptional activation of uPAR and dependent angioinvasion. We used human pancreatic cancer cells and a model of parental and derived HIF-1β-deficient mouse liver cancer cell lines and performed Northern blot analysis, nuclear runoff assays, electrophoretic mobility shift assay, polymerase chain reaction-generated deletion mutants, luciferase assays, Matrigel invasion assays, and in vivo angioinvasion assays in the chorioallantoic membrane of fertilized chicken eggs. Urokinase-type plasminogen activator receptor promoter analysis resulted in four putative HIF binding sites. Hypoxia strongly induced de novo transcription of uPAR mRNA. With sequential deletion mutants of the uPAR promoter, it was possible to identify one HIF binding site causing a nearly 200-fold increase in luciferase activity. Hypoxia enhanced the number of invading tumor cells in vitro and in vivo. In contrast, HIF-1β-deficient cells failed to upregulate uPAR expression, to activate luciferase activity, and to invade on hypoxia. Taken together, we show for the first time that uPAR is under transcriptional control of HIF and that this is important for hypoxia-induced metastasis.

  8. Prognostic value analysis of urokinase-type plasminogen activator receptor in oral squamous cell carcinoma: an immunohistochemical study

    Rocchetti Romina

    2008-08-01

    Full Text Available Abstract Background Oral squamous cell carcinoma (OSCC represents the most common oral malignancy. Despite recent advances in therapy, up to 50% of the cases have relapse and/or metastasis. There is therefore a strong need for the identification of new biological markers able to predict the clinical behaviour of these lesions in order to improve quality of life and overall survival. Among tumour progression biomarkers, already known for their involvement in other neoplasia, a crucial role is ascribed to the urokinase-type plasminogen activator receptor (uPAR, which plays a multiple role in extracellular proteolysis, cell migration and tissue remodelling not only as a receptor for the zymogen pro-uPA but also as a component for cell adhesion and as a chemoattractant. The purpose of this study was to gain information on the expression of uPAR in OSCC and to verify whether this molecule can have a role as a prognostic/predictive marker for this neoplasia. Methods In a retrospective study, a cohort of 189 OSCC patients was investigated for uPAR expression and its cellular localization by immunohistochemistry. As standard controls, 8 normal oral mucosal tissues free of malignancy, obtained from patients with no evidence or history of oral cavity tumours, were similarly investigated. After grouping for uPAR expression, OSCCs were statistically analyzed for the variables age, gender, histological grading (G, tumour size, recurrence, TNM staging and overall survival rate. Results In our immunohistochemical study, 74 cases (39.1% of OSCC showed a mostly cytoplasmic positivity for uPAR, whereas 115 were negative. uPAR expression correlated with tumour differentiation grade and prognosis: percentage of positive cases was the greatest in G3 (70.4% and patients positives for uPAR expression had an expectation of life lower than those for uPAR negatives. Conclusion The results obtained in this study suggest a role of uPAR as a potential biomarker useful to

  9. 68Ga-labeling and in vivo evaluation of a uPAR binding DOTA- and NODAGA-conjugated peptide for PET imaging of invasive cancers

    Persson, Morten; Madsen, Jacob; Østergaard, Søren;

    2012-01-01

    INTRODUCTION: The urokinase-type plasminogen activator receptor (uPAR) is a well-established biomarker for tumor aggressiveness and metastatic potential. DOTA-AE105 and DOTA-AE105-NH(2) labeled with (64)Cu have previously been demonstrated to be able to noninvasively monitor uPAR expression using...... positron emission tomography (PET) in human cancer xenograft mice models. Here we introduce (68)Ga-DOTA-AE105-NH(2) and (68)Ga-NODAGA-AE105-NH(2) and evaluate their imaging properties using small-animal PET. METHODS: Synthesis of DOTA-AE105-NH(2) and NODAGA-AE105-NH(2) was based on solid-phase peptide......, uPAR binding affinity and cell uptake were determined. To characterize the in vivo properties, dynamic microPET imaging was carried out in nude mice bearing human glioma U87MG tumor xenograft. RESULTS: In vitro experiments revealed uPAR binding affinities in the lower nM range for both conjugated...

  10. Human Secreted Ly-6/uPAR Related Protein-1 (SLURP-1) Is a Selective Allosteric Antagonist of α7 Nicotinic Acetylcholine Receptor

    Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kudryavtsev, Denis

    2016-01-01

    SLURP-1 is a secreted toxin-like Ly-6/uPAR protein found in epithelium, sensory neurons and immune cells. Point mutations in the slurp-1 gene cause the autosomal inflammation skin disease Mal de Meleda. SLURP-1 is considered an autocrine/paracrine hormone that regulates growth and differentiation......AChRs. Exposure of Xenopus oocytes expressing α7-nAChRs to rSLURP-1 caused a significant non-competitive inhibition of the response to acetylcholine (up to ~ 70%, IC50 ~ 1 μM). It was shown that rSLURP-1 binds to α7-nAChRs overexpressed in GH4Cl cells, but does not compete with 125I-α-bungarotoxin for binding...

  11. PET imaging of urokinase-type plasminogen activator receptor (uPAR) in prostate cancer

    Skovgaard, Dorthe; Persson, Morten; Kjaer, Andreas

    2016-01-01

    (intact/cleaved forms)-provides independent additional clinical information to that contributed by PSA, Gleason score, and other relevant pathological and clinical parameters. In this respect, non-invasive molecular imaging by positron emission tomography (PET) offers a very attractive technology platform...

  12. Method and tool for prognosticating HIV infection in a subject by measuring soluble urokinase plasminogen activator receptor, degradation products thereof, and urokinase plasminogen activator receptor

    2000-01-01

    Method of diagnosing and/or prognosticating HIV infection in a subject comprising the steps of: (a) performing in vitro a measurement of the level of a marker in the form of (i) urokinase plasminogen activator receptor (uPAR), (ii) soluble urokinase plasminogen activator receptor (suPAR), (iii......) urokinase-type plasminogen activator (uPA), (iv) one or more degradation products of (i), (ii), or (iii), and/or (v) an mRNA for (i), (ii) or (iii), in a biological fluid sample from a subject, and (b) using the measurement value obtained to evaluate the state of the subject....

  13. Immunoglobulin and enzyme-conjugated dextran polymers enhance u-PAR staining intensity of carcinoma cells in peripheral blood smears

    Werther, K; Normark, M; Hansen, B F;

    1999-01-01

    phenotyping of disseminated carcinoma cells in bone marrow and peripheral blood smears. In the first step, the cells were incubated with antibodies against urokinase plasminogen activator receptor (u-PAR) and subsequently with secondary antibodies conjugated to peroxidase-labeled dextran polymers. A brown...... color reaction was developed with diaminobenzidine as chromogen. In the second step, the cells were incubated with alkaline phosphatase-conjugated murine monoclonal antibodies against a common cytokeratin epitope and a red color reaction was developed with new fuchsin as substrate. This method allows...

  14. Urokinase plasminogen receptor and the fibrinolytic complex play a role in nerve repair after nerve crush in mice, and in human neuropathies.

    Cristina Rivellini

    Full Text Available Remodeling of extracellular matrix (ECM is a critical step in peripheral nerve regeneration. In fact, in human neuropathies, endoneurial ECM enriched in fibrin and vitronectin associates with poor regeneration and worse clinical prognosis. Accordingly in animal models, modification of the fibrinolytic complex activity has profound effects on nerve regeneration: high fibrinolytic activity and low levels of fibrin correlate with better nerve regeneration. The urokinase plasminogen receptor (uPAR is a major component of the fibrinolytic complex, and binding to urokinase plasminogen activator (uPA promotes fibrinolysis and cell movement. uPAR is expressed in peripheral nerves, however, little is known on its potential function on nerve development and regeneration. Thus, we investigated uPAR null mice and observed that uPAR is dispensable for nerve development, whereas, loss of uPAR affects nerve regeneration. uPAR null mice showed reduced nerve repair after sciatic nerve crush. This was a consequence of reduced fibrinolytic activity and increased deposition of endoneurial fibrin and vitronectin. Exogenous fibrinolysis in uPAR null mice rescued nerve repair after sciatic nerve crush. Finally, we measured the fibrinolytic activity in sural nerve biopsies from patients with peripheral neuropathies. We showed that neuropathies with defective regeneration had reduced fibrinolytic activity. On the contrary, neuropathies with signs of active regeneration displayed higher fibrinolytic activity. Overall, our results suggest that enforced fibrinolysis may facilitate regeneration and outcome of peripheral neuropathies.

  15. Urokinase-type Plasminogen Activator-like Proteases in Teleosts Lack Genuine Receptor-binding Epidermal Growth Factor-like Domains

    Bager, René; Kristensen, Thomas Kielsgaard; Jensen, Jan Kristian

    2012-01-01

    Plasminogen activation catalyzed by urokinase-type plasminogen activator (uPA) plays an important role in normal and pathological tissue remodeling processes. Since its discovery in the mid-1980s, the cell membrane-anchored urokinase-type plasminogen activator receptor (uPAR) has been believed...... mammals and uPA-catalyzed plasminogen activation in fish may occur mainly in solution. Studies with nonmammalian vertebrate species are needed to obtain a comprehensive understanding of the mechanism of plasminogen activation....

  16. uPA, uPAR and TGFβ₁ expression during early and late post myocardial infarction period in rat myocardium.

    Stavropoulou, Anastasia; Philippou, Anastassios; Halapas, Antonios; Sourla, Antigone; Pissimissis, Nikolaos; Koutsilieris, Michael

    2010-01-01

    The expression patterns of transforming growth factor beta 1 (TGFβ₁), urokinase-type plasminogen activator (uPA) and uPA receptor (uPAR) were analysed after artery ligation-induced myocardial infarction (MI) in the rat myocardium. uPA and uPAR expressions were significantly increased both at transcriptional and protein level during early phase post MI period (uPA at 1 hour and uPAR at 24 hours post infarction). TGFβ1 mRNA expression profile revealed a significant increase of TGFβ1 expression from day 4 up to 8 weeks post infarction. These data suggest that the need for an increasing TGFβ₁ bioavailability during the post-infarction period in rat myocardium is achieved in the early post MI period by an increased expression of uPA/uPAR proteolytic system (indirect activation of latent TGFβ₁) and in the late post MI period by direct regulation of TGFβ₁ expression. It is therefore concluded that differential regulation of the TGFβ₁ bioavailability may be a crucial step of the repair mechanisms during the post MI infarction period in the rat myocardium.

  17. The prognostic value of the suPARnosticTM ELISA assay in HIV-1 infected individuals is not affected by uPAR promoter polymorphisms

    Schneider, Uffe; Nielsen, Rikke; Pedersen, Court;

    2007-01-01

    ABSTRACT: BACKGROUND: High blood levels of soluble urokinase Plasminogen Activator Receptor (suPAR) are associated with poor outcomes in human immunodeficiency-1 (HIV-1) infected individuals. Research on the clinical value of suPAR in HIV-1 infection led to the development of the su......PARnosticTM assay for commercial use in 2006. The aim of this study was to: 1) Evaluate the prognostic value of the new suPARnosticTM assay and 2) Determine whether polymorphisms in the active promoter of uPAR influences survival and/or suPAR values in HIV-1 patients who are antiretroviral therapy (ART) naive...

  18. The urokinase receptor associated protein (uPARAP/endo180)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While...

  19. Conformational regulation of urokinase receptor function

    Gårdsvoll, Henrik; Jacobsen, Benedikte; Kriegbaum, Mette C

    2011-01-01

    PA per se into the hydrophobic ligand binding cavity of uPAR that modulates the function of this receptor. Based on these data, we now propose a model in which the inherent interdomain mobility in uPAR plays a major role in modulating its function. Particularly one uPAR conformation, which is stabilized...

  20. Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate

    Picone, R; Kajtaniak, E L; Nielsen, L S

    1989-01-01

    A specific surface receptor for urokinase plasminogen activator (uPA) recognizes the amino-terminal growth factor-like sequence of uPA, a region independent from and not required for the catalytic activity of this enzyme. The properties of the uPA receptor (uPAR) and the localization and distribu...

  1. Inhibition of establishment of primary and micrometastatic tumors by a urokinase plasminogen activator receptor antagonist.

    Ignar, D M; Andrews, J L; Witherspoon, S M; Leray, J D; Clay, W C; Kilpatrick, K; Onori, J; Kost, T; Emerson, D L

    1998-01-01

    Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 microg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.

  2. The structure and function of the urokinase receptor, a membrane protein governing plasminogen activation on the cell surface

    Behrendt, N; Rønne, E; Danø, K

    1995-01-01

    PA receptor, uPAR, is a cell-surface protein which plays an important role in the localization and regulation of these processes. In the present article a number of established conclusions concerning the structure and function of uPAR are presented, and in addition various models are discussed which might...... explain additional observations for which the mechanisms involved have not yet been clarified experimentally. uPAR is a highly glycosylated, 3-domain protein, anchored in the plasma membrane by a glycolipid moiety. The domain organization is important for efficient ligand-binding, and the NH2-terminal...... to an interplay between uPAR and other, unidentified components. In addition to the function in the regulation of proteolysis, uPAR seems to play a role in internalization processes and in cellular signal transduction and adhesion. A few reagents have been identified which are capable to inhibit the interaction...

  3. ELISA for complexes between urokinase-type plasminogen activator and its receptor in lung cancer tissue extracts

    de Witte, H; Pappot, H; Brünner, N;

    1997-01-01

    A sandwich-type ELISA has been developed for the assessment of complexes between urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in extracts of squamous cell lung carcinomas. The assay is based on a combination of rabbit polyclonal anti-uPA antibodies and a biotinylated mouse...... extraction of uPAR yields the highest amounts of uPA:uPAR complexes. Absorption of tumor extracts with anti-uPA or anti-uPAR MAbs results in a complete disappearance of the ELISA signal, demonstrating the specificity of the ELISA. The recovery of chemically cross-linked uPA:uPAR complexes added to tumor...... extracts varies between 80% and 105%. The intra- and inter-assay variation coefficients are 5.3% and 9.8%, respectively. Furthermore, a peptide antagonist for uPAR was employed to evaluate de novo uPA:uPAR complex formation during tumor tissue extraction and the immunoassay procedure. Our results strongly...

  4. Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and α2-antiplasmin in human corneal perforation: a case report

    Sugioka Koji

    2012-11-01

    Full Text Available Abstract Background Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA, u-PA receptor (u-PAR and α2-antiplasmin (α2AP in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, α2AP, CD68, and α-smooth muscle actin (α-SMA in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or α-SMA and α2AP with CD68 or α-SMA to detect the localization of u-PA and α2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some α-SMA positive cells. On the other hand, α2AP was not expressed in CD68-positive cells, but was expressed in α-SMA positive cells. Conclusion We identified expression of the u-PA/u-PAR complex and α2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.

  5. Urokinase receptor mediates osteoclastogenesis via M-CSF release from osteoblasts and the c-Fms/PI3K/Akt/NF-κB pathway in osteoclasts.

    Kalbasi Anaraki, Parnian; Patecki, Margret; Tkachuk, Sergey; Kiyan, Yulia; Haller, Hermann; Dumler, Inna

    2015-02-01

    Bone remodeling is a dynamic process based on a fine-tuned balance between formation and degradation of bone. Osteoblasts (OBLs) are responsible for bone formation and bone resorption is mediated by osteoclasts (OCLs). The mechanisms regulating the OBL-OCL balance are critical in health and disease; however, they are still far from being understood. We reported recently that the multifunctional urokinase receptor (uPAR) mediates osteogenic differentiation of mesenchymal stem cells (MSCs) to OBLs and vascular calcification in atherosclerosis. Here, we address the question of whether uPAR may also be engaged in regulation of osteoclastogenesis. We show that uPAR mediates this process in a dual fashion. Thus, uPAR affected OBL-OCL interplay. We observed that osteoclastogenesis was significantly impaired in co-culture of monocyte-derived OCLs and in OBLs derived from MSCs lacking uPAR. We show that expression and release, from OBLs, of macrophage colony-stimulating factor (M-CSF), which is indispensable for OCL differentiation, was inhibited by uPAR loss. We further found that uPAR, on the other hand, controlled formation, differentiation, and functional properties of macrophage-derived OCLs. Expression of osteoclastogenic markers, such as tartrate-resistant acid phosphatase (TRAP) and cathepsin K, was impaired in OCLs derived from uPAR-deficient macrophages. The requirement of uPAR for osteoclastogenesis was further confirmed by immunocytochemistry and in bone resorption assay. We provide evidence that the underlying signaling mechanisms involve uPAR association with the M-CSF binding receptor c-Fms followed by c-Fms phosphorylation and activation of the PI3K/Akt/NF-κB pathway in OCLs. We further show that uPAR uses this pathway to regulate a balance between OCL differentiation, apoptosis, and cell proliferation. Our study identified uPAR as an important and multifaceted regulator of OBL-OCL molecular interplay that may serve as an attractive target in bone disease

  6. Urokinase receptor mRNA level and gene transcription are strongly and rapidly increased by phorbol myristate acetate in human monocyte-like U937 cells

    Lund, L R; Rønne, E; Roldan, A L;

    1991-01-01

    We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-fold...... enhancement after 24 h of treatment. Half-maximal stimulation occurs at approximately 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 micrograms/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments...... show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNA is paralleled by a time-dependent increase in u-PAR protein as detected by cross-linking studies with radiolabeled ligand. We conclude that PMA stimulates...

  7. New peptide receptor radionuclide therapy of invasive cancer cells: in vivo studies using 177Lu-DOTA-AE105 targeting uPAR in human colorectal cancer xenografts

    Persson, Morten; Rasmussen, Palle; Madsen, Jacob;

    2012-01-01

    -of-concept for a theranostic approach as treatment modality in a human xenograft colorectal cancer model. MethodsA DOTA-conjugated 9-mer high affinity uPAR binding peptide (DOTA-AE105) was radiolabeled with 64Cu and 177Lu, for PET imaging and targeted radionuclide therapy study, respectively. Human uPAR-positive CRC HT-29...... by recording mouse weight and by H&E staining of kidneys in each treatment group. ResultsuPAR-positive HT-29 xenograft was clearly visualized by PET/CT imaging using 64Cu-DOTA-AE105. Subsequently, these xenograft transplants were locally irradiated using 177Lu-DOTA-AE105, where a significant effect on tumor...... size and the number of uPAR-positive cells in the tumor was found (p

  8. Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

    Gårdsvoll, Henrik; Kjærgaard, Magnus; Jacobsen, Benedikte;

    2011-01-01

    The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the ......The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed...

  9. The Inhibitory Effects of an Antisense u-PAR Vector on Invasion of Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones

    廖国宁; 李清芬; 冯友梅; 邓耀祖; 李卓娅; 龚非力; 马丁

    2003-01-01

    Summary: To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly inva-sive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highlyinvasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer in-vasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasionsby the cell subclones with or without the antisense u-PAR were observed in nude mice. It was foundthat in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PARwas declined, and the ability of anchorage-independent growth of those cell subclones was found de-creased sharply, with the inhibiting rate becoming 79 % and 60 %, respectively. Although the anti-sense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 ofhighly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with theantisense u-PAR decreased and the growth of a neoplasm also slowed down. Thet tests showed thedifference between experimental and control groups was statistically significant (P<0. 01). The anti-sense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclonesin vitro but also restrain the growth of those cell subclones in vivo.

  10. HIV-1 infected lymphoid organs upregulate expression and release of the cleaved form of uPAR that modulates chemotaxis and virus expression.

    Manuela Nebuloni

    Full Text Available Cell-associated receptor for urokinase plasminogen activator (uPAR is released as both full-length soluble uPAR (suPAR and cleaved (c-suPAR form that maintain ability to bind to integrins and other receptors, thus triggering and modulating cell signaling responses. Concerning HIV-1 infection, plasma levels of suPAR have been correlated with the severity of disease, levels of immune activation and ineffective immune recovery also in individuals receiving combination anti-retroviral therapy (cART. However, it is unknown whether and which suPAR forms might contribute to HIV-1 induced pathogenesis and to the related state of immune activation. In this regard, lymphoid organs represent an import site of chronic immune activation and virus persistence even in individuals receiving cART. Lymphoid organs of HIV-1(+ individuals showed an enhanced number of follicular dendritic cells, macrophages and endothelial cells expressing the cell-associated uPAR in comparison to those of uninfected individuals. In order to investigate the potential role of suPAR forms in HIV-1 infection of secondary lymphoid organs, tonsil histocultures were established from HIV-1 seronegative individuals and infected ex vivo with CCR5- and CXCR4-dependent HIV-1 strains. The levels of suPAR and c-suPAR were significantly increased in HIV-infected tonsil histocultures supernatants in comparison to autologous uninfected histocultures. Supernatants from infected and uninfected cultures before and after immunodepletion of suPAR forms were incubated with the chronically infected promonocytic U1 cell line characterized by a state of proviral latency in unstimulated conditions. In the contest of HIV-conditioned supernatants we established that c-suPAR, but not suPAR, inhibited chemotaxis and induced virus expression in U1 cells. In conclusion, lymphoid organs are an important site of production and release of both suPAR and c-suPAR, this latter form being endowed with the capacity of

  11. Identification and characterization of the murine cell surface receptor for the urokinase-type plasminogen activator

    Solberg, H; Løber, D; Eriksen, J;

    1992-01-01

    -hybridization and pronounced sequence similarity with human u-PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115, 1763-1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand...... of this variant yielded a polypeptide with an apparent M(r) of about 30,000, which corresponds to the Mr calculated from the cDNA derived protein sequence of mouse u-PAR. Receptor-bound mouse u-PA could be released by phosphatidylinositol-specific phospholipase C treatment, indicating that mouse u-PAR is attached...... by ligand-blotting analysis.(ABSTRACT TRUNCATED AT 400 WORDS)...

  12. Prognostic value of intact and cleaved forms of the urokinase plasminogen activator receptor in a retrospective study of 518 colorectal cancer patients

    Lomholt, Anne Fog; Christensen, Ib J; Høyer-Hansen, Gunilla

    2010-01-01

    The levels of the soluble urokinase plasminogen activator receptor (suPAR) in blood have been shown to correlate with prognosis in various cancers. Plasma levels of the combined suPAR forms have previously shown to be a strong prognostic marker in the present cohort of CRC patients and could...... potentially identify high-risk patients among those with early stage disease. In order to investigate whether the individual suPAR forms are stronger prognostic markers than the combined amount we measured the different uPAR forms in serum from the same cohort and evaluated their prognostic significance....

  13. Markers of angiogenesis and epidermal growth factor receptor signalling in patients with pancreatic and gastroesophageal junction cancer.

    Rohrberg, Kristoffer Staal; Skov, Birgit Guldhammer; Lassen, Ulrik; Christensen, Ib Jarle; Høyer-Hansen, Gunilla; Buysschaert, Ian; Pappot, Helle

    2010-01-01

    The epidermal growth factor receptor (EGFR) and angiogenesis are well established targets in anti-cancer therapy. Several targeted anti-cancer therapies are in clinical trials in pancreatic and gastroesophageal (GEJ) cancer. However, many patients do not respond to these targeted therapies and there is therefore an increasing need for biomarkers for selection of patients to these therapies. We investigated the expression of EGFR, vascular endothelial growth factor A (VEGF-A), and VEGF receptor 2 (VEGFR-2) in tumour tissue by immunohistochemistry, and soluble EGFR (sEGFR), soluble VEGFR-2 (sVEGFR-2), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), plasminogen activator inhibitor 1 (PAI-1), and different forms of the urokinase plasminogen activator receptor (uPAR): uPAR (I), uPAR (I-III), and uPAR (I-III)+(II-III) in plasma by quantitative immunoassays in 14 patients with pancreatic and GEJ cancer. We found expression in tumour tissue and the plasma levels to be similar to those found in patients with other tumour types. No correlation was found between the blood levels of soluble receptors and the corresponding tumour tissue levels. We conclude that these markers are present in pancreatic and GEJ cancer patients, and could be investigated further as predictive biomarkers in such patients treated with EGFR or angiogenesis targeted therapies.

  14. Evaluation of alpha 1-antitrypsin and the levels of mRNA expression of matrix metalloproteinase 7, urokinase type plasminogen activator receptor and COX-2 for the diagnosis of colorectal cancer.

    Luis Bujanda

    Full Text Available BACKGROUND: Colorectal cancer (CRC is the second most common cause of death from cancer in both men and women in the majority of developed countries. Molecular tests of blood could potentially provide this ideal screening tool. AIM: Our objective was to assess the usefulness of serum markers and mRNA expression levels in the diagnosis of CRC. METHODS: In a prospective study, we measured mRNA expression levels of 13 markers (carbonic anhydrase, guanylyl cyclase C, plasminogen activator inhibitor, matrix metalloproteinase 7 (MMP7, urokinase-type plasminogen activator receptor (uPAR, urokinase-type plasminogen activator, survivin, tetranectin, vascular endothelial growth factor (VEGF, cytokeratin 20, thymidylate synthase, cyclooxygenase 2 (COX-2, and CD44 and three proteins in serum (alpha 1 antitrypsin, carcinoembryonic antigen (CEA and activated C3 in 42 patients with CRC and 33 with normal colonoscopy results. RESULTS: Alpha 1-antitrypsin was the serum marker that was most useful for CRC diagnosis (1.79 ± 0.25 in the CRC group vs 1.27 ± 0.25 in the control group, P<0.0005. The area under the ROC curve for alpha 1-antitrypsin was 0.88 (0.79-0.96. The mRNA expression levels of five markers were statistically different between CRC cases and controls: those for which the ROC area was over 75% were MMP7 (0.81 and tetranectin (0.80, COX-2 (0.78, uPAR (0.78 and carbonic anhydrase (0.77. The markers which identified early stage CRC (Stages I and II were alpha 1-antitrypsin, uPAR, COX-2 and MMP7. CONCLUSIONS: Serum alpha 1-antitrypsin and the levels of mRNA expression of MMP7, COX-2 and uPAR have good diagnostic accuracy for CRC, even in the early stages.

  15. First-in-human uPAR PET

    Persson, Morten; Skovgaard, Dorthe; Brandt-Larsen, Malene;

    2015-01-01

    of invasion, metastasis and indicates poor prognosis. uPAR PET imaging therefore holds promise to be a new and innovative method for improved cancer diagnosis, staging and individual risk stratification. The uPAR specific peptide AE105 was conjugated to the macrocyclic chelator DOTA and labeled with (64)Cu...... for targeted molecular imaging with PET. The safety, pharmacokinetic, biodistribution profile and radiation dosimetry after a single intravenous dose of (64)Cu-DOTA-AE105 were assessed by serial PET and computed tomography (CT) in 4 prostate, 3 breast and 3 bladder cancer patients. Safety assessment...

  16. Downregulation of uPAR and cathepsin B induces apoptosis via regulation of Bcl-2 and Bax and inhibition of the PI3K/Akt pathway in gliomas.

    Ramarao Malla

    Full Text Available BACKGROUND: Glioma is the most commonly diagnosed primary brain tumor and is characterized by invasive and infiltrative behavior. uPAR and cathepsin B are known to be overexpressed in high-grade gliomas and are strongly correlated with invasive cancer phenotypes. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we observed that simultaneous downregulation of uPAR and cathepsin B induces upregulation of some pro-apoptotic genes and suppression of anti-apoptotic genes in human glioma cells. uPAR and cathepsin B (pCU-downregulated cells exhibited decreases in the Bcl-2/Bax ratio and initiated the collapse of mitochondrial membrane potential. We also observed that the broad caspase inhibitor, Z-Asp-2, 6-dichlorobenzoylmethylketone rescued pCU-induced apoptosis in U251 cells but not in 5310 cells. Immunoblot analysis of caspase-9 immunoprecipitates for Apaf-1 showed that uPAR and cathepsin B knockdown activated apoptosome complex formation in U251 cells. Downregulation of uPAR and cathepsin B also retarded nuclear translocation and interfered with DNA binding activity of CREB in both U251 and 5310 cells. Further western blotting analysis demonstrated that downregulation of uPAR and cathepsin B significantly decreased expression of the signaling molecules p-PDGFR-β, p-PI3K and p-Akt. An increase in the number of TUNEL-positive cells, increased Bax expression, and decreased Bcl-2 expression in nude mice brain tumor sections and brain tissue lysates confirm our in vitro results. CONCLUSIONS/SIGNIFICANCE: In conclusion, RNAi-mediated downregulation of uPAR and cathepsin B initiates caspase-dependent mitochondrial apoptosis in U251 cells and caspase-independent mitochondrial apoptosis in 5310 cells. Thus, targeting uPAR and cathepsin B-mediated signaling using siRNA may serve as a novel therapeutic strategy for the treatment of gliomas.

  17. Probing binding and cellular activity of pyrrolidinone and piperidinone small molecules targeting the urokinase receptor.

    Mani, Timmy; Liu, Degang; Zhou, Donghui; Li, Liwei; Knabe, William Eric; Wang, Fang; Oh, Kyungsoo; Meroueh, Samy O

    2013-12-01

    The urokinase receptor (uPAR) is a cell-surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of pyrrolidinone and piperidinone compounds, along with derivatives of previously-identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147-FAM) with inhibition constant (Ki ) values ranging from 6 to 63 μM. Structure-based computational pharmacophore analysis followed by extensive explicit-solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole-based and piperidinone-based compounds adopt different binding modes, despite their similar two-dimensional structures. In cells, pyrazole-based compounds showed significant inhibition of breast adenocarcinoma (MDA-MB-231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone-containing compounds exhibited no cytotoxicity even at concentrations of 100 μM. One pyrazole-based compound impaired MDA-MB-231 invasion, adhesion, and migration in a concentration-dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease-9 (gelatinase) activity in a concentration-dependent manner, while the piperidinone showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF-κB signaling, while pyrrolidinones and piperidinones had no effect. Annexin V staining suggested that the effect of the pyrazole-based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and

  18. Levels of plasminogen activator inhibitor type 1 and urokinase plasminogen activator receptor in non-small cell lung cancer as measured by quantitative ELISA and semiquantitative immunohistochemistry

    Pappot, Helle; Skov, Birgit Guldhammer; Pyke, Charles;

    1997-01-01

    , results in non-small cell lung cancer (NSCLC) studies obtained by quantitative ELISA and semiquantitative immunohistochemistry differ. If the prognostic value of the components of the plasminogen activation system is to be exploited clinically in the future, it is important to choose an easy and valid......PAR with the aim of predicting prognosis. In conclusion, a larger comparative study is needed to clarify the relationship between ELISA and immunohistochemical results, before a methodology for clinical use can be chosen in non-small cell lung cancer....... methodology. In the present study we investigated levels of plasminogen activator inhibitor type 1 (PAI-I) and urokinase plasminogen activator receptor (uPAR), as quantitated by ELISA in tumour extracts from 64 NSCLC patients (38 squamous cell carcinomas, 26 adenocarcinomas), and compared them to staining...

  19. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification

    Nielsen, L S; Kellerman, G M; Behrendt, N;

    1988-01-01

    The iodinated Mr approximately equal to 15,000 amino-terminal fragment (ATF) of the urokinase-type plasminogen activator (u-PA) molecule bound specifically to the cell surface of all of seven cultured human tumor cell lines studied. Cross-linking of iodinated ATF to the cell surface using...... a bifunctional amino-reactive reagent followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed with the four cell lines studied the occurrence of a single band migrating with an Mr of 70,000-75,000, indicating complex formation with an Mr of 55,000-60,000 u-PA receptor......-PA-R consists of one polypeptide chain. Two forms of u-PA-R, which differed with respect to affinity to concanavalin A, were identified. u-PA-R retained its ability to bind to ATF after cell lysis, and it was purified approximately 2,200-fold from biosynthetically labeled U937 cells by affinity chromatography...

  20. TISSUE INHIBITOR OF METALLOPROTEINASE 1, MATRIX METALLOPROTEINASE 9, ALPHA-1 ANTITRYPSIN, METALLOTHIONEIN AND UROKINASE TYPE PLASMINOGEN ACTIVATOR RECEPTOR IN SKIN BIOPSIES FROM PATIENTS AFFECTED BY AUTOIMMUNE BLISTERING DISEASES

    Ana Maria Abreu Velez

    2013-07-01

    Full Text Available Introduction: Proteinases and proteinase inhibitors have been described to play a role in autoimmune skin blistering diseases. We studied skin lesional biopsies from patients affected by several autoimmune skin blistering diseases for proteinases and proteinase inhibitors. Methods: We utilized immunohistochemistry to evaluate biopsies for alpha-1-antitrypsin, human matrix metalloproteinase 9 (MMP9, human tissue inhibitor of metalloproteinases 1 (TIMP-1, metallothionein and urokinase type plasminogen activator receptor (uPAR. We tested 30 patients affected by endemic pemphigus, 30 controls from the endemic area, and 15 normal controls. We also tested 30 biopsies from patients with bullous pemphigoid (BP, 20 with pemphigus vulgaris (PV, 8 with pemphigus foliaceus, and 14 with dermatitis herpetiformis (DH. Results: Contrary to findings in the current literature, most autoimmune skin blistering disease biopsies were negative for uPAR and MMP9. Only some chronic patients with El Bagre-EPF were positive to MMP9 in the dermis, in proximity to telocytes. TIMP-1 and metallothionein were positive in half of the biopsies from BP patients at the basement membrane of the skin, within several skin appendices, in areas of dermal blood vessel inflammation and within dermal mesenchymal-epithelial cell junctions.

  1. Bacterial endotoxin enhances colorectal cancer cell adhesion and invasion through TLR-4 and NF-kappaB-dependent activation of the urokinase plasminogen activator system.

    Killeen, S D

    2009-05-19

    Perioperative exposure to lipopolysaccharide (LPS) is associated with accelerated metastatic colorectal tumour growth. LPS directly affects cells through Toll-like receptor 4 (TLR-4) and the transcription factor NF-kappaB. The urokinase plasminogen activator (u-PA) system is intimately implicated in tumour cell extracellular matrix (ECM) interactions fundamental to tumour progression. Thus we sought to determine if LPS directly induces accelerated tumour cell ECM adhesion and invasion through activation of the u-PA system and to elucidate the cellular pathways involved. Human colorectal tumour cell lines were stimulated with LPS. u-PA concentration, u-PA activity, active u-PA, surface urokinase plasminogen activator receptor (u-PAR) and TLR-4 expression were assessed by ELISA, colorimetric assay, western blot analysis and flow cytometry respectively. In vitro tumour cell vitronectin adhesion and ECM invasion were analysed by vitronectin adhesion assay and ECM invasion chambers. u-PA and u-PAR function was inhibited with anti u-PA antibodies or the selective u-PA inhibitors amiloride or WXC-340, TLR-4 by TLR-4-blocking antibodies and NF-kappaB by the selective NF-kappaB inhibitor SN-50. LPS upregulates u-PA and u-PAR in a dose-dependent manner, enhancing in vitro tumour cell vitronectin adhesion and ECM invasion by >40% (P<0.01). These effects were ameliorated by u-PA and u-PAR inhibition. LPS activates NF-kappaB through TLR-4. TLR-4 and NF-kappaB inhibition ameliorated LPS-enhanced u-PA and u-PAR expression, tumour cell vitronectin adhesion and ECM invasion. LPS promotes tumour cell ECM adhesion and invasion through activation of the u-PA system in a TLR-4- and NF-kappaB-dependent manner.

  2. Modulation of cellular migration and survival by c-Myc through the downregulation of urokinase (uPA) and uPA receptor.

    Alfano, Daniela; Votta, Giuseppina; Schulze, Almut; Downward, Julian; Caputi, Mario; Stoppelli, Maria Patrizia; Iaccarino, Ingram

    2010-04-01

    It has been proposed that c-Myc proapoptotic activity accounts for most of its restraint of tumor formation. We established a telomerase-immortalized human epithelial cell line expressing an activatable c-Myc protein. We found that c-Myc activation induces, in addition to increased sensitivity to apoptosis, reductions in cell motility and invasiveness. Transcriptome analysis revealed that urokinase (uPA) and uPA receptor (uPAR) were strongly downregulated by c-Myc. Evidence is provided that the repression of uPA and uPAR may account for most of the antimigratory and proapoptotic activities of c-Myc. c-Myc is known to cooperate with Ras in cellular transformation. We therefore investigated if this cooperation could converge in the control of uPA/uPAR expression. We found that Ras is able to block the effects of c-Myc activation on apoptosis and cellular motility but not on cell invasiveness. Accordingly, the activation of c-Myc in the context of Ras expression had only minor influence on uPAR expression but still had a profound repressive effect on uPA expression. Thus, the differential regulation of uPA and uPAR by c-Myc and Ras correlates with the effects of these two oncoproteins on cell motility, invasiveness, and survival. In conclusion, we have discovered a novel link between c-Myc and uPA/uPAR. We propose that reductions of cell motility and invasiveness could contribute to the inhibition of tumorigenesis by c-Myc and that the regulation of uPA and uPAR expression may be a component of the ability of c-Myc to reduce motility and invasiveness.

  3. Administration of Recombinant Soluble Urokinase Receptor Per Se Is Not Sufficient to Induce Podocyte Alterations and Proteinuria in Mice

    Cathelin, Dominique; Placier, Sandrine; Ploug, Michael

    2014-01-01

    Circulating levels of soluble forms of urokinase-type plasminogen activator receptor (suPAR) are generally elevated in sera from children and adults with FSGS compared with levels in healthy persons or those with other types of kidney disease. In mice lacking the gene encoding uPAR, forced increa...... in increased glomerular proteinuria or altered podocyte architecture. Our findings suggest that glomerular deposits of suPAR caused by elevated plasma levels are not sufficient to engender albuminuria....

  4. Characterization and function of human Ly-6/uPAR molecules

    Hyun Kyung Kong & Jong Hoon Park*

    2012-11-01

    Full Text Available Human Ly-6/uPAR molecules are a superfamily composed oftwo subfamilies; one is the membrane bound proteins with aGPI-anchor and the other are secreted proteins without theGPI-anchor. Ly-6/uPAR molecules have remarkable amino acidhomology through a distinctive 8-10 cysteine-rich domain thatis associated predominantly with O-linked glycans. Thesemolecules are encoded by multiple tightly linked genes locatedon Chr. 8q23, and have a conserved genomic organization.Ly-6/uPAR molecules have an interesting expression patternduring hematopoiesis and on specific tumors indicating thatLy-6/uPAR molecules are associated with development of theimmune system and carcinogenesis. Thus, Ly-6/uPAR moleculesare useful antigens for diagnostic and therapeutic targets.This review summarizes our understanding of human Ly-6/uPAR molecules with regard to molecular structure as well aswhat is known about their function in normal and malignanttissues and suggest Ly-6/uPAR molecules as target antigens forcancer immunotherapy.

  5. Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures.

    Ge, Yifan; Siegel, Amanda P; Jordan, Rainer; Naumann, Christoph A

    2014-11-01

    Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

  6. Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor

    Høyer-Hansen, G; Pessara, U; Holm, A;

    2001-01-01

    Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this ...

  7. Structure-based design of an urokinase-type plasminogen activator receptor-derived peptide inhibiting cell migration and lung metastasis.

    Carriero, Maria Vincenza; Longanesi-Cattani, Immacolata; Bifulco, Katia; Maglio, Ornella; Lista, Liliana; Barbieri, Antonio; Votta, Giuseppina; Masucci, Maria Teresa; Arra, Claudio; Franco, Renato; De Rosa, Mario; Stoppelli, Maria Patrizia; Pavone, Vincenzo

    2009-09-01

    The urokinase-type plasminogen activator receptor (uPAR) plays a central role in sustaining the malignant phenotype and promoting tumor metastasis. The Ser(88)-Arg-Ser-Arg-Tyr(92) is the minimum chemotactic sequence of uPAR required to induce the same intracellular signaling as its ligand uPA. Here, we describe the generation of new peptide inhibitors of cell migration and invasion derived from SRSRY by a drug design approach. Ac-Arg-Glu-Arg-Phe-NH(2) (i.e., RERF), which adopts a turned structure in solution, was selected for its ability to potently prevent SRSRY-directed cell migration. Fluorescein-RERF associates with very high affinity to RBL-2H3 rat basophilic leukemia cells expressing the human formyl peptide receptor (FPR). Accordingly, femtomolar concentrations of RERF prevent agonist-dependent internalization of FPR and inhibit N-formyl-Met-Leu-Phe-dependent migration in a dose-dependent manner. In the absence of FPR, fluorescein-RERF binds to cell surface at picomolar concentrations in an alphav integrin-dependent manner. The involvement of vitronectin receptor is further supported by the findings that 100 pmol/L RERF selectively inhibits vitronectin-dependent RBL-2H3 cell migration and prevents SRSRY-triggered uPAR/alphav association. Furthermore, RERF reduces the speed of wound closure and the extent of Matrigel invasion by human fibrosarcoma HT1080 cells without affecting cell proliferation. Finally, a 3- to 5-fold reduction of lung metastasis number and size in nude mice following i.v. injection of green fluorescent protein-expressing HT1080 cells in the presence of 3.32 mg/kg RERF is observed. Our findings indicate that RERF effectively prevents malignant cell invasion in vivo with no signs of toxicity and may represent a promising prototype drug for anticancer therapy.

  8. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases

    Illemann, Martin; Bird, Nigel; Majeed, Ali;

    2009-01-01

    Metastatic growth and invasion by colon cancer cells in the liver requires the ability of the cancer cells to interact with the new tissue environment. Plasmin(ogen) is activated on cell surfaces by urokinase-type PA (uPA), and is regulated by uPAR and plasminogen activator inhibitor-1 (PAI-1......). To compare the expression patterns of uPA, uPAR and PAI-1 in colon cancer with that in their liver metastases, we analysed matched samples from 14 patients. In all 14 primary colon cancers, we found upregulation of uPAR, uPA mRNA and PAI-1 in primarily stromal cells at the invasive front. In 5 of the 14......, whereas 8 of the remaining 9 showed direct contact between the cancer cells and the liver parenchyma. We conclude that there are 2 distinct patterns of expression of uPAR, uPA and PAI-1 in colon cancer liver metastases and that these correlate closely with 2 morphological growth patterns. These findings...

  9. Chapter 8. Activation mechanisms of chemokine receptors

    Jensen, Pia C; Rosenkilde, Mette M

    2009-01-01

    Chemokine receptors belong to the large family of 7-transmembrane (7TM) G-protein-coupled receptors. These receptors are targeted and activated by a variety of different ligands, indicating that activation is a result of similar molecular mechanisms but not necessarily similar modes of ligand bin...

  10. Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR.

    Berg, Daniela; Wolff, Claudia; Malinowsky, Katharina; Tran, Kai; Walch, Axel; Bronger, Holger; Schuster, Tibor; Höfler, Heinz; Becker, Karl-Friedrich

    2012-01-01

    In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.

  11. Mechanism for the activation of glutamate receptors

    Scientists at the NIH have used a technique called cryo-electron microscopy to determine a molecular mechanism for the activation and desensitization of ionotropic glutamate receptors, a prominent class of neurotransmitter receptors in the brain and spina

  12. CERAPP: Collaborative Estrogen Receptor Activity Prediction Project

    U.S. Environmental Protection Agency — Data from a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project) demonstrating using predictive computational...

  13. Improved PET Imaging of uPAR expression using new Cu-64-labeled cross-bridged peptide ligands

    Persson, Morten; Hosseini, Masood; Madsen, Jacob;

    2013-01-01

    The correlation between uPAR expression, cancer cell invasion and metastases is now well-established and has prompted the development of a number of uPAR PET imaging agents, which could potentially identify cancer patients with invasive and metastatic lesions. In the present study, we synthesized...

  14. Urokinase receptor promotes ovarian cancer cell dissemination through its 84-95 sequence.

    Bifulco, Katia; Votta, Giuseppina; Ingangi, Vincenzo; Di Carluccio, Gioconda; Rea, Domenica; Losito, Simona; Montuori, Nunzia; Ragno, Pia; Stoppelli, Maria Patrizia; Arra, Claudio; Carriero, Maria Vincenza

    2014-06-30

    The clinical relevance of the urokinase receptor (uPAR) as a prognostic marker in ovarian cancer is well documented. We had shown that the uPAR sequence corresponding to 84-95 residues, linking D1 and D2 domains (uPAR84-95), drives cell migration and angiogenesis in a protease-independent manner. This study was aimed at defining the contribution of uPAR84-95 sequence to invasion of ovarian cancer cells. Now, we provide evidence that the ability of uPAR-expressing ovarian cancer cells to cross extra-cellular matrix and mesothelial monolayers is prevented by specific inhibitors of the uPAR84-95 sequence. To specifically investigate uPAR84-95 function, uPAR-negative CHO-K1 cells were stably transfected with cDNAs coding for uPAR D2 and D3 regions exposing (uPARD2D3) or lacking (uPAR∆D2D3) the 84-95 sequence. CHO-K1/D2D3 cells were able to cross matrigel, mesothelial and endothelial monolayers more efficiently than CHO-K1/∆D2D3 cells, which behave as CHO-K1 control cells. When orthotopically implanted in nude mice, tumor nodules generated by CHO-K1/D2D3 cells spreading to peritoneal cavity were more numerous as compared to CHO-K1/∆D2D3 cells. Ovarian tumor size and intra-tumoral microvessel density were significantly reduced in the absence of uPAR84-95. Our results indicate that cell associated uPAR promotes growth and abdominal dissemination of ovarian cancer cells mainly through its uPAR84-95 sequence.

  15. Serum uPAR as Biomarker in Breast Cancer Recurrence: A Mathematical Model.

    Hao, Wenrui; Friedman, Avner

    2016-01-01

    There are currently over 2.5 million breast cancer survivors in the United States and, according to the American Cancer Society, 10 to 20 percent of these women will develop recurrent breast cancer. Early detection of recurrence can avoid unnecessary radical treatment. However, self-examination or mammography screening may not discover a recurring cancer if the number of surviving cancer cells is small, while biopsy is too invasive and cannot be frequently repeated. It is therefore important to identify non-invasive biomarkers that can detect early recurrence. The present paper develops a mathematical model of cancer recurrence. The model, based on a system of partial differential equations, focuses on tissue biomarkers that include the plasminogen system. Among them, only uPAR is known to have significant correlation to its concentration in serum and could therefore be a good candidate for serum biomarker. The model includes uPAR and other associated cytokines and cells. It is assumed that the residual cancer cells that survived primary cancer therapy are concentrated in the same location within a region with a very small diameter. Model simulations establish a quantitative relation between the diameter of the growing cancer and the total uPAR mass in the cancer. This relation is used to identify uPAR as a potential serum biomarker for breast cancer recurrence.

  16. NMDA receptor activity in neuropsychiatric disorders

    Shaheen E Lakhan

    2013-06-01

    Full Text Available N-Methyl-D-aspartate (NMDA receptors play a variety of physiologic roles and their proper signaling is essential for cellular homeostasis. Any disruption in this pathway, leading to either enhanced or decreased activity, may result in the manifestation of neuropsychiatric pathologies such as schizophrenia, mood disorders, substance induced psychosis, Huntington's disease, Alzheimer's disease, and neuropsychiatric systemic lupus erythematosus. Here, we explore the notion that the overlap in activity of at least one biochemical pathway, the NMDA receptor pathway, may be the link to understanding the overlap in psychotic symptoms between diseases. This review intends to present a broad overview of those neuropsychiatric disorders for which alternations in NMDA receptor activity is prominent thus suggesting that continued direction of pharmaceutical intervention to this pathway may present a viable option for managing symptoms.

  17. Mechanism of FGF receptor dimerization and activation

    Sarabipour, Sarvenaz; Hristova, Kalina

    2016-01-01

    Fibroblast growth factors (fgfs) are widely believed to activate their receptors by mediating receptor dimerization. Here we show, however, that the FGF receptors form dimers in the absence of ligand, and that these unliganded dimers are phosphorylated. We further show that ligand binding triggers structural changes in the FGFR dimers, which increase FGFR phosphorylation. The observed effects due to the ligands fgf1 and fgf2 are very different. The fgf2-bound dimer structure ensures the smallest separation between the transmembrane (TM) domains and the highest possible phosphorylation, a conclusion that is supported by a strong correlation between TM helix separation in the dimer and kinase phosphorylation. The pathogenic A391E mutation in FGFR3 TM domain emulates the action of fgf2, trapping the FGFR3 dimer in its most active state. This study establishes the existence of multiple active ligand-bound states, and uncovers a novel molecular mechanism through which FGFR-linked pathologies can arise.

  18. Activation of Neuropeptide FF Receptors by Kisspeptin Receptor Ligands.

    Oishi, Shinya; Misu, Ryosuke; Tomita, Kenji; Setsuda, Shohei; Masuda, Ryo; Ohno, Hiroaki; Naniwa, Yousuke; Ieda, Nahoko; Inoue, Naoko; Ohkura, Satoshi; Uenoyama, Yoshihisa; Tsukamura, Hiroko; Maeda, Kei-Ichiro; Hirasawa, Akira; Tsujimoto, Gozoh; Fujii, Nobutaka

    2011-01-13

    Kisspeptin is a member of the RFamide neuropeptide family that is implicated in gonadotropin secretion. Because kisspeptin-GPR54 signaling is implicated in the neuroendocrine regulation of reproduction, GPR54 ligands represent promising therapeutic agents against endocrine secretion disorders. In the present study, the selectivity profiles of GPR54 agonist peptides were investigated for several GPCRs, including RFamide receptors. Kisspeptin-10 exhibited potent binding and activation of neuropeptide FF receptors (NPFFR1 and NPFFR2). In contrast, short peptide agonists bound with much lower affinity to NPFFRs while showing relatively high selectivity toward GPR54. The possible localization of secondary kisspeptin targets was also demonstrated by variation in the levels of GnRH release from the median eminence and the type of GPR54 agonists used. Negligible affinity of the reported NPFFR ligands to GPR54 was observed and indicates the unidirectional cross-reactivity between both ligands.

  19. 全身炎症反应综合征患者血浆尿激酶型纤溶酶原激活物及其特异性受体动态变化的研究%Dynamic changes in plasma levels of urokinase type plasminogen activator and urokinase type plasminogen activator receptor in patients with systemic inflammatory response syndrome

    武晓灵; 喻莉; 龙鼎; 杨军辉; 张远超; 耿峰

    2011-01-01

    目的 观察全身炎症反应综合征(SIRS)患者血浆尿激酶型纤溶酶原激活物(uPA)及其特异性受体(uPAR)的动态变化以及对预后的影响.方法 采用前瞻性病例对照研究设计方法,将85例住院患者按SIRS诊断标准分为SIRS组(50例)和非SlRS组(35例);SIRS组再按病情分为单纯SIRS组(26例)和合并多器官功能障碍综合征(MODS)组(24例),按预后分为生存组(35例)和死亡组(15例);另选择同期30例健康体检者作为对照.非SIRS组于入院当日,SIRS组于发生SIRS后的1、3、5、7 d,健康对照组于体检时采集空腹静脉血2 ml,用双抗体夹心酶联免疫吸附法(ELISA)测定血浆uPA和uPAR含量,并分析SIRS患者血浆uPAR含量与急性生理学与慢性健康状况评分系统I(APACHE I)评分的相关性.结果 单纯SIRS组、合并MODS组患者血浆uPA和uPAR含量均较非SIRS组和健康对照组显著升高[uPA(μg/L):1.208±0.264、1.120±0.276比0.744±0.190、0.782±0.257;uPAR(μg/L):3.704±1.018、4.970±1.284比1.892±0.476、1.823±0.797,均P<0.01],且合并MODS组uPAR含量明显高于单纯SIRS组(P<0.01).与生存组比较,死亡组5 d、7 d血浆uPA含量(μg/L)明显升高(5 d:1.177±0.185比0.856±0.223,7 d:1.377±0.185比0.836±0.223,均P<0.01),1、3、5、7 d血浆uPAR含量(μg/L)显著增高(1 d:5.301±1.410比3.888±1.015,3 d:4.017±0.898比2.994±0.638,5 d:5.032±1.238比2.536±1.017,7 d:5.232±1.238比3.536±1.017,均P<0.01).SIRS患者血浆uPAR含量与APACHE I评分呈显著正相关(r=0.640,P<0.O1).结论 SIRS患者存在凝血功能障碍,血浆uPA、uPAR含量显著增高,uPAR含量的升高提示预后不良.%Objective To study the dynamic changes in plasma levels of urokinase type plasminogen activator (uPA) and urokinase type plasminogen activator receptor (uPAR) and their influence on prognosis in patients with systemic inflammatory response syndrome (SIRS). Methods In this study, a prospective clinical case-control study was

  20. CERAPP: Collaborative estrogen receptor activity prediction project

    Mansouri, Kamel; Abdelaziz, Ahmed; Rybacka, Aleksandra

    2016-01-01

    Background: Humans are exposed to thousands of man-made chemicals in the environment. Some chemicals mimic natural endocrine hormones and, thus, have the potential to be endocrine disruptors. Most of these chemicals have never been tested for their ability to interact with the estrogen receptor (ER......). Risk assessors need tools to prioritize chemicals for evaluation in costly in vivo tests, for instance, within the U.S. EPA Endocrine Disruptor Screening Program. oBjectives: We describe a large-scale modeling project called CERAPP (Collaborative Estrogen Receptor Activity Prediction Project...

  1. Phenobarbital and Insulin Reciprocate Activation of the Nuclear Receptor Constitutive Androstane Receptor through the Insulin Receptor.

    Yasujima, Tomoya; Saito, Kosuke; Moore, Rick; Negishi, Masahiko

    2016-05-01

    Phenobarbital (PB) antagonized insulin to inactivate the insulin receptor and attenuated the insulin receptor downstream protein kinase B (AKT)-forkhead box protein O1 and extracellular signal-regulated kinase 1/2 signals in mouse primary hepatocytes and HepG2 cells. Hepatic AKT began dephosphorylation in an early stage of PB treatment, and blood glucose levels transiently increased in both wild-type and constitutive androstane receptor (CAR) knockout (KO) mice. On the other hand, blood glucose levels increased in wild-type mice, but not KO mice, in later stages of PB treatment. As a result, PB, acting as an insulin receptor antagonist, elicited CAR-independent increases and CAR-dependent decreases of blood glucose levels at these different stages of treatment, respectively. Reciprocally, insulin activation of the insulin receptor repressed CAR activation and induction of its target CYP2B6 gene in HepG2 cells. Thus, PB and insulin cross-talk through the insulin receptor to regulate glucose and drug metabolism reciprocally.

  2. Intact and cleaved forms of the urokinase receptor enhance discrimination of cancer from non-malignant conditions in patients presenting with symptoms related to colorectal cancer

    Lomholt, A F; Høyer-Hansen, G; Nielsen, H J;

    2009-01-01

    plasminogen activator receptor (suPAR) was proposed as a marker in CRC patients. This study was undertaken to evaluate the individual molecular forms of suPAR as discriminators in a group of patients undergoing endoscopical examination following symptoms related to colorectal cancer. METHODS: In a case......-control study comprising 308 patients undergoing endoscopical examination following CRC-related symptoms, 77 CRC patients with adenocarcinoma were age and gender matched to: 77 patients with adenomas; 77 with other non-malignant findings, and 77 with no findings. The different uPAR forms were measured...

  3. Urokinase receptor-associated protein (uPARAP) is expressed in connection with malignant as well as benign lesions of the human breast and occurs in specific populations of stromal cells

    Schnack Nielsen, Boye; Rank, Fritz; Engelholm, Lars H;

    2002-01-01

    The urokinase-type plasminogen activator (uPA) and the uPA receptor (uPAR) are key components in the plasminogen activation system, serving to promote specific events of extracellular matrix degradation in connection with tissue remodeling and cancer invasion. We recently described a new uPAR-ass...... lesions. Whereas the normal breast tissue was uPARAP-negative, all benign lesions and ductal carcinoma in situ lesions showed immunoreactivity in fibroblast-like cells and myoepithelial cells associated with the lesion. In invasive carcinoma, uPARAP immunoreactivity was limited to tumor...

  4. Structure-driven design of radionuclide tracers for non-invasive imaging of uPAR and targeted radiotherapy. The tale of a synthetic peptide antagonist

    Ploug, Michael

    2013-01-01

    as for monitoring the effects of such treatments by non-invasive imaging using e.g. positron emission tomography. This mini-review will focus on recent advancements in translational research devoted to non-invasive targeting of uPAR, with a view to molecular imaging of its expression in live individuals as well......-function relationships in uPAR has been refined to such a level that a rational design of uPAR function as well as compounds specifically targeting defined functions of uPAR are now realistic options. This knowledge opens new avenues for developing therapeutic intervention regimens targeting uPAR as well...... as specific eradication of these cells by targeted radiotherapy....

  5. Peroxisome proliferator-activated receptors for hypertension

    Daisuke; Usuda; Tsugiyasu; Kanda

    2014-01-01

    Peroxisome proliferator-activated receptors(PPARs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily, which is composed of four members encoded by distinct genes(α, β, γ, and δ). The genes undergo transactivation or transrepression under specific mechanisms that lead to the induction or repression of target gene expression. As is the case with other nuclear receptors, all four PPAR isoforms contain five or six structural regions in four functional domains; namely, A/B, C, D, and E/F. PPARs have many functions, particularly functions involving control of vascular tone, inflammation, and energy homeostasis, and are, therefore, important targets for hypertension, obesity, obesity-induced inflammation, and metabolic syndrome in general. Hence, PPARs also represent drug targets, and PPARα and PPARγ agonists are used clinically in the treatment of dyslipidemia and type 2 diabetes mellitus, respectively. Because of their pleiotropic effects, they have been identified as active in a number of diseases and are targets for the development of a broad range of therapies for a variety of diseases. It is likely that the range of PPARγ agonist therapeutic actions will result in novel approaches to lifestyle and other diseases. The combination of PPARs with reagents or with other cardiovascular drugs, such as diuretics and angiotensin Ⅱ receptor blockers, should be studied.This article provides a review of PPAR isoform characteristics, a discussion of progress in our understanding of the biological actions of PPARs, and a summary of PPAR agonist development for patient management. We also include a summary of the experimental and clinical evidence obtained from animal studies and clinical trials conducted to evaluate the usefulness and effectiveness of PPAR agonists in the treatment of lifestyle-related diseases.

  6. Expression of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor in synovial fluid of patients with temporomandibular disorders%颞下颌关节紊乱病关节液中尿纤溶酶原激活物及其受体的表达

    胡蕾; 梁新华; 朱桂全; 胡静; 史宗道

    2008-01-01

    Objective To investigate the level of urokinase-type plasminogen activator(uPA)and urokinase-type plasminogen activator receptor(uPAR)in synovial fluid of patients with temporomandibular disorders and to analyze their relation with temporomandibular disorders(TMD).Methods Synovial fluid was obtained from 64 sides of 56 TMD patients and from 16 sides of 10 asymptomatic healthy volunteers(control).The concentrations of uPA and uPAR in the synovial fluid were measured by ELISA.Forty-eight sides of TMD were divided into 3 groups:arthrosis,structure disorder and osteoarthrosis,each including 16 sides.Resuits The levels of uPA and uPAR were significantly higher in the synovial fluid of TMD patients than that in the control group(P<0.05),and the level of uPA and uPAR in osteoarthrosis group was significantly higher than that in arthrosis and structure disorder group(P<0.05).However,there was no difference in expression of uPA and uPAR between arthrosis and structure disorder groups(P>0.05).Conclusions uPA and uPAR in the synovial fluid may play a role in the pathogenesis of TMD.and the lever of uPA and uPAR in synovial fluid of TMD could be used as a biochemical markers to reflect pathological degree of TMD.%目的 检测颞下颌关节(TMJ)关节液中尿纤溶酶原激活物(urokinase-type plasminogen activator,uPA)及受体(urokinase-type plasminogen activator receptor,uPAR)的分泌量,探讨TMJ液中uPA及uPAR与颞下颌关节紊乱病(TMD)的关系.方法 采用酶联免疫吸附实验法检测56例TMD患者的64侧关节和10名健康志愿者的16侧关节的关节液标本中的uPA及uPAR的量.将符合纳入标准的48侧TMD患者的关节液标本根据临床诊断分为关节炎性组(A组)、结构紊乱组(B组)、骨关节病组(C组),每组16侧;10名健康志愿者的16侧关节液设为对照组(D组).结果 TMD中A组、B组、C组、D组uPA的检出量分别为(51.200±8.786)ng/L、(53.667±11.894)ng/L、(81.278±25.828)ng/L、(17.960±9.859)ng

  7. Towards universal approach for bacterial production of three-finger Ly6/uPAR proteins: Case study of cytotoxin I from cobra N. oxiana.

    Shulepko, M A; Lyukmanova, E N; Shenkarev, Z O; Dubovskii, P V; Astapova, M V; Feofanov, A V; Arseniev, A S; Utkin, Y N; Kirpichnikov, M P; Dolgikh, D A

    2017-02-01

    Cytotoxins or cardiotoxins is a group of polycationic toxins from cobra venom belonging to the 'three-finger' protein superfamily (Ly6/uPAR family) which includes small β-structural proteins (60-90 residues) with high disulfide bond content (4-5 disulfides). Due to a high cytotoxic activity for cancer cells, cytotoxins are considered as potential anticancer agents. Development of the high-throughput production methods is required for the prospective applications of cytotoxins. Here, efficient approach for bacterial production of recombinant analogue of cytotoxin I from N. oxiana containing additional N-terminal Met-residue (rCTX1) was developed. rCTX1 was produced in the form of E. coli inclusion bodies. Refolding in optimized conditions provided ∼6 mg of correctly folded protein from 1 L of bacterial culture. Cytotoxicity of rCTX1 for C6 rat glioma cells was found to be similar to the activity of wild type CTX1. The milligram quantities of (13)C,(15)N-labeled rCTX1 were obtained. NMR study confirmed the similarity of the spatial structures of recombinant and wild-type toxins. Additional Met residue does not perturb the overall structure of the three-finger core. The analysis of available data for different Ly6/uPAR proteins of snake and human origin revealed that efficiency of their folding in vitro is correlated with the number of proline residues in the third loop and the surface area of hydrophobic residues buried within the protein interior. The obtained data indicate that hydrophobic core is important for the folding of proteins with high disulfide bond content. Developed expression method opens new possibilities for structure-function studies of CTX1 and other related three-finger proteins.

  8. Peroxisome Proliferator Activated Receptors and Lipoprotein Metabolism

    Sander Kersten

    2008-01-01

    Full Text Available Plasma lipoproteins are responsible for carrying triglycerides and cholesterol in the blood and ensuring their delivery to target organs. Regulation of lipoprotein metabolism takes place at numerous levels including via changes in gene transcription. An important group of transcription factors that mediates the effect of dietary fatty acids and certain drugs on plasma lipoproteins are the peroxisome proliferator activated receptors (PPARs. Three PPAR isotypes can be distinguished, all of which have a major role in regulating lipoprotein metabolism. PPARα is the molecular target for the fibrate class of drugs. Activation of PPARα in mice and humans markedly reduces hepatic triglyceride production and promotes plasma triglyceride clearance, leading to a clinically significant reduction in plasma triglyceride levels. In addition, plasma high-density lipoprotein (HDL-cholesterol levels are increased upon PPARα activation in humans. PPARγ is the molecular target for the thiazolidinedione class of drugs. Activation of PPARγ in mice and human is generally associated with a modest increase in plasma HDL-cholesterol and a decrease in plasma triglycerides. The latter effect is caused by an increase in lipoprotein lipase-dependent plasma triglyceride clearance. Analogous to PPARα, activation of PPARβ/δ leads to increased plasma HDL-cholesterol and decreased plasma triglyceride levels. In this paper, a fresh perspective on the relation between PPARs and lipoprotein metabolism is presented. The emphasis is on the physiological role of PPARs and the mechanisms underlying the effect of synthetic PPAR agonists on plasma lipoprotein levels.

  9. Identification of Gene Markers for Activation of the Nuclear Receptor Pregnane X Receptor

    Many environmentally-relevant chemicals and drugs activate the nuclear receptor pregnane X receptor (PXR). Activation of PXR in the mouse liver can lead to increases in liver weight in part through increased hepatocyte replication similar to chemicals that activate other nuclear ...

  10. Model for growth hormone receptor activation based on subunit rotation within a receptor dimer

    Brown, Richard J.; Adams, Julian J.; Pelekanos, Rebecca A.; Wan, Yu; McKinstry, William J.; Palethorpe, Kathryn; Seeber, Ruth M.; Monks, Thea A.; Eidne, Karin A.; Parker, Michael W.; Waters, Michael J. (UWA); (St. Vincent); (Queensland)

    2010-07-13

    Growth hormone is believed to activate the growth hormone receptor (GHR) by dimerizing two identical receptor subunits, leading to activation of JAK2 kinase associated with the cytoplasmic domain. However, we have reported previously that dimerization alone is insufficient to activate full-length GHR. By comparing the crystal structure of the liganded and unliganded human GHR extracellular domain, we show here that there is no substantial change in its conformation on ligand binding. However, the receptor can be activated by rotation without ligand by inserting a defined number of alanine residues within the transmembrane domain. Fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET) and coimmunoprecipitation studies suggest that receptor subunits undergo specific transmembrane interactions independent of hormone binding. We propose an activation mechanism involving a relative rotation of subunits within a dimeric receptor as a result of asymmetric placement of the receptor-binding sites on the ligand.

  11. Interleukin-6 infusion during human endotoxaemia inhibits in vitro release of the urokinase receptor from peripheral blood mononuclear cells

    Ostrowski, S R; Plomgaard, P; Fischer, C P

    2005-01-01

    Leucocyte expression of the urokinase receptor [urokinase-type plasminogen activator receptor (uPAR)] is regulated by inflammatory mediators. This study investigated the in vivo effect of endotoxin, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha on uPAR-release in vivo and in vitro...... in humans. Healthy subjects received intravenous endotoxin injection [high-dose, 2 ng/kg (n=8) and low-dose, 0.06 ng/kg (n=7)], coadministration of 0.06 ng/kg endotoxin and 3 h recombinant human (rh)IL-6 infusion (n=7) or 3 h infusion of rhIL-6 (n=6), rhTNF-alpha (n=6) or NaCl (n=5). Soluble uPAR (su......PAR) was measured by enzyme-linked immunosorbent assay in plasma and supernatants from unstimulated and phytohaemagglutinin and lipopolysaccharide-stimulated peripheral blood mononuclear cell (PBMC) cultures incubated for 24 h. The spontaneous and stimulated uPAR-release from PBMC cultures was enhanced 5 h after...

  12. Design, synthesis, biochemical studies, cellular characterization, and structure-based computational studies of small molecules targeting the urokinase receptor.

    Wang, Fang; Eric Knabe, W; Li, Liwei; Jo, Inha; Mani, Timmy; Roehm, Hartmut; Oh, Kyungsoo; Li, Jing; Khanna, May; Meroueh, Samy O

    2012-08-01

    The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.

  13. Research progress of the relationship between urokinase receptor in tumor progression and development%尿激酶受体与肿瘤转移的关系研究进展

    王合兵; 肖坚; 陈文新; 杨炳林

    2014-01-01

    Elevated level of urokinase receptor (uPAR) is detected in various aggressive cancer types and is closely associated with poor prognosis of cancers. Binding of uPA to uPAR triggers the conversion of plasminogen to plasmin and the subsequent activation of metalloproteinases. These events confer tumor cells with the capability to degrade the components of the surrounding extracellular matrix, thus contributing to tumor cell invasion and metastasis. uPA-uPAR interaction also elicits signals that stimulate cell proliferation/survival and the expression of tumor-promoting genes, thus assisting tumor development. In addition to its interaction with uPA, uPAR also interacts with vitronectin and this interaction promotes cancer metastasis by activating Rac and stimulating cell migration. Although underlying mechanisms are yet to be fully elucidated, uPAR has been shown to facilitate epithelial-mesenchymal transition (EMT) and induce cancer stem cell-like properties in breast cancer cells. The fact that uPAR lacks intracellular domain suggests that its signaling must be mediated through its co-receptors. Indeed, uPAR interacts with diverse transmembrane proteins including integrins, ENDO180, G protein-coupled receptors and growth factor receptors in cancer cells and these interactions are proven to be critical for the role of uPAR in tumorigenesis. Inhibitory peptide that prevents uPA-uPAR interaction has shown the promise to prolong patients’ survival in the early stage of clinical trial. The importance of uPAR’s co-receptor in uPAR’s tumor-promoting effects implicate that anti-cancer therapeutic agents may also be developed by disrupting the interactions between uPAR and its functional partners.%尿激酶受体(uPAR)水平升高在不同类型的浸润性肿瘤中大多可检测到,且与肿瘤预后差密切相关。尿激酶(uPA)与uPAR结合触发纤溶酶原转换成纤维蛋白溶酶,继而出现基质金属蛋白酶被激活,降低周围的细

  14. Recruitment of activation receptors at inhibitory NK cell immune synapses.

    Nicolas Schleinitz

    Full Text Available Natural killer (NK cell activation receptors accumulate by an actin-dependent process at cytotoxic immune synapses where they provide synergistic signals that trigger NK cell effector functions. In contrast, NK cell inhibitory receptors, including members of the MHC class I-specific killer cell Ig-like receptor (KIR family, accumulate at inhibitory immune synapses, block actin dynamics, and prevent actin-dependent phosphorylation of activation receptors. Therefore, one would predict inhibition of actin-dependent accumulation of activation receptors when inhibitory receptors are engaged. By confocal imaging of primary human NK cells in contact with target cells expressing physiological ligands of NK cell receptors, we show here that this prediction is incorrect. Target cells included a human cell line and transfected Drosophila insect cells that expressed ligands of NK cell activation receptors in combination with an MHC class I ligand of inhibitory KIR. The two NK cell activation receptors CD2 and 2B4 accumulated and co-localized with KIR at inhibitory immune synapses. In fact, KIR promoted CD2 and 2B4 clustering, as CD2 and 2B4 accumulated more efficiently at inhibitory synapses. In contrast, accumulation of KIR and of activation receptors at inhibitory synapses correlated with reduced density of the integrin LFA-1. These results imply that inhibitory KIR does not prevent CD2 and 2B4 signaling by blocking their accumulation at NK cell immune synapses, but by blocking their ability to signal within inhibitory synapses.

  15. 新型抗uPAR人源化抗体的表达和活性检测%Expression and activity detection of the humanized anti-uPAR antibody

    孙梦梅; 靳彦文; 李平; 曹诚; 张部昌

    2011-01-01

    目的 制备抗尿激酶型纤溶酶原激活物受体(uPAR)人源化抗体并初步检测它们与抗原的亲和能力.方法 通过计算机辅助设计的结果,合成新型抗uPAR抗体的轻链和重链可变区基因序列,通过重叠PCR方法,拼接成完整的轻链和重链基因并克隆入pIRES双向表达载体.瞬时转染293T细胞,收取细胞上清,rProtein A亲和层析法纯化目的 抗体,并进行SDS-PAGE和免疫印迹鉴定,采用Biacore3000技术检测抗体与抗原的结合能力.结果 成功构建5种表达载体S1~S5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和55×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合.Biacore3000实验结果表明,S2、S4和S5抗体与抗原具有良好的亲和活性,且素和活性分别为1.74×10-8,1.49×10-8和1.05×10-8 mol/L.结论 成功构建并表达了5种抗uPAR人源化抗体,其中S2、S4和S5具有良好的抗原结合能力.%Objective To prepare the humanized monoclonal antibodies against urokinase-type plasminogen activator receptor (uPAR), and preliminarily detect their affinity to uPAR. Methods L and VH genes of humanized monoclonal antibodies against uPAR were designed by the computer and synthesized by overlap PCR. Genes encoding L and H chains were connected and then cloned into vector pIRES,a bicistronic expression vector. The recombinant plasmids were transfected into 293T cells, purified antibodies through rProteinA affinity chromatography,and further confirmed by Westernblotting. The affinity of the humanized antibodies to the uPAR was assessed by Biacore assay. Results Five expression vectors were constructed and the humanized monoclonal antibodies against uPAR were expressed and purified successfully.In reducing SDS-PAGE, the antibodies exhibited two bands of approximately 25 × 103 and 55 × 103, respectively. Western blotting assay showed that the humanized antibodies had recognition specificity to

  16. Cell death sensitization of leukemia cells by opioid receptor activation

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A.; Debatin, Klaus-Michael; Miltner, Erich

    2013-01-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies. PMID:23633472

  17. Activation of glucocorticoid receptors increases 5-HT2A receptor levels

    Trajkovska, Viktorija; Kirkegaard, Lisbeth; Krey, Gesa;

    2009-01-01

    Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker...... of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish...... an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased...

  18. Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /

    2014-01-01

    G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequ...

  19. The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor

    Zhou, X. Edward; Suino-Powell, Kelly M.; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W.; Reynolds, Ross; Engel, James Douglas; Xu, H. Eric (Michigan-Med); (Van Andel)

    2015-11-30

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  20. The orphan nuclear receptor TR4 is a vitamin A-activated nuclear receptor.

    Zhou, X Edward; Suino-Powell, Kelly M; Xu, Yong; Chan, Cee-Wah; Tanabe, Osamu; Kruse, Schoen W; Reynolds, Ross; Engel, James Douglas; Xu, H Eric

    2011-01-28

    Testicular receptors 2 and 4 (TR2/4) constitute a subgroup of orphan nuclear receptors that play important roles in spermatogenesis, lipid and lipoprotein regulation, and the development of the central nervous system. Currently, little is known about the structural features and the ligand regulation of these receptors. Here we report the crystal structure of the ligand-free TR4 ligand binding domain, which reveals an autorepressed conformation. The ligand binding pocket of TR4 is filled by the C-terminal half of helix 10, and the cofactor binding site is occupied by the AF-2 helix, thus preventing ligand-independent activation of the receptor. However, TR4 exhibits constitutive transcriptional activity on multiple promoters, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, or ligand binding substantially reduce the transcriptional activity of this receptor. Importantly, both retinol and retinoic acid are able to promote TR4 to recruit coactivators and to activate a TR4-regulated reporter. These findings demonstrate that TR4 is a ligand-regulated nuclear receptor and suggest that retinoids might have a much wider regulatory role via activation of orphan receptors such as TR4.

  1. Tools and techniques to study ligand-receptor interactions and receptor activation by TNF superfamily members.

    Schneider, Pascal; Willen, Laure; Smulski, Cristian R

    2014-01-01

    Ligands and receptors of the TNF superfamily are therapeutically relevant targets in a wide range of human diseases. This chapter describes assays based on ELISA, immunoprecipitation, FACS, and reporter cell lines to monitor interactions of tagged receptors and ligands in both soluble and membrane-bound forms using unified detection techniques. A reporter cell assay that is sensitive to ligand oligomerization can identify ligands with high probability of being active on endogenous receptors. Several assays are also suitable to measure the activity of agonist or antagonist antibodies, or to detect interactions with proteoglycans. Finally, self-interaction of membrane-bound receptors can be evidenced using a FRET-based assay. This panel of methods provides a large degree of flexibility to address questions related to the specificity, activation, or inhibition of TNF-TNF receptor interactions in independent assay systems, but does not substitute for further tests in physiologically relevant conditions.

  2. Insect Repellents: Modulators of Mosquito Odorant Receptor Activity

    2010-08-01

    molecular pathways through allosteric regulation of various proteins including proteases [39,40], the cannabinoid receptor 1 (CB1) [41], the a7 nicotinic...41. Price MR, Baillie GL, Thomas A, Stevenson LA, Easson M, et al. (2005) Allosteric modulation of the cannabinoid CB1 receptor . Mol Pharmacol 68...Insect Repellents: Modulators of Mosquito Odorant Receptor Activity Jonathan D. Bohbot, Joseph C. Dickens* Invasive Insect Biocontrol and Behavior

  3. Cellular receptor for urokinase plasminogen activator. Carboxyl-terminal processing and membrane anchoring by glycosyl-phosphatidylinositol

    Ploug, M; Rønne, E; Behrendt, N

    1991-01-01

    analysis of u-PAR after micropurification by affinity chromatography and N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of 2-3 mol of ethanolamine/mol protein. 2) Membrane-bound u-PAR is efficiently released from the surface...

  4. Evaluation of single amino acid chelate derivatives and regioselective radiolabelling of a cyclic peptide for the urokinase plasminogen activator receptor

    Armstrong, Andrea F.; Lemon, Jennifer A. [McMaster Institute for Applied Radiation Sciences, McMaster University, ON, L8S 4M1 (Canada); Czorny, Shannon K. [McMaster Institute for Applied Radiation Sciences, McMaster University, ON, L8S 4M1 (Canada); Juravinski Cancer Centre, Hamilton, ON, L8V 5C2 (Canada); Singh, Gurmit [Juravinski Cancer Centre, Hamilton, ON, L8V 5C2 (Canada); Valliant, John F. [Department of Chemistry, McMaster University, Hamilton, ON, L8S 4M1 (Canada); Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, ON, L8S 4M1 (Canada)], E-mail: valliant@mcmaster.ca

    2009-11-15

    Introduction: The aim of this work was to investigate the relative radiolabelling kinetics and affinity of a series of ligands for the [{sup 99m}Tc(CO){sub 3}]{sup +} core, both in the absence and in the presence of competing donors. This information was used to select a suitable ligand for radiolabelling complex peptide-based targeting vectors in high yield under mild conditions. Methods: A series of {alpha}-N-Fmoc-protected lysine derivatives bearing two heterocyclic donor groups at the {epsilon}-amine (, 2-pyridyl; , quinolyl; , 6-methoxy-2-pyridyl; 1d, 2-thiazolyl; 1e, N-methylimidazolyl; , 3-pyridyl) were synthesized and labelled with {sup 99m}Tc. A resin-capture purification strategy for the separation of residual ligand from the radiolabelled product was also developed. The binding affinities of targeted peptides 4, 5a and 5b for uPAR were determined using flow cytometry. Results: Variable temperature radiolabelling reactions using - and [{sup 99m}Tc(CO){sub 3}]{sup +} revealed optimal kinetics and good selectivity for compounds and 1d; in the case of , 1d, and 1e, the labelling can be conducted at ambient temperature. The utility of this class of ligands was further demonstrated by the radiolabelling of a cyclic peptide that is known to target the serine protease receptor uPAR; essentially quantitative incorporation of {sup 99m}Tc occurred exclusively at the SAAC site, despite the presence of a His residue, and without disruption of the disulfide bond. Conclusion: A series of single amino acid chelate (SAAC) ligands have been evaluated for their ability to incorporate {sup 99m}Tc into peptides. The lead agent to emerge from this work is the thiazole SAAC derivative 1d which has demonstrated the ability to regioselectively label the widest range of peptides.

  5. Gene expression changes of urokinase plasminogen activator and urokinase receptor in rat testes at postnatal stages%出生后不同发育阶段大鼠睾丸uPA/uPAR基因表达的变化

    D.H. Huang; H. Zhao; Y.H.Tian; H. G.Li; X. F. Ding; C. L.Xiong

    2007-01-01

    目的:为探讨uPA/uPAR系统在大鼠精子发生中的作用,研究出生后不同发育阶段大鼠睾丸组织中uPA/uPARmRNA表达的变化.方法:分别取出生后0、5、10、15、21、28、35、42、49、56天大鼠的睾丸组织,用实时定量聚合酶链反应法(PCR)检测各年龄组uPA/uPAR mRNA的表达变化.结果:除了出生后0天外,在其它大多数时间点uPA与uPAR mRNA表达趋势很相似.在出生后0天,大鼠睾丸组织中uPAR mRNA表达水平相对来说高于uPA mRNA表达水平,后逐渐减少,在出生后21天表达水平最低,到28天急剧增加,35天到达高峰,42天迅速下降,之后维持一个低表达水平.结论:uPA/uPAR系统可能通过调节生精细胞迁移及增殖、促进精子排放及残余胞体从成熟精子中脱落等方式与精子排放和精子发生密切相关.%Aim: To investigate the gene expression changes of urokinase plasminogen activator (uPA)/urokinase receptor (uPAR)in rat testes at postnatal stages and explore the effects of uPA/uPAR system on the rat spermatogenesis. Methods:The mRNAs of uPA and uPAR in rat testes were measured by using real-time quantitative polymerase chain reaction (PCR) at postnatal days 0, 5, 10, 15, 21, 28, 35, 42, 49 and 56, respectively. Results: The tendencies of uPA and uPAR mRNA expression were similar at most postnatal stages except for D0. The expression of uPAR mRNA in rats testes was relatively higher than that of uPA at postnatal D0, and both were decreased until D21, increased obviously at postnatal D28, reached a peak at postnatal D35, then declined sharply at postnatal D42 and retained at a low level afterwards. Conclusion: The uPA/uPAR system may be strongly linked to spermiation and spermatogenesis via regulating germ cell migration and proliferation, as well as promoting the spermiation and detached residual bodies from the mature spermatids. (Asian J Androl 2007 Sep; 9: 679-683)

  6. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Maryam Rakhshandehroo; Bianca Knoch; Michael Müller; Sander Kersten

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPAR alpha binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPAR alpha governs biologi...

  7. [Regulation of G protein-coupled receptor kinase activity].

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  8. Structural basis for activation of G-protein-coupled receptors

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine

    2002-01-01

    -type and mutant beta2-adrenergic receptors purified from Sf-9 insect cells. Our studies have also raised important questions regarding kinetics of receptors activation. These questions should be addressed in the future by application of techniques that will allow for simultaneous measurement of conformational...

  9. Endomorphins fully activate a cloned human mu opioid receptor.

    Gong, J; Strong, J A; Zhang, S; Yue, X; DeHaven, R N; Daubert, J D; Cassel, J A; Yu, G; Mansson, E; Yu, L

    1998-11-13

    Endomorphins were recently identified as endogenous ligands with high selectivity for mu opioid receptors. We have characterized the ability of endomorphins to bind to and functionally activate the cloned human mu opioid receptor. Both endomorphin-1 and endomorphin-2 exhibited binding selectivity for the mu opioid receptor over the delta and kappa opioid receptors. Both agonists inhibited forskolin-stimulated increase of cAMP in a dose-dependent fashion. When the mu opioid receptor was coexpressed in Xenopus oocytes with G protein-activated K+ channels, application of either endomorphin activated an inward K+ current. This activation was dose-dependent and blocked by naloxone. Both endomorphins acted as full agonists with efficacy similar to that of [D-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO). These data indicate that endomorphins act as full agonists at the human mu opioid receptor, capable of stimulating the receptor to inhibit the cAMP/adenylyl cyclase pathway and activate G-protein-activated inwardly rectifying potassium (GIRK) channels.

  10. Activating Receptor Signals Drive Receptor Diversity in Developing Natural Killer Cells.

    Freund, Jacquelyn; May, Rebecca M; Yang, Enjun; Li, Hongchuan; McCullen, Matthew; Zhang, Bin; Lenvik, Todd; Cichocki, Frank; Anderson, Stephen K; Kambayashi, Taku

    2016-08-01

    It has recently been appreciated that NK cells exhibit many features reminiscent of adaptive immune cells. Considerable heterogeneity exists with respect to the ligand specificity of individual NK cells and as such, a subset of NK cells can respond, expand, and differentiate into memory-like cells in a ligand-specific manner. MHC I-binding inhibitory receptors, including those belonging to the Ly49 and KIR families, are expressed in a variegated manner, which creates ligand-specific diversity within the NK cell pool. However, how NK cells determine which inhibitory receptors to express on their cell surface during a narrow window of development is largely unknown. In this manuscript, we demonstrate that signals from activating receptors are critical for induction of Ly49 and KIR receptors during NK cell development; activating receptor-derived signals increased the probability of the Ly49 bidirectional Pro1 promoter to transcribe in the forward versus the reverse direction, leading to stable expression of Ly49 receptors in mature NK cells. Our data support a model where the balance of activating and inhibitory receptor signaling in NK cells selects for the induction of appropriate inhibitory receptors during development, which NK cells use to create a diverse pool of ligand-specific NK cells.

  11. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly......The human chemokine system comprises 19 seven-transmembrane helix (7TM) receptors and 45 endogenous chemokines that often interact with each other in a promiscuous manner. Due to the chemokine system's primary function in leukocyte migration, it has a central role in immune homeostasis...... and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity...

  12. Tonic activation of presynaptic GABAB receptors on rat pallidosubthalamic terminals

    Lei CHEN; Wing-ho YUNG

    2005-01-01

    Aim: The subthalamic nucleus plays a critical role in the regulation of movement,and abnormal activity of its neurons is associated with some basal ganglia motor symptoms. We examined the presence of functional presynaptic GABAB receptors on pallidosubthalamic terminals and tested whether they were tonically active in the in vitro subthalamic slices. Methods: Whole-cell patch-clamp recordings were applied to acutely prepared rat subthalamic nucleus slices. The effects of specific GABAB agonist and antagonist on action potential-independent inhibitory postsynapfic currents (IPSCs), as well as holding current, were examined.Results: Superfusion of baclofen, a GABAB receptor agonist, significantly reduced the frequency of GABAA receptor-mediated miniature IPSCs (mIPSCs), in a Cd2+-sensitive manner, with no effect on the amplitude, indicating presynaptic inhibition on GABA release. In addition, baclofen induced a weak outward current only in a minority of subthalamic neurons. Both the pre- and post-synaptic effects of baclofen were prevented by the specific GABAB receptor antagonist,CGP55845. Furthermore, CGP55845 alone increased the frequency of mIPSCs,but had no effect on the holding current. Conclusion: These findings suggest the functional dominance of presynaptic GABAB receptors on the pallidosubthalamic terminals over the postsynaptic GABAB receptors on subthalamic neurons.Furthermore, the presynaptic, but not the postsynaptic, GABAB receptors are tonically active, suggesting that the presynaptic GABAB receptors in the subthalamic nucleus are potential therapeutic target for the treatment of Parkinson disease.

  13. Structure and dynamics of a constitutively active neurotensin receptor

    Krumm, Brian E. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Lee, Sangbae [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Bhattacharya, Supriyo [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Botos, Istvan [National Inst. of Health (NIH), Bethesda, MD (United States). National Inst. of Diabetes and; White, Courtney F. [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Du, Haijuan [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services; Vaidehi, Nagarajan [Beckman Research Inst. of the City of Hope, Duarte, CA (United States). Dept. of Molecular Immunology; Grisshammer, Reinhard [National Inst. of Health (NIH), Rockville, MD (United States). National Inst. of Neurological Disorders and Stroke, Dept. of Health and Human Services

    2016-12-07

    Many G protein-coupled receptors show constitutive activity, resulting in the production of a second messenger in the absence of an agonist; and naturally occurring constitutively active mutations in receptors have been implicated in diseases. To gain insight into mechanistic aspects of constitutive activity, we report here the 3.3 Å crystal structure of a constitutively active, agonist-bound neurotensin receptor (NTSR1) and molecular dynamics simulations of agonist-occupied and ligand-free receptor. Comparison with the structure of a NTSR1 variant that has little constitutive activity reveals uncoupling of the ligand-binding domain from conserved connector residues, that effect conformational changes during GPCR activation. Furthermore, molecular dynamics simulations show strong contacts between connector residue side chains and increased flexibility at the intracellular receptor face as features that coincide with robust signalling in cells. The loss of correlation between the binding pocket and conserved connector residues, combined with altered receptor dynamics, possibly explains the reduced neurotensin efficacy in the constitutively active NTSR1 and a facilitated initial engagement with G protein in the absence of agonist.

  14. Activation of 5-HT7 receptors increases neuronal platelet-derived growth factor β receptor expression.

    Vasefi, Maryam S; Kruk, Jeff S; Liu, Hui; Heikkila, John J; Beazely, Michael A

    2012-03-09

    Several antipsychotics have a high affinity for 5-HT7 receptors yet despite intense interest in the 5-HT7 receptor as a potential drug target to treat psychosis, the function and signaling properties of 5-HT7 receptors in neurons remain largely uncharacterized. In primary mouse hippocampal and cortical neurons, as well as in the SH-SY5Y cell line, incubation with 5-HT, 5-carboxamidotryptamine (5-CT), or 5-HT7 receptor-selective agonists increases the expression of platelet-derived growth factor (PDGF)β receptors. The increased PDGFβ receptor expression is cyclic AMP-dependent protein kinase (PKA)-dependent, suggesting that 5-HT7 receptors couple to Gα(s) in primary neurons. Interestingly, up-regulated PDGFβ receptors display an increased basal phosphorylation state at the phospholipase Cγ-activating tyrosine 1021. This novel linkage between the 5-HT7 receptor and the PDGF system may be an important GPCR-neurotrophic factor signaling pathway in neurons.

  15. Multiple switches in G protein-coupled receptor activation.

    Ahuja, Shivani; Smith, Steven O

    2009-09-01

    The activation mechanism of G protein-coupled receptors has presented a puzzle that finally may be close to solution. These receptors have a relatively simple architecture consisting of seven transmembrane helices that contain just a handful of highly conserved amino acids, yet they respond to light and a range of chemically diverse ligands. Recent NMR structural studies on the active metarhodopsin II intermediate of the visual receptor rhodopsin, along with the recent crystal structure of the apoprotein opsin, have revealed multiple structural elements or 'switches' that must be simultaneously triggered to achieve full activation. The confluence of several required structural changes is an example of "coincidence counting", which is often used by nature to regulate biological processes. In ligand-activated G protein-coupled receptors, the presence of multiple switches may provide an explanation for the differences between full, partial and inverse agonists.

  16. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Anshula eSamarajeewa

    2014-11-01

    Full Text Available The serotonin (5-HT type 7 receptor is expressed throughout the CNS including cortical neurons. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA-induced toxicity. The tropomyosin-related kinase B (TrkB receptor is one of the receptors for brain-derived neurotrophic factor (BDNF and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins towards the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  17. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  18. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation.

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G; Beazely, Michael A

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands.

  19. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamatergic synapses.

    Wang, Xulong; Lippi, Giordano; Carlson, David M; Berg, Darwin K

    2013-12-01

    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert 'silent' glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. We find that activation of nicotinic receptors on astrocytes releases a component that specifically recruits AMPA receptors to glutamatergic synapses. The recruitment appears to occur preferentially at what may be 'silent synapses', that is, synapses that have all the components required for glutamatergic transmission (including NMDA receptors) but lack sufficient AMPA receptors to generate a response. The results are unexpected and open up new possibilities for mechanisms underlying network formation and synaptic plasticity.

  20. Helix 11 Dynamics is Critical for Constitutive Androstane Receptor Activity

    Wright, Edward; Busby, Scott A.; Wisecarver, Sarah; Vincent, Jeremy; Griffin, Patrick R.; Fernandez, Elias J.

    2011-01-01

    The constitutive androstane receptor (CAR) transactivation can occur in the absence of exogenous ligand and this activity is enhanced by agonists TCPOBOP and meclizine. We use biophysical and cell-based assays to show that increased activity of CAR(TCPOBOP) relative to CAR(meclizine) corresponds to a higher affinity of CAR(TCPOBOP) for the steroid receptor coactivator-1. Additionally, steady-state fluorescence spectra suggest conformational differences between CAR(TCPOBOP):RXR and CAR(meclizi...

  1. Activation of 5-HT6 receptors inhibits corticostriatal glutamatergic transmission.

    Tassone, Annalisa; Madeo, Graziella; Schirinzi, Tommaso; Vita, Daniela; Puglisi, Francesca; Ponterio, Giulia; Borsini, Franco; Pisani, Antonio; Bonsi, Paola

    2011-09-01

    We investigated the effect of 5-HT6 receptor subtype activation on glutamatergic transmission by means of whole-cell patch-clamp electrophysiological recordings from medium spiny neurons of the striatum and layer V pyramidal neurons of the prefrontal cortex. To this aim, we took advantage of a novel ligand, ST1936, showing nM affinity and agonist activity at the 5-HT6 receptor subtype. Our data show that 5-HT6 receptor activation by ST1936 reduces the frequency of spontaneous excitatory postsynaptic currents, with an IC50 of 1.3 μM. Moreover, 5-HT6 receptor activation also reduced the amplitude of spontaneous excitatory postsynaptic currents recorded from medium spiny neurons, suggesting a mechanism of action involving postsynaptic 5-HT6 receptors, as further confirmed by the paired-pulse analysis on evoked excitatory postsynaptic currents and by recordings of miniature glutamatergic events. The inhibitory effect of ST1936 on glutamatergic transmission was prevented by the selective 5-HT6 receptor antagonist SB258585 and mimicked by a different agonist, WAY-181187. Conversely, in the cortex ST1936 reduced the frequency, but not the amplitude, of spontaneous excitatory postsynaptic currents suggesting a presynaptic or indirect effect of the 5-HT6 receptor.

  2. Screening of selected pesticides for oestrogen receptor activation in vitro

    Vinggaard, Anne; Breinholt, Vibeke; Larsen, John Christian

    1999-01-01

    Twenty pesticides were tested for their ability to activate the oestrogen receptor in vitro using an,MCF7 cell proliferation assay and a Yeast Oestrogen Screen. The fungicides fenarimol, triadimefon, and triadimenol were identified as weak oestrogen receptor agonists, which at 10 mu M induces a 2...... published that support oestrogenic activity in the intact animal, Thus, from the present results Mie suggest that oestrogen receptor activation may not be an important mode of action for these compounds. The need to include at least two bioassays in a screening procedure and for combining in vitro.......0, 2.4, and 1.9-fold increase in proliferation of human MCF7 breast cancer cells (E3 clone). The relative proliferation efficiency (RPE) was 43-69%, indicating partial agonism at the oestrogen receptor. Several pesticides did not have any effect oil the proliferation response after 6 days of exposure...

  3. Nicotinic Receptor Activity Alters Synaptic Plasticity

    John A. Dani

    2001-01-01

    Full Text Available Studies using specific agonists, antagonists, and lesions have shown that nicotinic cholinergic systems participate in attention, learning, and memory[1,2]. The nicotinic manipulations usually have the greatest influence on difficult tasks or on cognitively impaired subjects[2]. For example, Alzheimer's disease is characterized by a loss of cholinergic projections and nicotinic acetylcholine receptors (nAChRs in the cortex and hippocampus[3]. Nicotine skin patches can improve learning rates and attention in Alzheimer's patients[4].

  4. Modulation of β-catenin signaling by glucagon receptor activation.

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  5. Nuclear receptor corepressor-dependent repression of peroxisome-proliferator-activated receptor delta-mediated transactivation

    Krogsdam, Anne-M; Nielsen, Curt A F; Neve, Søren

    2002-01-01

    delta-RXR alpha heterodimer bound to an acyl-CoA oxidase (ACO)-type peroxisome-proliferator response element recruited a glutathione S-transferase-NCoR fusion protein in a ligand-independent manner. Contrasting with most other nuclear receptors, PPAR delta was found to interact equally well......The nuclear receptor corepressor (NCoR) was isolated as a peroxisome-proliferator-activated receptor (PPAR) delta interacting protein using the yeast two-hybrid system. NCoR interacted strongly with the ligand-binding domain of PPAR delta, whereas interactions with the ligand-binding domains...

  6. Analyzing the activation of the melanocortin-2 receptor of tetrapods.

    Dores, Robert M; Liang, Liang

    2014-07-01

    Following the biochemical characterization of the pituitary hormone, adrenocorticotropin (ACTH), in the 1950's, a number of structure/function studies were done which identifies two amino acid motifs in ACTH, the HFRW motif and KKRR motif, as critical for the activation of the "ACTH" receptor on adrenal cortex cells. In the 1990's the "ACTH" receptor was identified as a member of the melanocortin receptor gene family, and given the name melanocortin-2 receptor (MC2R). Since that time a number of studies on both tetrapod and teleost MC2R orthologs have established that these orthologs can only be activated by ACTH, but not by any of the MSH-sized melanocortin ligands, and these orthologs require interaction with the melanocortin-2 receptor accessory protein (MRAP) for functional expression. This review summarizes recent structure/function studies on human ACTH, and points out the importance of the GKPVG motif in ACTH for the activation of the receptor. In this regard, a multiple-step model for the activation of tetrapod and teleost MC2R orthologs is presented, and the evolution of gnathostome MC2R ligand selectivity and the requirement for MRAP interaction is discussed in light of a recent study on a cartilaginous fish MC2R ortholog. This review contains excerpts from the Gorbman/Bern Lecture presented at the Second Meeting of the North American Society for Comparative Endocrinology (NASCE).

  7. Monitoring leptin activity using the chicken leptin receptor.

    Hen, Gideon; Yosefi, Sera; Ronin, Ana; Einat, Paz; Rosenblum, Charles I; Denver, Robert J; Friedman-Einat, Miriam

    2008-05-01

    We report on the construction of a leptin bioassay based on the activation of chicken leptin receptor in cultured cells. A human embryonic kidney (HEK)-293 cell line, stably transfected with the full-length cDNA of chicken leptin receptor together with a STAT3-responsive reporter gene specifically responded to recombinant human and Xenopus leptins. The observed higher sensitivity of chicken leptin receptor to the former is in agreement with the degree of sequence similarity among these species (about 60 and 38% identical amino acids between humans and chickens, and between humans and Xenopus respectively). The specific activation of signal transduction through the chicken leptin receptor, shown here for the first time, suggests that the transition of Gln269 (implicated in the Gln-to-Pro Zucker fatty mutation in rats) to Glu in chickens does not impair its activity. Analysis of leptin-like activity in human serum samples of obese and lean subjects coincided well with leptin levels determined by RIA. Serum samples of pre- and post partum cows showed a tight correlation with the degree of adiposity. However, specific activation of the chicken leptin receptor in this assay was not observed with serum samples from broiler or layer chickens (representing fat and lean phenotypes respectively) or with those from turkey. Similar leptin receptor activation profiles were observed with cells transfected with human leptin receptor. Further work is needed to determine whether the lack of leptin-like activity in the chicken serum samples is due to a lack of leptin in this species or simply to a serum level of leptin that is below the detection threshold.

  8. A human vitamin D receptor mutant activated by cholecalciferol.

    Ousley, Amanda M; Castillo, Hilda S; Duraj-Thatte, Anna; Doyle, Donald F; Azizi, Bahareh

    2011-07-01

    The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.

  9. UROKINASE-TYPE PLASMINOGEN ACTIVATOR, ITS RECEPTOR AND INHIBITOR EXPRESSION IN HEPATOCELLULAR CARCINOMA RELATION TO CANCER INVASIVENESS AND PROGNOSIS

    Zheng Qi; Tang Zhaoyou; Wu Zhiquan; Shi Daren; Tang Huibin; Zhu Yunsong; Song Houyan

    1998-01-01

    Objective:To study the relevance of uPA, uPAR and PAI-1 to hepatocellular carcinoma (HCC). Methods:The expression at protein level of uPA, uPAR and PAI-1was determined in 48 cases of HCC and 12 cases of benign tumors of liver (as control) by immunohistochemistry.Results: When compared to cancer-adjacent liver tissue and the control, positive rate of immune staining for uPA,uPAR and PAI-1 on cell membrane were significantly higher in HCC cells (P<0.05). Positive staining of uPA and uPAR was seen in 16 of 22 and 19 of 22 cases of HCC with invasion, respectively (P<0.01 and P<0.001). In 8 of 8cases with cancer embolus, and in 6 of 6 cases with lymph node metastasis was the expression of positive uPAR.Compared with 2 of 17 cases without recurrence, uPAR was positive in 15 of 17 recurrent cases (P<0.01). In 36cases who survived, 17 was positive uPAR and 15 positive PAI-1, while in 12 cases who died 2 years after surgery, 12were positive for uPAR and 9 positive PAI-1, respectively (P<0.01 and P<0.05). In 15 positive cases for all three parameters, 11 had cancer invasion and 7 died within 2 years, while in negative cases, 2 had invasion and none died within 2 years (P<0.05). Conclusion: Expression of.uPA, uPAR and PAI-1 is increased in HCC, uPA and uPAR may contribute significantly to HCC invasion and metastasis. uPAR and PAI-1 are associated with poor prognosis of HCC.

  10. Activation and dynamic network of the M2 muscarinic receptor

    Miao, Yinglong; Nichols, Sara E.; Gasper, Paul M.; Metzger, Vincent T; McCammon, J. Andrew

    2013-01-01

    G-protein-coupled receptors (GPCRs) mediate cellular responses to various hormones and neurotransmitters and are important targets for treating a wide spectrum of diseases. Although significant advances have been made in structural studies of GPCRs, details of their activation mechanism remain unclear. The X-ray crystal structure of the M2 muscarinic receptor, a key GPCR that regulates human heart rate and contractile forces of cardiomyocytes, was determined recently in an inactive antagonist...

  11. uPAR Targeted Radionuclide Therapy with 177Lu-DOTA-AE105 Inhibits Dissemination of Metastatic Prostate Cancer

    Persson, Morten; Juhl, Karina; Rasmussen, Palle

    2014-01-01

    value of 100 nM in a competitive binding experiment. In vivo, uPAR targeted radionuclide therapy significantly reduced the number of metastatic lesions in the disseminated metastatic prostate cancer model, when compared to vehicle and nontargeted 177Lu groups (p bioluminescence imaging...... with bioluminescence imaging in a cohort of animals during the treatment study. In conclusion, uPAR targeted radiotherapy resulted in a significant reduction in the number of metastatic lesions in a human metastatic prostate cancer model. Furthermore, we have provided the first evidence of the potential...

  12. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Rakhshandehroo, M.; Knoch, B.; Müller, M.R.; Kersten, A.H.

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPAR alpha) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPAR alpha serves as a molecular target for hypolip

  13. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  14. Extended Synaptotagmin Interaction with the Fibroblast Growth Factor Receptor Depends on Receptor Conformation, Not Catalytic Activity.

    Tremblay, Michel G; Herdman, Chelsea; Guillou, François; Mishra, Prakash K; Baril, Joëlle; Bellenfant, Sabrina; Moss, Tom

    2015-06-26

    We previously demonstrated that ESyt2 interacts specifically with the activated FGF receptor and is required for a rapid phase of receptor internalization and for functional signaling via the ERK pathway in early Xenopus embryos. ESyt2 is one of the three-member family of Extended Synaptotagmins that were recently shown to be implicated in the formation of endoplasmic reticulum (ER)-plasma membrane (PM) junctions and in the Ca(2+) dependent regulation of these junctions. Here we show that ESyt2 is directed to the ER by its putative transmembrane domain, that the ESyts hetero- and homodimerize, and that ESyt2 homodimerization in vivo requires a TM adjacent sequence but not the SMP domain. ESyt2 and ESyt3, but not ESyt1, selectively interact in vivo with activated FGFR1. In the case of ESyt2, this interaction requires a short TM adjacent sequence and is independent of receptor autophosphorylation, but dependent on receptor conformation. The data show that ESyt2 recognizes a site in the upper kinase lobe of FGFR1 that is revealed by displacement of the kinase domain activation loop during receptor activation.

  15. Implications of compound heterozygous insulin receptor mutations in congenital muscle fibre type disproportion myopathy for the receptor kinase activation

    Klein, H H; Müller, R; Vestergaard, H

    1999-01-01

    % of the receptors to become insulin-dependently activated. The mother carries a point mutation at the last base pair in exon 17 which, due to abnormal alternative splicing, could lead to normally transcribed receptor or truncated receptor lacking the kinase region. Kinase activation was normal in the mother......We studied insulin receptor kinase activation in two brothers with congenital muscle fibre type disproportion myopathy and compound heterozygous mutations of the insulin receptor gene, their parents, and their unaffected brother. In the father who has a heterozygote Arg1174-->Gln mutation, in situ......'s skeletal muscle, suggesting that virtually no truncated receptor was expressed. Receptor kinase activity was, however, reduced by 95 and 91% in the compound heterozygous brothers. This suggests that the mother's mutated allele contributes little to the generation of functional receptor protein...

  16. Epidermis-type lipoxygenase 3 regulates adipocyte differentiation and peroxisome proliferator-activated receptor gamma activity

    Hallenborg, Philip; Jørgensen, Claus; Petersen, Rasmus K;

    2010-01-01

    The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differenti...

  17. PSD-95 regulates D1 dopamine receptor resensitization, but not receptor-mediated Gs-protein activation

    Peihua Sun; Jingru Wang; Weihua Gu; Wei Cheng; Guo-zhang Jin; Eitan Friedman; Jie Zheng; Xuechu Zhen

    2009-01-01

    The present study aims to define the role of postsynaptic density (PSD)-95 in the regulation of dopamine (DA) receptor function. We found that PSD-95 physically associates with either D1 or D2 DA receptors in co-transfected HEK-293 cells. Stimulation of DA receptors altered the association between D1 receptor and PSD-95 in a time-depen-dent manner. Functional assays indicated that PSD-95 co-expression did not affect D1 receptor-stimulated cAMP pro-duction, Gs-protein activation or receptor desensitization. However, PSD-95 accelerated the recovery of internalized membrane receptors by promoting receptor recycling, thus resulting in enhanced resensitization of internalized D1 receptors. Our results provide a novel mechanism for regulating DA receptor recycling that may play an important role in postsynaptic DA functional modulation and synaptic neuroplasticity.

  18. Activities of nicotinic acetylcholine receptors modulate neurotransmission and synaptic architecture

    Akira Oda; Hidekazu Tanaka

    2014-01-01

    The cholinergic system is involved in a broad spectrum of brain function, and its failure has been implicated in Alzheimer’s disease. Acetylcholine transduces signals through muscarinic and nicotinic acetylcholine receptors, both of which inlfuence synaptic plasticity and cognition. However, the mechanisms that relate the rapid gating of nicotinic acetylcholine receptors to per-sistent changes in brain function have remained elusive. Recent evidence indicates that nicotinic acetylcholine receptors activities affect synaptic morphology and density, which result in per-sistent rearrangements of neural connectivity. Further investigations of the relationships between nicotinic acetylcholine receptors and rearrangements of neural circuitry in the central nervous system may help understand the pathogenesis of Alzheimer’s disease.

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

    Li-Jun Li

    2016-10-01

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

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

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

    2016-01-01

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

  1. Ghrelin receptor conformational dynamics regulate the transition from a preassembled to an active receptor:Gq complex.

    Damian, Marjorie; Mary, Sophie; Maingot, Mathieu; M'Kadmi, Céline; Gagne, Didier; Leyris, Jean-Philippe; Denoyelle, Séverine; Gaibelet, Gérald; Gavara, Laurent; Garcia de Souza Costa, Mauricio; Perahia, David; Trinquet, Eric; Mouillac, Bernard; Galandrin, Ségolène; Galès, Céline; Fehrentz, Jean-Alain; Floquet, Nicolas; Martinez, Jean; Marie, Jacky; Banères, Jean-Louis

    2015-02-03

    How G protein-coupled receptor conformational dynamics control G protein coupling to trigger signaling is a key but still open question. We addressed this question with a model system composed of the purified ghrelin receptor assembled into lipid discs. Combining receptor labeling through genetic incorporation of unnatural amino acids, lanthanide resonance energy transfer, and normal mode analyses, we directly demonstrate the occurrence of two distinct receptor:Gq assemblies with different geometries whose relative populations parallel the activation state of the receptor. The first of these assemblies is a preassembled complex with the receptor in its basal conformation. This complex is specific of Gq and is not observed with Gi. The second one is an active assembly in which the receptor in its active conformation triggers G protein activation. The active complex is present even in the absence of agonist, in a direct relationship with the high constitutive activity of the ghrelin receptor. These data provide direct evidence of a mechanism for ghrelin receptor-mediated Gq signaling in which transition of the receptor from an inactive to an active conformation is accompanied by a rearrangement of a preassembled receptor:G protein complex, ultimately leading to G protein activation and signaling.

  2. Allosteric activation mechanism of the cys-loop receptors

    Yong-chang CHANG; Wen WU; Jian-liang ZHANG; Yao HUANG

    2009-01-01

    Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors (nAChRs). With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy (EM), and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of amino-terminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.

  3. Immunomodulatory effects of endogenous and synthetic peptides activating opioid receptors.

    Pomorska, Dorota K; Gach, Katarzyna; Janecka, Anna

    2014-01-01

    The main role of endogenous opioid peptides is the modulation of pain. Opioid peptides exert their analgesic activity by binding to the opioid receptors distributed widely in the central nervous system (CNS). However, opioid receptors are also found on tissues and organs outside the CNS, including the cells of the immune system, indicating that opioids are capable of exerting additional effects in periphery. Morphine, which is a gold standard in the treatment of chronic pain, is well-known for its immunosuppressive effects. Much less is known about the immunomodulatory effects exerted by endogenous (enkephalins, endorphins, dynorphins and endomorphins) and synthetic peptides activating opioid receptors. In this review we tried to summarize opioid peptide-mediated modulation of immune cell functions which can be stimulatory as well as inhibitory.

  4. A bacterial tyrosine phosphatase inhibits plant pattern recognition receptor activation.

    Macho, Alberto P; Schwessinger, Benjamin; Ntoukakis, Vardis; Brutus, Alexandre; Segonzac, Cécile; Roy, Sonali; Kadota, Yasuhiro; Oh, Man-Ho; Sklenar, Jan; Derbyshire, Paul; Lozano-Durán, Rosa; Malinovsky, Frederikke Gro; Monaghan, Jacqueline; Menke, Frank L; Huber, Steven C; He, Sheng Yang; Zipfel, Cyril

    2014-03-28

    Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.

  5. Ah receptor agonist activity in frequently consumed food items

    Waard, de W.J.; Aarts, J.M.M.J.G.; Peijnenburg, A.A.C.M.; Kok, de T.M.C.M.; Schooten, van F.J.; Hoogenboom, L.A.P.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) receives much attention for its role in the toxicity of dioxins and dioxin-like polychlorinated biphenyls. However, many other compounds have also been reported to bind and activate AhR, of which natural food components are of special interest from a human health

  6. The cardiovascular effects of peroxisome proliferator-activated receptor agonists.

    Friedland, Sayuri N; Leong, Aaron; Filion, Kristian B; Genest, Jacques; Lega, Iliana C; Mottillo, Salvatore; Poirier, Paul; Reoch, Jennifer; Eisenberg, Mark J

    2012-02-01

    Although peroxisome proliferator-activated receptor agonists are prescribed to improve cardiovascular risk factors, their cardiovascular safety is controversial. We therefore reviewed the literature to identify landmark randomized controlled trials evaluating the effect of peroxisome proliferator-activated receptor gamma agonists (pioglitazone and rosiglitazone), alpha agonists (fenofibrate and gemfibrozil), and pan agonists (bezafibrate, muraglitazar, ragaglitazar, tesaglitazar, and aleglitazar) on cardiovascular outcomes. Pioglitazone may modestly reduce cardiovascular events but also may increase the risk of bladder cancer. Rosiglitazone increases the risk of myocardial infarction and has been withdrawn in European and restricted in the United States. Fibrates improve cardiovascular outcomes only in select subgroups: fenofibrate in diabetic patients with metabolic syndrome, gemfibrozil in patients with dyslipidemia, and bezafibrate in patients with diabetes or metabolic syndrome. The cardiovascular safety of the new pan agonist aleglitazar, currently in phase II trials, remains to be determined. The heterogenous effects of peroxisome proliferator-activated receptor agonists to date highlight the importance of postmarketing surveillance. The critical question of why peroxisome proliferator-activated receptor agonists seem to improve cardiovascular risk factors without significantly improving cardiovascular outcomes requires further investigation.

  7. Urokinase-Type Plasminogen Activator Receptor as a Potential PET Biomarker in Glioblastoma

    Persson, Morten; Nedergaard, Mette K; Brandt-Larsen, Malene

    2016-01-01

    an orthotopic xenograft model of glioblastoma. Tumor growth was monitored using bioluminescence imaging. Five to six weeks after inoculation, all mice were scanned with small-animal PET/CT using two new uPAR PET ligands ((64)Cu-NOTA-AE105 and (68)Ga-NOTA-AE105) and, for comparison, O-(2-(18)F...

  8. Identification of prostaglandin E2 receptor subtype 2 as a receptor activated by OxPAPC.

    Li, Rongsong; Mouillesseaux, Kevin P; Montoya, Dennis; Cruz, Daniel; Gharavi, Navid; Dun, Martin; Koroniak, Lukasz; Berliner, Judith A

    2006-03-17

    Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC), which has been shown to accumulate in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to bind monocytes by activation of endothelial beta1 integrin and subsequent deposition of fibronectin on the apical surface. Our previous studies suggest this function of OxPAPC is mediated via a Gs protein-coupled receptor (GPCR). PEIPC (1-palmitoyl-2-epoxyisoprostane E2-sn-glycero-3-phosphorylcholine) is the most active lipid in OxPAPC that activates this pathway. We screened a number of candidate GPCRs for their interaction with OxPAPC and PEIPC, using a reporter gene assay; we identified prostaglandin E2 receptor EP2 and prostaglandin D2 receptor DP as responsive to OxPAPC. We focused on EP2, which is expressed in ECs, monocytes, and macrophages. OxPAPC component PEIPC, but not POVPC, activated EP2 with an EC50 of 108.6 nmol/L. OxPAPC and PEIPC were also able to compete with PGE2 for binding to EP2 in a ligand-binding assay. The EP2 specific agonist butaprost was shown to mimic the effect of OxPAPC on the activation of beta1 integrin and the stimulation of monocyte binding to endothelial cells. Butaprost also mimicked the effect of OxPAPC on the regulation of tumor necrosis factor-alpha and interleukin-10 in monocyte-derived cells. EP2 antagonist AH6809 blocked the activation of EP2 by OxPAPC in HEK293 cells and blocked the interleukin-10 response to PEIPC in monocytic THP-1 cells. These results suggest that EP2 functions as a receptor for OxPAPC and PEIPC, either as the phospholipid ester or the released fatty acid, in both endothelial cells and macrophages.

  9. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Patricia C. Brum; Rolim, Natale P. L.; BACURAU, Aline V. N.; Alessandra Medeiros

    2006-01-01

    Heart failure (HF) is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardia...

  10. Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

    2012-01-01

    Abstract Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV) and active zone (AZ) protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imag...

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

    Sato, Shoko, E-mail: satosho@rs.tus.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan); Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp [Division of Molecular Pharmacology, Department of Pathophysiological and Therapeutic Science, Yonago 683-8503 (Japan); Tohkin, Masahiro, E-mail: tohkin@phar.nagoya-cu.ac.jp [Department of Medical Safety Science, Graduate School of Pharmaceutical Science, Nagoya City University, Nagoya 267-8603 (Japan); Gonzalez, Frank J., E-mail: gonzalef@mail.nih.gov [Laboratory of Metabolism, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 (United States); Komai, Michio, E-mail: mkomai@m.tohoku.ac.jp [Laboratory of Nutrition, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555 (Japan)

    2013-11-15

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

  12. Influence of phasic and tonic dopamine release on receptor activation

    Dreyer, Jakob Kristoffer Kisbye; Herrik, Kjartan F; Berg, Rune W

    2010-01-01

    Tonic and phasic dopamine release is implicated in learning, motivation, and motor functions. However, the relationship between spike patterns in dopaminergic neurons, the extracellular concentration of dopamine, and activation of dopamine receptors remains unresolved. In the present study, we...... develop a computational model of dopamine signaling that give insight into the relationship between the dynamics of release and occupancy of D(1) and D(2) receptors. The model is derived from first principles using experimental data. It has no free parameters and offers unbiased estimation...

  13. An improved ivermectin-activated chloride channel receptor for inhibiting electrical activity in defined neuronal populations

    Lynagh, Timothy Peter; Lynch, Joseph W

    2010-01-01

    for surgically implanted stimulus delivery methods and their use of nonhuman receptors. A third silencing method, an invertebrate glutamate-gated chloride channel receptor (GluClR) activated by ivermectin, solves the stimulus delivery problem as ivermectin is a safe, well tolerated drug that reaches the brain...

  14. Activation of D4 dopamine receptor decreases angiotensin II type 1 receptor expression in rat renal proximal tubule cells.

    Chen, Ken; Deng, Kun; Wang, Xiaoyan; Wang, Zhen; Zheng, Shuo; Ren, Hongmei; He, Duofen; Han, Yu; Asico, Laureano D; Jose, Pedro A; Zeng, Chunyu

    2015-01-01

    The dopaminergic and renin-angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal angiotensin type 1 (AT1) receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubule cells from Wistar-Kyoto (WKY) rats, but the D4 receptor regulation of AT1 receptor is aberrant in renal proximal tubule cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time- and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na(+)-K(+) ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na(+)-K(+) ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na(+)-K(+) ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na(+)-K(+) ATPase activity in SHR cells. This was confirmed in vivo; pretreatment with PD128077 for 1 week augmented the antihypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension.

  15. Peroxisome proliferator-activated receptor alpha target genes.

    Rakhshandehroo, Maryam; Knoch, Bianca; Müller, Michael; Kersten, Sander

    2010-01-01

    The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  16. Peroxisome Proliferator-Activated Receptor Alpha Target Genes

    Maryam Rakhshandehroo

    2010-01-01

    Full Text Available The peroxisome proliferator-activated receptor alpha (PPARα is a ligand-activated transcription factor involved in the regulation of a variety of processes, ranging from inflammation and immunity to nutrient metabolism and energy homeostasis. PPARα serves as a molecular target for hypolipidemic fibrates drugs which bind the receptor with high affinity. Furthermore, PPARα binds and is activated by numerous fatty acids and fatty acid-derived compounds. PPARα governs biological processes by altering the expression of a large number of target genes. Accordingly, the specific role of PPARα is directly related to the biological function of its target genes. Here, we present an overview of the involvement of PPARα in lipid metabolism and other pathways through a detailed analysis of the different known or putative PPARα target genes. The emphasis is on gene regulation by PPARα in liver although many of the results likely apply to other organs and tissues as well.

  17. Glycine Receptor α2 Subunit Activation Promotes Cortical Interneuron Migration

    Ariel Avila

    2013-08-01

    Full Text Available Glycine receptors (GlyRs are detected in the developing CNS before synaptogenesis, but their function remains elusive. This study demonstrates that functional GlyRs are expressed by embryonic cortical interneurons in vivo. Furthermore, genetic disruption of these receptors leads to interneuron migration defects. We discovered that extrasynaptic activation of GlyRs containing the α2 subunit in cortical interneurons by endogenous glycine activates voltage-gated calcium channels and promotes calcium influx, which further modulates actomyosin contractility to fine-tune nuclear translocation during migration. Taken together, our data highlight the molecular events triggered by GlyR α2 activation that control cortical tangential migration during embryogenesis.

  18. Hyaluronic acid induces activation of the κ-opioid receptor.

    Barbara Zavan

    Full Text Available INTRODUCTION: Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function. METHODS: We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM. RESULTS: Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr. CONCLUSIONS: HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  19. Pyrimidinergic Receptor Activation Controls Toxoplasma gondii Infection in Macrophages.

    Aline Cristina Abreu Moreira-Souza

    Full Text Available Infection by the protozoan parasite Toxoplasma gondii is highly prevalent worldwide and may have serious clinical manifestations in immunocompromised patients. T. gondii is an obligate intracellular parasite that infects almost any cell type in mammalian hosts, including immune cells. The immune cells express purinergic P2 receptors in their membrane--subdivided into P2Y and P2X subfamilies--whose activation is important for infection control. Here, we examined the effect of treatment with UTP and UDP in mouse peritoneal macrophages infected with T. gondii tachyzoites. Treatment with these nucleotides reduced parasitic load by 90%, but did not increase the levels of the inflammatory mediators NO and ROS, nor did it modulate host cell death by apoptosis or necrosis. On the other hand, UTP and UDP treatments induced early egress of tachyzoites from infected macrophages, in a Ca2+-dependent manner, as shown by scanning electron microscopy analysis, and videomicroscopy. In subsequent infections, prematurely egressed parasites had reduced infectivity, and could neither replicate nor inhibit the fusion of lysosomes to the parasitophorous vacuole. The use of selective agonists and antagonists of the receptor subtypes P2Y2 and P2Y4 and P2Y6 showed that premature parasite egress may be mediated by the activation of these receptor subtypes. Our results suggest that the activity of P2Y host cell receptors controls T. gondii infection in macrophages, highlighting the importance of pyrimidinergic signaling for innate immune system response against infection. Finally the P2Y receptors should be considered as new target for the development of drugs against T. gondii infection.

  20. Neurohumoral activation in heart failure: the role of adrenergic receptors

    Patricia C. Brum

    2006-09-01

    Full Text Available Heart failure (HF is a common endpoint for many forms of cardiovascular disease and a significant cause of morbidity and mortality. The development of end-stage HF often involves an initial insult to the myocardium that reduces cardiac output and leads to a compensatory increase in sympathetic nervous system activity. Acutely, the sympathetic hyperactivity through the activation of beta-adrenergic receptors increases heart rate and cardiac contractility, which compensate for decreased cardiac output. However, chronic exposure of the heart to elevated levels of catecholamines released from sympathetic nerve terminals and the adrenal gland may lead to further pathologic changes in the heart, resulting in continued elevation of sympathetic tone and a progressive deterioration in cardiac function. On a molecular level, altered beta-adrenergic receptor signaling plays a pivotal role in the genesis and progression of HF. beta-adrenergic receptor number and function are decreased, and downstream mechanisms are altered. In this review we will present an overview of the normal beta-adrenergic receptor pathway in the heart and the consequences of sustained adrenergic activation in HF. The myopathic potential of individual components of the adrenergic signaling will be discussed through the results of research performed in genetic modified animals. Finally, we will discuss the potential clinical impact of beta-adrenergic receptor gene polymorphisms for better understanding the progression of HF.A insuficiência cardíaca (IC é a via final comum da maioria das doenças cardiovasculares e uma das maiores causas de morbi-mortalidade. O desenvolvimento do estágio final da IC freqüentemente envolve um insulto inicial do miocárdio, reduzindo o débito cardíaco e levando ao aumento compensatório da atividade do sistema nervoso simpático (SNS. Existem evidências de que apesar da exposição aguda ser benéfica, exposições crônicas a elevadas concentra

  1. Activation of Penile Proadipogenic Peroxisome Proliferator-Activated Receptor with an Estrogen: Interaction with Estrogen Receptor Alpha during Postnatal Development

    Mahmoud M. Mansour

    2008-01-01

    Full Text Available Exposure to the estrogen receptor alpha (ER ligand diethylstilbesterol (DES between neonatal days 2 to 12 induces penile adipogenesis and adult infertility in rats. The objective of this study was to investigate the in vivo interaction between DES-activated ER and the proadipogenic transcription factor peroxisome proliferator-activated receptor gamma (PPAR. Transcripts for PPARs , , and and 1a splice variant were detected in Sprague-Dawley normal rat penis with PPAR predominating. In addition, PPAR1b and PPAR2 were newly induced by DES. The PPAR transcripts were significantly upregulated with DES and reduced by antiestrogen ICI 182, 780. At the cellular level, PPAR protein was detected in urethral transitional epithelium and stromal, endothelial, neuronal, and smooth muscular cells. Treatment with DES activated ER and induced adipocyte differentiation in corpus cavernosum penis. Those adipocytes exhibited strong nuclear PPAR expression. These results suggest a biological overlap between PPAR and ER and highlight a mechanism for endocrine disruption.

  2. Visualising androgen receptor activity in male and female mice.

    D Alwyn Dart

    Full Text Available Androgens, required for normal development and fertility of males and females, have vital roles in the reproductive tract, brain, cardiovascular system, smooth muscle and bone. Androgens function via the androgen receptor (AR, a ligand-dependent transcription factor. To assay and localise AR activity in vivo we generated the transgenic "ARE-Luc" mouse, expressing a luciferase reporter gene under the control of activated endogenous AR. In vivo imaging of androgen-mediated luciferase activity revealed several strongly expressing tissues in the male mouse as expected and also in certain female tissues. In males the testes, prostate, seminal vesicles and bone marrow all showed high AR activity. In females, strong activity was seen in the ovaries, uterus, omentum tissue and mammary glands. In both sexes AR expression and activity was also found in salivary glands, the eye (and associated glands, adipose tissue, spleen and, notably, regions of the brain. Luciferase protein expression was found in the same cell layers as androgen receptor expression. Additionally, mouse AR expression and activity correlated well with AR expression in human tissues. The anti-androgen bicalutamide reduced luciferase signal in all tissues. Our model demonstrates that androgens can act in these tissues directly via AR, rather than exclusively via androgen aromatisation to estrogens and activation of the estrogen receptor. Additionally, it visually demonstrates the fundamental importance of AR signalling outside the normal role in the reproductive organs. This model represents an important tool for physiological and developmental analysis of androgen signalling, and for characterization of known and novel androgenic or antiandrogenic compounds.

  3. Monocyte Signal Transduction Receptors in Active and Latent Tuberculosis

    Magdalena Druszczynska

    2013-01-01

    Full Text Available The mechanisms that promote either resistance or susceptibility to TB disease remain insufficiently understood. Our aim was to compare the expression of cell signaling transduction receptors, CD14, TLR2, CD206, and β2 integrin LFA-1 on monocytes from patients with active TB or nonmycobacterial lung disease and healthy individuals with M.tb latency and uninfected controls to explain the background of the differences between clinical and subclinical forms of M.tb infection. A simultaneous increase in the expression of the membrane bound mCD14 receptor and LFA-1 integrin in patients with active TB may be considered a prodrome of breaking immune control by M.tb bacilli in subjects with the latent TB and absence of clinical symptoms.

  4. Manipulation of P2X Receptor Activities by Light Stimulation

    Sang Seong Kim

    2016-01-01

    Full Text Available P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels.

  5. Estrogen receptor- and aryl hydrocarbon receptor- mediated activities of a coal-tar creosote

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R. [Michigan State University, East Lansing, MI (USA). Dept. of Biochemistry

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol- equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyp 1a1-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O- depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal- tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic response in vivo.

  6. Estrogen receptor- and aryl hydrocarbon receptor-mediated activities of a coal-tar creosote

    Fielden, M.R.; Wu, Z.F.; Sinal, C.J.; Jury, H.H.; Bend, J.R.; Hammond, G.L.; Zacharewski, T.R.

    2000-05-01

    A coal-tar creosote was examined for estrogen receptor (ER)- and aryl hydrocarbon receptor (AhR)-mediated activity using a battery of mechanistically based assays. In vitro, creosote was found to bind to the mouse ER, bind to the human sex hormone-binding globulin, and elicit partial agonist activity in reporter gene assays in transiently transfected MCF-7 cells. Based on competitive binding to the mouse ER, creosote contains approximately 165 mg/L of estradiol-equivalents. Creosote effectively transformed the AhR in vitro and induced a Cyplal-regulated luciferase reporter gene in transiently transfected Hepa 1c1c7 cells. Based on dose-response curves, creosote contains approximately 730 mg/L of dioxin-equivalents. Creosote did not exhibit any AhR-mediated antiestrogenic activity in vitro. In vivo, creosote significantly induced liver pentoxyresorufin O-depentylation and ethoxyresorufin-O-deethylation (EROD) in a dose-dependent manner in ovariectomized (OVX) ICR mice, but did not increase uterine weight wet or vaginal cornification, due possibly to AhR-mediated antiestrogenic activity. In OVX DBA/2 mice, a strain less responsive to AhR ligands, creosote induced liver EROD to a lesser extent, but still did not show an increase in uterine wet weight or vaginal cornification. These results demonstrate that coal-tar creosote exhibits AhR- and ER-mediated activity in vitro, but its dioxinlike activity may suppress estrogenic responses in vivo.

  7. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  8. Discovery of novel protease activated receptors 1 antagonists with potent antithrombotic activity in vivo.

    Perez, Michel; Lamothe, Marie; Maraval, Catherine; Mirabel, Etienne; Loubat, Chantal; Planty, Bruno; Horn, Clemens; Michaux, Julien; Marrot, Sebastien; Letienne, Robert; Pignier, Christophe; Bocquet, Arnaud; Nadal-Wollbold, Florence; Cussac, Didier; de Vries, Luc; Le Grand, Bruno

    2009-10-08

    Protease activated receptors (PARs) or thrombin receptors constitute a class of G-protein-coupled receptors (GPCRs) implicated in the activation of many physiological mechanisms. Thus, thrombin activates many cell types such as vascular smooth muscle cells, leukocytes, endothelial cells, and platelets via activation of these receptors. In humans, thrombin-induced platelet aggregation is mediated by one subtype of these receptors, termed PAR1. This article describes the discovery of new antagonists of these receptors and more specifically two compounds: 2-[5-oxo-5-(4-pyridin-2-ylpiperazin-1-yl)penta-1,3-dienyl]benzonitrile 36 (F 16618) and 3-(2-chlorophenyl)-1-[4-(4-fluorobenzyl)piperazin-1-yl]propenone 39 (F 16357), obtained after optimization. Both compounds are able to inhibit SFLLR-induced human platelet aggregation and display antithrombotic activity in an arteriovenous shunt model in the rat after iv or oral administration. Furthermore, these compounds are devoid of bleeding side effects often observed with other types of antiplatelet drugs, which constitutes a promising advantage for this new class of antithrombotic agents.

  9. Methylthioadenosine reprograms macrophage activation through adenosine receptor stimulation.

    Peter A Keyel

    Full Text Available Regulation of inflammation is necessary to balance sufficient pathogen clearance with excessive tissue damage. Central to regulating inflammation is the switch from a pro-inflammatory pathway to an anti-inflammatory pathway. Macrophages are well-positioned to initiate this switch, and as such are the target of multiple therapeutics. One such potential therapeutic is methylthioadenosine (MTA, which inhibits TNFα production following LPS stimulation. We found that MTA could block TNFα production by multiple TLR ligands. Further, it prevented surface expression of CD69 and CD86 and reduced NF-KB signaling. We then determined that the mechanism of this action by MTA is signaling through adenosine A2 receptors. A2 receptors and TLR receptors synergized to promote an anti-inflammatory phenotype, as MTA enhanced LPS tolerance. In contrast, IL-1β production and processing was not affected by MTA exposure. Taken together, these data demonstrate that MTA reprograms TLR activation pathways via adenosine receptors to promote resolution of inflammation.

  10. DMPD: Receptor tyrosine kinases and the regulation of macrophage activation. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 14726496 Receptor tyrosine kinases and the regulation of macrophage activation. Cor...(.csml) Show Receptor tyrosine kinases and the regulation of macrophage activation. PubmedID 14726496 Title ...Receptor tyrosine kinases and the regulation of macrophage activation. Authors Co

  11. Bioluminescence imaging of estrogen receptor activity during breast cancer progression.

    Vantaggiato, Cristina; Dell'Omo, Giulia; Ramachandran, Balaji; Manni, Isabella; Radaelli, Enrico; Scanziani, Eugenio; Piaggio, Giulia; Maggi, Adriana; Ciana, Paolo

    2016-01-01

    Estrogen receptors (ER) are known to play an important regulatory role in mammary gland development as well as in its neoplastic transformation. Although several studies highlighted the contribution of ER signaling in the breast transformation, little is known about the dynamics of ER state of activity during carcinogenesis due to the lack of appropriate models for measuring the extent of receptor signaling in time, in the same animal. To this aim, we have developed a reporter mouse model for the non-invasive in vivo imaging of ER activity: the ERE-Luc reporter mouse. ERE-Luc is a transgenic mouse generated with a firefly luciferase (Luc) reporter gene driven by a minimal promoter containing an estrogen responsive element (ERE). This model allows to measure receptor signaling in longitudinal studies by bioluminescence imaging (BLI). Here, we have induced sporadic mammary cancers by treating systemically ERE-Luc reporter mice with DMBA (9,10-dimethyl 1,2-benzanthracene) and measured receptor signaling by in vivo imaging in individual animals from early stage until a clinically palpable tumor appeared in the mouse breast. We showed that DMBA administration induces an increase of bioluminescence in the whole abdominal area 6 h after treatment, the signal rapidly disappears. Several weeks later, strong bioluminescence is observed in the area corresponding to the mammary glands. In vivo and ex vivo imaging analysis demonstrated that this bioluminescent signal is localized in the breast area undergoing neoplastic transformation. We conclude that this non-invasive assay is a novel relevant tool to identify the activation of the ER signaling prior the morphological detection of the neoplastic transformation.

  12. UV ACTIVATION OF RECEPTOR TYROSINE KINASE-ACTIVITY

    COFFER, PJ; BURGERING, BMT; PEPPELENBOSCH, MP; BOS, JL; KRUIJER, W

    1995-01-01

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumour promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as

  13. Peroxisome Proliferator–Activated Receptors and The Metabolic Syndrome

    Anna Meiliana

    2009-04-01

    Full Text Available BACKGROUND: Obesity is a growing threat to global health by virtue of its association with insulin resistance, inflammation, hypertension, and dyslipidemia, collectively known as the metabolic syndrome (MetS. The nuclear receptors PPARα and PPARγ are therapeutic targets for hypertriglyceridemia and insulin resistance, respectively, and drugs that modulate these receptors are currently in clinical use. More recent work on the PPARδ has uncovered a dual benefit for both hypertriglyceridemia and insulin resistance, highlighting the broad potential of PPARs in the treatment of metabolic disease. CONTENT: We have learned much about PPARs, the metabolic fat sensors, and the molecular pathways they regulate. Through their distinct tissue distribution and specific target gene activation, the three PPARs together control diverse aspects of fatty acid metabolism, energy balance, insulin sensitivity glucose homeostasis, inflammation, hypertension and atherosclerosis. These studies have advanced our understanding of the etiology for the MetS. Mechanisms revealed by these studies highlight the importance of emerging concepts, such as the endocrine function of adipose tissue, tissue-tissue cross-talk and lipotoxicity, in the pathogenesis of type 2 diabetes mellitus and CVD. SUMMARY: The elucidation of key regulators of energy balance and insulin signaling have revolutionized our understanding of fat and sugar metabolism and their intimate link. The three ‘lipidsensing’ (PPARα, PPARγ and PPARδ exemplify this connection, regulating diverse aspects of lipid and glucose homeostasis, and serving as bonafide therapeutic targets. KEYWORDS: peroxisome proliferator, activated receptor, metabolic syndrome.

  14. Persistently active cannabinoid receptors mute a subpopulation of hippocampal interneurons.

    Losonczy, Attila; Biró, Agota A; Nusser, Zoltan

    2004-02-03

    Cortical information processing requires an orchestrated interaction between a large number of pyramidal cells and albeit fewer, but highly diverse GABAergic interneurons (INs). The diversity of INs is thought to reflect functional and structural specializations evolved to control distinct network operations. Consequently, specific cortical functions may be selectively modified by altering the input-output relationship of unique IN populations. Here, we report that persistently active cannabinoid receptors, the site of action of endocannabinoids, and the psychostimulants marijuana and hashish, switch off the output (mute) of a unique class of hippocampal INs. In paired recordings between cholecystokinin-immunopositive, mossy fiber-associated INs, and their target CA3 pyramidal cells, no postsynaptic currents could be evoked with single presynaptic action potentials or with repetitive stimulations at frequencies <25 Hz. Cannabinoid receptor antagonists converted these "mute" synapses into high-fidelity ones. The selective muting of specific GABAergic INs, achieved by persistent presynaptic cannabinoid receptor activation, provides a state-dependent switch in cortical networks.

  15. Receptor conformation and constitutive activity in CCR5 chemokine receptor function and HIV infection.

    Flanagan, Colleen A

    2014-01-01

    The CCR5 chemokine receptor mediates the effects of proinflammatory β-chemokines that stimulate chemotaxis, activation, and proliferation of macrophages and T cells. CCR5 is also the major coreceptor that mediates HIV infection in combination with CD4. Chemokine agonists of CCR5 stimulate the activation of cellular calcium and protein kinase signaling pathways that depend on the activation of Gαi and probably also Gαq in some cells. Chemokines also stimulate the recruitment of β-arrestin, which is required for clathrin-dependent receptor internalization and acts as a scaffold protein for the chemotaxis signaling complex that mobilizes the actin cytoskeleton. CCR5 is partially constitutively active for the activation of Gαi, but the physiological significance has not been studied. HIV binding to CCR5 also activates G protein and protein kinase signaling but, in addition, stimulates the production of proinflammatory cytokines, including TNF-α, and mobilizes the actin cytoskeleton to form the fusion pore that allows viral entry and subsequently supports viral replication in the cell. The CCR5 conformation that mediates the fusion of the viral and cell membranes is unknown, but it is probably distinct from the conformation that mediates G protein signaling. Nonpeptide CCR5 blockers are allosteric inverse agonists that increase dissociation of both chemokines and HIV envelope proteins, but this does not correlate with their ability to inhibit HIV infection. Nevertheless, the inverse agonist activity may ameliorate the immune activation that exacerbates AIDS pathogenesis. Inverse agonists of CCR5 have established efficacy for the treatment of AIDS, but may also be useful in preventing HIV infection.

  16. Common structural basis for constitutive activity of the ghrelin receptor family

    Holst, Birgitte; Holliday, Nicholas D; Bach, Anders

    2004-01-01

    -independent signaling activity. The structurally homologous motilin receptor served as a constitutively silent control; upon agonist stimulation, however, it signaled with a similar efficacy to the three related receptors. The constitutive activity of the ghrelin receptor and of neurotensin receptor 2 through the G...... demonstrated that the epitope-tagged ghrelin receptor was constitutively internalized but could be trapped at the cell surface by an inverse agonist, whereas GPR39 remained at the cell surface. Mutational analysis showed that the constitutive activity of both the ghrelin receptor and GPR39 could systematically...

  17. Activation profiles of opioid ligands in HEK cells expressing δ opioid receptors

    Clark J; Demirci Hasan; Gharagozlou Parham; Lameh Jelveh

    2002-01-01

    Abstract Background The aim of the present study was to characterize the activation profiles of 15 opioid ligands in transfected human embryonic kidney cells expressing only δ opioid receptors. Activation profiles of most of these ligands at δ opioid receptors had not been previously characterized in vitro. Receptor activation was assessed by measuring the inhibition of forskolin-stimulated cAMP production. Results Naltrexone and nalorphine were classified as antagonists at δ opioid receptor....

  18. Dual activities of odorants on olfactory and nuclear hormone receptors.

    Pick, Horst; Etter, Sylvain; Baud, Olivia; Schmauder, Ralf; Bordoli, Lorenza; Schwede, Torsten; Vogel, Horst

    2009-10-30

    We have screened an odorant compound library and discovered molecules acting as chemical signals that specifically activate both G-protein-coupled olfactory receptors (ORs) on the cell surface of olfactory sensory neurons and the human nuclear estrogen receptor alpha (ER) involved in transcriptional regulation of cellular differentiation and proliferation in a wide variety of tissues. Hence, these apparent dual active odorants induce distinct signal transduction pathways at different subcellular localizations, which affect both neuronal signaling, resulting in odor perception, and the ER-dependent transcriptional control of specific genes. We demonstrate these effects using fluorescence-based in vitro and cellular assays. Among these odorants, we have identified synthetic sandalwood compounds, an important class of molecules used in the fragrance industry. For one estrogenic odorant we have also identified the cognate OR. This prompted us to compare basic molecular recognition principles of odorants on the two structurally and apparent functionally non-related receptors using computational modeling in combination with functional assays. Faced with the increasing evidence that ORs may perform chemosensory functions in a number of tissues outside of the nasal olfactory epithelium, the unraveling of these molecular ligand-receptor interaction principles is of critical importance. In addition the evidence that certain olfactory sensory neurons naturally co-express ORs and ERs may provide a direct functional link between the olfactory and hormonal systems in humans. Our results are therefore useful for defining the structural and functional characteristics of ER-specific odorants and the role of odorant molecules in cellular processes other than olfaction.

  19. Phagocytic receptors activate and immune inhibitory receptor SIRPα inhibits phagocytosis through paxillin and cofilin.

    Gitik, Miri; Kleinhaus, Rachel; Hadas, Smadar; Reichert, Fanny; Rotshenker, Shlomo

    2014-01-01

    The innate immune function of phagocytosis of apoptotic cells, tissue debris, pathogens, and cancer cells is essential for homeostasis, tissue repair, fighting infection, and combating malignancy. Phagocytosis is carried out in the central nervous system (CNS) by resident microglia and in both CNS and peripheral nervous system by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a "do not eat me" message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue debris "degenerated myelin" which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a) the cytoskeleton generates the mechanical forces that drive phagocytosis and (b) both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation, and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the

  20. Phagocytic receptors activate and immune inhibitory receptor SIRPalpha inhibits phagocytosis through paxillin and cofilin

    Miri eGitik

    2014-04-01

    Full Text Available The innate-immune function of phagocytosis of apoptotic cells, tissue-debris, pathogens and cancer cells is essential for homeostasis, tissue repair, fighting infection and combating malignancy. Phagocytosis is carried out in the CNS by resident microglia and in both CNS and PNS by recruited macrophages. While phagocytosis proceeds, bystander healthy cells protect themselves by sending a do not eat me message to phagocytes as CD47 on their surface ligates immune inhibitory receptor SIRPα on the surface of phagocytes and SIRPα then produces the signaling which inhibits phagocytosis. This helpful mechanism becomes harmful when tissue-debris and unhealthy cells inhibit their own phagocytosis by employing the same mechanism. However, the inhibitory signaling that SIRPα produces has not been fully revealed. We focus here on how SIRPα inhibits the phagocytosis of the tissue-debris degenerated-myelin which hinders repair in axonal injury and neurodegenerative diseases. We tested whether SIRPα inhibits phagocytosis by regulating cytoskeleton function through paxillin and cofilin since (a the cytoskeleton generates the mechanical forces that drive phagocytosis and (b both paxillin and cofilin control cytoskeleton function. Paxillin and cofilin were transiently activated in microglia as phagocytosis was activated. In contrast, paxillin and cofilin were continuously activated and phagocytosis augmented in microglia in which SIRPα expression was knocked-down by SIRPα-shRNA. Further, levels of phagocytosis, paxillin activation and cofilin activation positively correlated with one another. Taken together, these observations suggest a novel mechanism whereby paxillin and cofilin are targeted to control phagocytosis by both the activating signaling that phagocytic receptors produce by promoting the activation of paxillin and cofilin and the inhibiting signaling that immune inhibitory SIRPα produces by promoting the inactivation of paxillin and cofilin.

  1. Stimulation of cannabinoid receptor 2 (CB2 suppresses microglial activation

    Fernandez Francisco

    2005-12-01

    Full Text Available Abstract Background Activated microglial cells have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD, multiple sclerosis (MS, and HIV dementia. It is well known that inflammatory mediators such as nitric oxide (NO, cytokines, and chemokines play an important role in microglial cell-associated neuron cell damage. Our previous studies have shown that CD40 signaling is involved in pathological activation of microglial cells. Many data reveal that cannabinoids mediate suppression of inflammation in vitro and in vivo through stimulation of cannabinoid receptor 2 (CB2. Methods In this study, we investigated the effects of a cannabinoid agonist on CD40 expression and function by cultured microglial cells activated by IFN-γ using RT-PCR, Western immunoblotting, flow cytometry, and anti-CB2 small interfering RNA (siRNA analyses. Furthermore, we examined if the stimulation of CB2 could modulate the capacity of microglial cells to phagocytise Aβ1–42 peptide using a phagocytosis assay. Results We found that the selective stimulation of cannabinoid receptor CB2 by JWH-015 suppressed IFN-γ-induced CD40 expression. In addition, this CB2 agonist markedly inhibited IFN-γ-induced phosphorylation of JAK/STAT1. Further, this stimulation was also able to suppress microglial TNF-α and nitric oxide production induced either by IFN-γ or Aβ peptide challenge in the presence of CD40 ligation. Finally, we showed that CB2 activation by JWH-015 markedly attenuated CD40-mediated inhibition of microglial phagocytosis of Aβ1–42 peptide. Taken together, these results provide mechanistic insight into beneficial effects provided by cannabinoid receptor CB2 modulation in neurodegenerative diseases, particularly AD.

  2. Facilitation of neocortical presynaptic terminal development by NMDA receptor activation

    Sceniak Michael P

    2012-02-01

    Full Text Available Abstract Background Neocortical circuits are established through the formation of synapses between cortical neurons, but the molecular mechanisms of synapse formation are only beginning to be understood. The mechanisms that control synaptic vesicle (SV and active zone (AZ protein assembly at developing presynaptic terminals have not yet been defined. Similarly, the role of glutamate receptor activation in control of presynaptic development remains unclear. Results Here, we use confocal imaging to demonstrate that NMDA receptor (NMDAR activation regulates accumulation of multiple SV and AZ proteins at nascent presynaptic terminals of visual cortical neurons. NMDAR-dependent regulation of presynaptic assembly occurs even at synapses that lack postsynaptic NMDARs. We also provide evidence that this control of presynaptic terminal development is independent of glia. Conclusions Based on these data, we propose a novel NMDAR-dependent mechanism for control of presynaptic terminal development in excitatory neocortical neurons. Control of presynaptic development by NMDARs could ultimately contribute to activity-dependent development of cortical receptive fields.

  3. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    Teodorov, E. [Centro de Matemática, Computação e Cognição, Universidade Federal do ABC, São Paulo, SP (Brazil); Ferrari, M.F.R. [Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Fior-Chadi, D.R. [Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, São Paulo, SP (Brazil); Camarini, R. [Departamento de Farmacologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP (Brazil); Felício, L.F. [Departamento de Patologia, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-06-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  4. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    E. Teodorov

    2012-10-01

    Full Text Available The periaqueductal gray (PAG has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc or 0.9% saline (up to 1 mL/kg and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05 because a lower percentage of kappa group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR. A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05 and lactating female rats (P < 0.01, with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in

  5. Peroxisome proliferator-activated receptor delta activation leads to increased transintestinal cholesterol efflux

    Vrins, Carlos L. J.; van der Velde, Astrid E.; van den Oever, Karin; Levels, Johannes H. M.; Huet, Stephane; Elferink, Ronald P. J. Oude; Kuipers, Folkert; Groen, Albert K.

    2009-01-01

    Peroxisome proliferator-activated receptor delta (PPAR delta) is involved in regulation of energy homeostasis. Activation of PPAR delta markedly increases fecal neutral sterol secretion, the last step in reverse cholesterol transport. This phenomenon can neither be explained by increased hepatobilia

  6. Sulfonylureas and glinides exhibit peroxisome proliferator-activated receptor gamma activity: A combined virtual screening and biological assay approach

    Scarsi, M.; Podvinec, M.; Roth, A.; Hug, H.; Kersten, A.H.; Albrecht, H.; Schwede, T.; Meyer, U.A.; Rucker, C.

    2007-01-01

    Most drugs currently employed in the treatment of type 2 diabetes either target the sulfonylurea receptor stimulating insulin release (sulfonylureas, glinides), or target the peroxisome proliferator-activated receptor (PPAR) improving insulin resistance (thiazolidinediones). Our work shows that sulf

  7. Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

    Seiji Hayashizaki; Shinobu Hirai; Yumi Ito; Yoshiko Honda; Yosefu Arime; Ichiro Sora; Haruo Okado; Tohru Kodama; Masahiko Takada

    2013-01-01

    Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behaviora...

  8. FATTY ACIDS MODULATE TOLL-LIKE RECEPTOR 4 ACTIVATION THROUGH REGULATION OF RECEPTOR DIMERIZATION AND RECRUITMENT INTO LIPID RAFTS

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies (J Biol Chem 2003, 2004) demonstrated that saturated ...

  9. Dopamine receptor activation increases HIV entry into primary human macrophages.

    Peter J Gaskill

    Full Text Available Macrophages are the primary cell type infected with HIV in the central nervous system, and infection of these cells is a major component in the development of neuropathogenesis and HIV-associated neurocognitive disorders. Within the brains of drug abusers, macrophages are exposed to increased levels of dopamine, a neurotransmitter that mediates the addictive and reinforcing effects of drugs of abuse such as cocaine and methamphetamine. In this study we examined the effects of dopamine on HIV entry into primary human macrophages. Exposure to dopamine during infection increased the entry of R5 tropic HIV into macrophages, irrespective of the concentration of the viral inoculum. The entry pathway affected was CCR5 dependent, as antagonizing CCR5 with the small molecule inhibitor TAK779 completely blocked entry. The effect was dose-dependent and had a steep threshold, only occurring above 108 M dopamine. The dopamine-mediated increase in entry required dopamine receptor activation, as it was abrogated by the pan-dopamine receptor antagonist flupenthixol, and could be mediated through both subtypes of dopamine receptors. These findings indicate that the effects of dopamine on macrophages may have a significant impact on HIV pathogenesis. They also suggest that drug-induced increases in CNS dopamine may be a common mechanism by which drugs of abuse with distinct modes of action exacerbate neuroinflammation and contribute to HIV-associated neurocognitive disorders in infected drug abusers.

  10. Characterization of human endothelial cell urokinase-type plasminogen activator receptor protein and messenger RNA

    Barnathan, E S; Kuo, A; Karikó, K

    1990-01-01

    Human umbilical vein endothelial cells in culture (HUVEC) express receptors for urokinase-type plasminogen activators (u-PA). The immunochemical nature of this receptor and its relationship to u-PA receptors expressed by other cell types is unknown. Cross-linking active site-blocked u-PA to HUVEC...

  11. Activation of μ-opioid receptor and Toll-like receptor 4 by plasma from morphine-treated mice.

    Xie, Nan; Gomes, Fabio P; Deora, Vandana; Gregory, Kye; Vithanage, Tharindu; Nassar, Zeyad D; Cabot, Peter J; Sturgess, David; Shaw, Paul N; Parat, Marie-Odile

    2017-03-01

    In this study, we quantified the ability of opioids present in biological samples to activate the μ-opioid receptor and TLR4 using cell-based assays. Each assay was standardised, in the presence of plasma, using morphine, its μ receptor-active metabolite morphine-6 glucuronide (M6G) and its μ receptor-inactive, but TLR4-active metabolite morphine-3 glucuronide (M3G). Specificity was verified using antagonists. Morphine- and M6G-spiked plasma samples exhibited μ receptor activation, which M3G-spiked plasma lacked. In contrast, M3G showed moderate but consistent activation of TLR-4. Plasma samples were collected at a number of time points from mice administered morphine (1 or 10mg/kg every 12h for 3days) or saline. Morphine administration led to intermittent μ receptor activation, reversed by μ receptor antagonists, and to TRL4 activation at time points where M3G is measured in plasma. Interestingly, this protocol of morphine administration also led to TLR4-independent NF-κB activation, at time points where M3G was not detected, presumably via elevation of circulating cytokines including, but not limited to, TNFα. Circulating TNFα was increased after three days of morphine administration, and TNFα mRNA elevated in the spleen of morphine-treated mice.

  12. Protease activated receptor-2 contributes to heart failure.

    Silvio Antoniak

    Full Text Available Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs, in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure.

  13. Evidence that adiponectin receptor 1 activation exacerbates ischemic neuronal death

    Thundyil John

    2010-08-01

    Full Text Available Abstract Background- Adiponectin is a hormone produced in and released from adipose cells, which has been shown to have anti-diabetic and anti-inflammatory actions in peripheral cells. Two cell surface adiponectin receptors (ADRs mediate the majority of the known biological actions of adiponectin. Thus far, ADR expression in the brain has been demonstrated in the arcuate and the paraventricular nucleus of hypothalamus, where its activation affects food intake. Recent findings suggest that levels of circulating adiponectin increase after an ischemic stroke, but the role of adiponectin receptor activation in stroke pathogenesis and its functional outcome is unclear. Methods- Ischemic stroke was induced in C57BL/6 mice by middle cerebral artery occlusion (MCAO for 1 h, followed by reperfusion. Primary cortical neuronal cultures were established from individual embryonic neocortex. For glucose deprivation (GD, cultured neurons were incubated in glucose-free Locke's medium for 6, 12 or 24 h. For combined oxygen and glucose deprivation (OGD, neurons were incubated in glucose-free Locke's medium in an oxygen-free chamber with 95% N2/5% CO2 atmosphere for either 3, 6, 9, 12 or 24 h. Primary neurons and brain tissues were analysed for Adiponectin and ADRs using reverse transcriptase polymerase chain reaction (RT-PCR, immunoblot and immunochemistry methods. Results- Cortical neurons express ADR1 and ADR2, and that the levels of ADR1 are increased in neurons in response to in vitro or in vivo ischemic conditions. Neurons treated with either globular or trimeric adiponectin exhibited increased vulnerability to oxygen and glucose deprivation which was associated with increased activation of a pro-apoptotic signaling cascade involving p38 mitogen-activated protein kinase (p38MAPK and AMP-activated protein kinase (AMPK. Conclusions- This study reveals a novel pathogenic role for adiponectin and adiponectin receptor activation in ischemic stroke. We show that

  14. Effect of highly bioaccumulated polychlorinated biphenyl congeners on estrogen and androgen receptor activity

    Bonefeld-Jørgensen, E.C.; Andersen, H. R.; Rasmussen, T.H.;

    2001-01-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental persistent contaminants giving rise to potential health hazard. Some PCBs exert dioxin-like activities mediated through the aryl hydrocarbon receptor. Although reports on interaction with other nuclear receptors are sparce, some...

  15. Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for platelet-activating factor.

    Smiley, P L; Stremler, K E; Prescott, S M; Zimmerman, G A; McIntyre, T M

    1991-06-15

    Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) activates neutrophils (polymorphonuclear leukocytes, PMN) through a receptor that specifically recognizes short sn-2 residues. We oxidized synthetic [2-arachidonoyl]phosphatidylcholine to fragment and shorten the sn-2 residue, and then examined the phospholipid products for the ability to stimulate PMN. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine was fragmented by ozonolysis to 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine. This phospholipid activated human neutrophils at submicromolar concentrations, and is effects were inhibited by specific PAF receptor antagonists WEB2086, L659,989, and CV3988. 1-Palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine next was fragmented by an uncontrolled free radical-catalyzed reaction: it was treated with soybean lipoxygenase to form its sn-2 15-hydroperoxy derivative (which did not activate neutrophils) and then allowed to oxidize under air. The secondary oxidation resulted in the formation of numerous fragmented phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103), some of which activated PMN. Hydrolysis of sn-2 residues with phospholipase A2 destroyed biologic activity, as did hydrolysis with PAF acetylhydrolase. PAF acetylhydrolase is specific for short or intermediate length sn-2 residues and does not hydrolyze the starting material (Stremler, K. E., Stafforini, D. M., Prescott, S. M., and McIntyre, T. M. (1991) J. Biol. Chem. 266, 11095-11103). Neutrophil activation was completely blocked by L659,989, a specific PAF receptor antagonist. We conclude that diacylphosphatidylcholines containing an sn-2 polyunsaturated fatty acyl residue can be oxidatively fragmented to species with sn-2 residues short enough to activate the PAF receptor of neutrophils. This suggests a new mechanism for the appearance of biologically active phospholipids, and shows

  16. The glucagon-like peptide 1 receptor agonist enhances intrinsic peroxisome proliferator-activated receptor γ activity in endothelial cells

    Onuma, Hirohisa; Inukai, Kouichi, E-mail: kinukai@ks.kyorin-u.ac.jp; Kitahara, Atsuko; Moriya, Rie; Nishida, Susumu; Tanaka, Toshiaki; Katsuta, Hidenori; Takahashi, Kazuto; Sumitani, Yoshikazu; Hosaka, Toshio; Ishida, Hitoshi

    2014-08-22

    Highlights: • PPARγ activation was involved in the GLP-1-mediated anti-inflammatory action. • Exendin-4 enhanced endogenous PPARγ transcriptional activity in HUVECs. • H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement. • The anti-inflammatory effects of GLP-1 may be explained by PPARγ activation. - Abstract: Recent studies have suggested glucagon-like peptide-1 (GLP-1) signaling to exert anti-inflammatory effects on endothelial cells, although the precise underlying mechanism remains to be elucidated. In the present study, we investigated whether PPARγ activation is involved in the GLP-1-mediated anti-inflammatory action on endothelial cells. When we treated HUVEC cells with 0.2 ng/ml exendin-4, a GLP-1 receptor agonist, endogenous PPARγ transcriptional activity was significantly elevated, by approximately 20%, as compared with control cells. The maximum PPARγ activity enhancing effect of exendin-4 was observed 12 h after the initiation of incubation with exendin-4. As H89, a PKA inhibitor, abolished GLP-1-induced PPARγ enhancement, the signaling downstream from GLP-1 cross-talk must have been involved in PPARγ activation. In conclusion, our results suggest that GLP-1 has the potential to induce PPARγ activity, partially explaining the anti-inflammatory effects of GLP-1 on endothelial cells. Cross-talk between GLP-1 signaling and PPARγ activation would have major impacts on treatments for patients at high risk for cardiovascular disease.

  17. The novel plasminogen receptor, plasminogen receptor(KT) (Plg-R(KT)), regulates catecholamine release.

    Bai, Hongdong; Baik, Nagyung; Kiosses, William B; Krajewski, Stan; Miles, Lindsey A; Parmer, Robert J

    2011-09-23

    Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

  18. Cloning and expression of the receptor for human urokinase plasminogen activator, a central molecule in cell surface, plasmin dependent proteolysis

    Roldan, A L; Cubellis, M V; Masucci, M T

    1990-01-01

    , and therefore the capacity of cells to migrate and invade neighboring tissues. We have isolated a 1.4 kb cDNA clone coding for the entire human uPAR. An oligonucleotide synthesized on the basis of the N-terminal sequence of the purified protein was used to screen a cDNA library made from SV40 transformed human......, a size very close to that of the cloned cDNA. Expression of the uPAR cDNA in mouse cells confirms that the clone is complete and expresses a functional uPA binding protein, located on the cell surface and with properties similar to the human uPAR. Caseinolytic plaque assay, immunofluorescence analysis...

  19. In vitro neuronal network activity in NMDA receptor encephalitis

    Jantzen Sabine U

    2013-02-01

    Full Text Available Abstract Background Anti-NMDA-encephalitis is caused by antibodies against the N-methyl-D-aspartate receptor (NMDAR and characterized by a severe encephalopathy with psychosis, epileptic seizures and autonomic disturbances. It predominantly occurs in young women and is associated in 59% with an ovarian teratoma. Results We describe effects of cerebrospinal fluid (CSF from an anti-N-methyl-D-aspartate receptor (NMDAR encephalitis patient on in vitro neuronal network activity (ivNNA. In vitro NNA of dissociated primary rat cortical populations was recorded by the microelectrode array (MEA system. The 23-year old patient was severely affected but showed an excellent recovery following multimodal immunomodulatory therapy and removal of an ovarian teratoma. Patient CSF (pCSF taken during the initial weeks after disease onset suppressed global spike- and burst rates of ivNNA in contrast to pCSF sampled after clinical recovery and decrease of NMDAR antibody titers. The synchrony of pCSF-affected ivNNA remained unaltered during the course of the disease. Conclusion Patient CSF directly suppresses global activity of neuronal networks recorded by the MEA system. In contrast, pCSF did not regulate the synchrony of ivNNA suggesting that NMDAR antibodies selectively regulate distinct parameters of ivNNA while sparing their functional connectivity. Thus, assessing ivNNA could represent a new technique to evaluate functional consequences of autoimmune encephalitis-related CSF changes.

  20. Antitussive activity of Withania somnifera and opioid receptors.

    Nosálová, Gabriela; Sivová, Veronika; Ray, Bimalendu; Fraňová, Soňa; Ondrejka, Igor; Flešková, Dana

    2015-01-01

    Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

  1. Vasopeptidase-activated latent ligands of the histamine receptor-1.

    Gera, Lajos; Roy, Caroline; Charest-Morin, Xavier; Marceau, François

    2013-11-01

    Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases.

  2. Relationship between somatostatin receptors and activation of hepatic stellate cells

    潘勤; 李定国; 陆汉明; 陆良勇; 尤汉宁; 徐芹芳

    2004-01-01

    Background Somafostatin receptors (SSTRs) have been suggested to involve in mediating the effect of somatostatin on hepatic stellate cells (HSCs) in an activation-dependent way. We, therefore, try to investigate the relationship between expression of SSTRs and activation of rat HSCs.Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation.SSTR1-5 mRNA levels in the differentiated first passage HSCs were detected by means of a reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1-5 in normal as well as fibrotic livers was measured by immunohistochemical staining.Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs or normal rat liver. However, SSTR1-3 mRNA appeared as HSCs became wholly activated, and could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc.Conclusion The expression of SSTR1-3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  3. Activity-dependent neurotransmitter-receptor matching at the neuromuscular junction.

    Borodinsky, Laura N; Spitzer, Nicholas C

    2007-01-02

    Signaling in the nervous system requires matching of neurotransmitter receptors with cognate neurotransmitters at synapses. The vertebrate neuromuscular junction is the best studied cholinergic synapse, but the mechanisms by which acetylcholine is matched with acetylcholine receptors are not fully understood. Because alterations in neuronal calcium spike activity alter transmitter specification in embryonic spinal neurons, we hypothesized that receptor expression in postsynaptic cells follows changes in transmitter expression to achieve this specific match. We find that embryonic vertebrate striated muscle cells normally express receptors for glutamate, GABA, and glycine as well as for acetylcholine. As maturation progresses, acetylcholine receptor expression prevails. Receptor selection is altered when early neuronal calcium-dependent activity is perturbed, and remaining receptor populations parallel changes in transmitter phenotype. In these cases, glutamatergic, GABAergic, and glycinergic synaptic currents are recorded from muscle cells, demonstrating that activity regulates matching of transmitters and their receptors in the assembly of functional synapses.

  4. Involvement of Activating NK Cell Receptors and Their Modulation in Pathogen Immunity

    Francesco Marras

    2011-01-01

    Full Text Available Natural Killer (NK cells are endowed with cell-structure-sensing receptors providing inhibitory protection from self-destruction (inhibitory NK receptors, iNKRs, including killer inhibitory receptors and other molecules and rapid triggering potential leading to functional cell activation by Toll-like receptors (TLRs, cytokine receptors, and activating NK cell receptors including natural cytotoxicity receptors (NCRs, i.e., NKp46, NKp46, and NKp44. NCR and NKG2D recognize ligands on infected cells which may be endogenous or may directly bind to some structures derived from invading pathogens. In this paper, we address the known direct or indirect interactions between activating receptors and pathogens and their expression during chronic HIV and HCV infections.

  5. ERK5 activation by Gq-coupled muscarinic receptors is independent of receptor internalization and β-arrestin recruitment.

    Guzmán Sánchez-Fernández

    Full Text Available G-protein-coupled receptors (GPCRs are known to activate both G protein- and β-arrestin-dependent signalling cascades. The initiation of mitogen-activated protein kinase (MAPK pathways is a key downstream event in the control of cellular functions including proliferation, differentiation, migration and apoptosis. Both G proteins and β-arrestins have been reported to mediate context-specific activation of ERK1/2, p38 and JNK MAPKs. Recently, the activation of ERK5 MAPK by Gq-coupled receptors has been described to involve a direct interaction between Gαq and two novel effectors, PKCζ and MEK5. However, the possible contribution of β-arrestin towards this pathway has not yet been addressed. In the present work we sought to investigate the role of receptor internalization processes and β-arrestin recruitment in the activation of ERK5 by Gq-coupled GPCRs. Our results show that ERK5 activation is independent of M1 or M3 muscarinic receptor internalization. Furthermore, we demonstrate that phosphorylation-deficient muscarinic M1 and M3 receptors are still able to fully activate the ERK5 pathway, despite their reported inability to recruit β-arrestins. Indeed, the overexpression of Gαq, but not that of β-arrestin1 or β-arrestin2, was found to potently enhance ERK5 activation by GPCRs, whereas silencing of β-arrestin2 expression did not affect the activation of this pathway. Finally, we show that a β-arrestin-biased mutant form of angiotensin II (SII; Sar1-Ile4-Ile8 AngII failed to promote ERK5 phosphorylation in primary cardiac fibroblasts, as compared to the natural ligand. Overall, this study shows that the activation of ERK5 MAPK by model Gq-coupled GPCRs does not depend on receptor internalization, β-arrestin recruitment or receptor phosphorylation but rather is dependent on Gαq-signalling.

  6. Selective 5-HT7 Receptor Activation May Enhance Synaptic Plasticity Through N-methyl-D-aspartate (NMDA) Receptor Activity in the Visual Cortex.

    Xiang, Kangjian; Zhao, Xuefei; Li, Youjun; Zheng, Liang; Wang, Jue; Li, Yan-Hai

    2016-01-01

    Serotonin (5-hydroxytryptamine, 5-HT) is an important neurotransmitter that modulates N-methyl-D-aspartate (NMDA) receptor activity by binding to several different 5-HT receptor subtypes. In the present study, we used whole-cell patch-clamp recordings in transverse slice preparations to test the role of 5-HT receptors in modulating the NMDA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) in layer II/III pyramidal neurons of the rat visual cortex. We found that the NMDA receptor-mediated component of mEPSCs could be potentiated by exogenously applied 5-HT. Similar results were obtained by exogenously applied 5-CT or 8-OH-DPAT (the 5-HT1A and 5-HT7 receptor agonist). A specific antagonist for the 5-HT7 receptor, SB-269970, completely blocked the increase in NMDA receptor-mediated component of mEPSCs by 5-CT or 8- OH-DPAT. Moreover, the selective 5-HT1A receptor antagonist, WAY-100135, displayed no influence on the enhancement in NMDA receptor-mediated component of mEPSCs by 5-CT or 8-OHDPAT. These results indicated that the increase in NMDA receptor-mediated component of mEPSCs by 5-HT in layer II/III pyramidal neurons of the young rat visual cortex requires activation of 5-HT7 receptors, but not 5-HT1A receptors. These observations might be clinically relevant to schizophrenia and Alzheimer's disease (AD), where enhancing NMDA receptor-mediated neurotransmission is considered to be a promising strategy for treatment of these diseases.

  7. Activation of cardiac ryanodine receptors by cardiac glycosides.

    Sagawa, Toshio; Sagawa, Kazuko; Kelly, James E; Tsushima, Robert G; Wasserstrom, J Andrew

    2002-03-01

    This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of approximately 0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 microM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 microM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

  8. RELATIONSHIP BETWEEN SOMATOSTATIN RECEPTORS AND ACTIVATION OF HEPATIC STELLATE CELL

    潘勤; 李定国; 陆汉明; 尤汉宁; 徐芹芳; 陆良勇

    2004-01-01

    Objective To investigate the relationship between expression of somatostatin receptors (SSTRs) and activation of rat hepatic stellate cell (HSC). Methods HSCs were isolated from rats by in situ perfusion and single-step density gradient centrifugation, and then SSTR1 ~5 mRNA levels in the differentiated first passage HSCs were detected by means of reverse transcription polymerase chain reaction. On the other hand, hepatic fibrosis was induced in adult male Sprague-Dawley rats by carbon tetrachloride intoxication, and the expression of SSTR1 ~5 in normal as well as fibrotic liver was measured by immunohistochemical staining. Results SSTR mRNA and SSTR could not be found in freshly isolated rat HSCs and normal rat liver. But SSTR1~3 mRNA appeared as HSCs became wholly activated, and SSTR1 ~3 could also be identified on the membrane of activated HSCs in the perisinusoid space, fibrous septa, etc Conclusion The expression of SSTR1~3 in the rat HSC is closely related to its activation. This may reflect one of the main negative regulation mechanisms in the course of HSC activation.

  9. Guidance Receptors in the Nervous and Cardiovascular Systems.

    Rubina, K A; Tkachuk, V A

    2015-10-01

    Blood vessels and nervous fibers grow in parallel, for they express similar receptors for chemokine substances. Recently, much attention is being given to studying guidance receptors and their ligands besides the growth factors, cytokines, and chemokines necessary to form structures in the nervous and vascular systems. Such guidance molecules determine trajectory for growing axons and vessels. Guidance molecules include Ephrins and their receptors, Neuropilins and Plexins as receptors for Semaphorins, Robos as receptors for Slit-proteins, and UNC5B receptors binding Netrins. Apart from these receptors and their ligands, urokinase and its receptor (uPAR) and T-cadherin are also classified as guidance molecules. The urokinase system mediates local proteolysis at the leading edge of cells, thereby providing directed migration. T-cadherin is a repellent molecule that regulates the direction of growing axons and blood vessels. Guidance receptors also play an important role in the diseases of the nervous and cardiovascular systems.

  10. Increased peroxisome proliferator-activated receptor-gamma activity reduces imatinib uptake and efficacy in chronic myeloid leukemia mononuclear cells.

    Wang, Jueqiong; Lu, Liu; Kok, Chung H; Saunders, Verity A; Goyne, Jarrad M; Dang, Phuong; Leclercq, Tamara M; Hughes, Timothy P; White, Deborah L

    2017-02-02

    Imatinib is actively transported by OCT-1 influx transporter, and low OCT-1 activity in diagnostic chronic myeloid leukemia blood mononuclear cells is significantly associated with poor molecular response to imatinib. Here we report that, in diagnostic chronic myeloid leukemia mononuclear cells and BCR-ABL1+ cell lines, peroxisome proliferator-activated receptor gamma agonists (GW1929, rosiglitazone, pioglitazone) significantly decrease OCT-1 activity; conversely, peroxisome proliferator-activated receptor gamma antagonists (GW9662, T0070907) increase OCT-1 activity. Importantly, these effects can lead to corresponding changes in sensitivity to Bcr-Abl kinase inhibition. Results were confirmed in peroxisome proliferator-activated receptor gamma-transduced K562 cells. Furthermore, we identified a strong negative correlation between OCT-1 activity and peroxisome proliferator-activated receptor gamma transcriptional activity in diagnostic chronic myeloid leukemia patients (n=84; preceptor gamma activation has a negative impact on the intracellular uptake of imatinib and consequent Bcr-Abl kinase inhibition. The inter-patient variability of peroxisome proliferator-activated receptor gamma activation likely accounts for the heterogeneity observed in patient OCT-1 activity at diagnosis. Recently, the peroxisome proliferator-activated receptor gamma agonist pioglitazone was reported to act synergistically with imatinib targeting the residual chronic myeloid leukemia stem cell pool. Our findings suggest that peroxisome proliferator-activated receptor gamma ligands have differential effects on circulating mononuclear cells compared to stem cells. Since the effect of peroxisome proliferator-activated receptor gamma activation on imatinib uptake in mononuclear cells may counteract the clinical benefit of this activation in stem cells, caution should be applied when combining these therapies, especially in patients with high peroxisome proliferator-activated receptor gamma

  11. New insights into the structural bases of activation of Cys-loop receptors.

    Bouzat, Cecilia

    2012-01-01

    Neurotransmitter receptors of the Cys-loop superfamily mediate rapid synaptic transmission throughout the nervous system, and include receptors activated by ACh, GABA, glycine and serotonin. They are involved in physiological processes, including learning and memory, and in neurological disorders, and they are targets for clinically relevant drugs. Cys-loop receptors assemble either from five copies of one type of subunit, giving rise to homomeric receptors, or from several types of subunits, giving rise to heteromeric receptors. Homomeric receptors are invaluable models for probing fundamental relationships between structure and function. Receptors contain a large extracellular domain that carries the binding sites and a transmembrane region that forms the ion pore. How the structural changes elicited by agonist binding are propagated through a distance of 50Å to the ion channel gate is central to understanding receptor function. Depending on the receptor subtype, occupancy of either two, as in the prototype muscle nicotinic receptor, or three binding sites, as in homomeric receptors, is required for full activation. The conformational changes initiated at the binding sites are propagated to the gate through the interface between the extracellular and transmembrane domains. This region forms a network that relays structural changes from the binding site towards the pore, and also contributes to open channel lifetime and rate of desensitization. Thus, this coupling region controls the beginning and duration of a synaptic response. Here we review recent advances in the molecular mechanism by which Cys-loop receptors are activated with particular emphasis on homomeric receptors.

  12. The role of GH receptor tyrosine phosphorylation in Stat5 activation

    Hansen, J A; Hansen, L H; Wang, X;

    1997-01-01

    Stimulation of GH receptors leads to rapid activation of Jak2 kinase and subsequent tyrosine phosphorylation of the GH receptor. Three specific tyrosines located in the C-terminal domain of the GH receptor have been identified as being involved in GH-stimulated transcription of the Spi 2.1 promoter....... Mutated GH receptors lacking all but one of these three tyrosines are able to mediate a transcriptional response when transiently transfected into CHO cells together with a Spi 2.1 promoter/luciferase construct. Similarly, these GH receptors were found to be able to mediate activation of Stat5 DNA......-binding activity, whereas the GH receptor mutant lacking all intracellular tyrosines was not. Synthetic tyrosine phosphorylated peptides corresponding to the GH receptor sequence around the three tyrosines inhibited Stat5 DNA-binding activity while their non-phosphorylated counterparts were ineffective. Tyrosine...

  13. The adipogenic acetyltransferase Tip60 targets activation function 1 of peroxisome proliferator-activated receptor gamma

    van Beekum, Olivier; Brenkman, Arjan B; Grøntved, Lars;

    2008-01-01

    The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) plays a key role in the regulation of lipid and glucose metabolism in adipocytes, by regulating their differentiation, maintenance, and function. The transcriptional activity of PPARgamma is dictated by the set...... of proteins with which this nuclear receptor interacts under specific conditions. Here we identify the HIV-1 Tat-interacting protein 60 (Tip60) as a novel positive regulator of PPARgamma transcriptional activity. Using tandem mass spectrometry, we found that PPARgamma and the acetyltransferase Tip60 interact...... in cells, and through use of chimeric proteins, we established that coactivation by Tip60 critically depends on the N-terminal activation function 1 of PPARgamma, a domain involved in isotype-specific gene expression and adipogenesis. Chromatin immunoprecipitation experiments showed that the endogenous Tip...

  14. SHP-1 phosphatase activity counteracts increased T cell receptor affinity.

    Hebeisen, Michael; Baitsch, Lukas; Presotto, Danilo; Baumgaertner, Petra; Romero, Pedro; Michielin, Olivier; Speiser, Daniel E; Rufer, Nathalie

    2013-03-01

    Anti-self/tumor T cell function can be improved by increasing TCR-peptide MHC (pMHC) affinity within physiological limits, but paradoxically further increases (K(d) affinity for the tumor antigen HLA-A2/NY-ESO-1, we investigated the molecular mechanisms underlying this high-affinity-associated loss of function. As compared with cells expressing TCR affinities generating optimal function (K(d) = 5 to 1 μM), those with supraphysiological affinity (K(d) = 1 μM to 15 nM) showed impaired gene expression, signaling, and surface expression of activatory/costimulatory receptors. Preferential expression of the inhibitory receptor programmed cell death-1 (PD-1) was limited to T cells with the highest TCR affinity, correlating with full functional recovery upon PD-1 ligand 1 (PD-L1) blockade. In contrast, upregulation of the Src homology 2 domain-containing phosphatase 1 (SHP-1/PTPN6) was broad, with gradually enhanced expression in CD8(+) T cells with increasing TCR affinities. Consequently, pharmacological inhibition of SHP-1 with sodium stibogluconate augmented the function of all engineered T cells, and this correlated with the TCR affinity-dependent levels of SHP-1. These data highlight an unexpected and global role of SHP-1 in regulating CD8(+) T cell activation and responsiveness and support the development of therapies inhibiting protein tyrosine phosphatases to enhance T cell-mediated immunity.

  15. Molecular pharmacological phenotyping of EBI2. An orphan seven-transmembrane receptor with constitutive activity

    Rosenkilde, Mette M; Benned-Jensen, Tau; Holst, Peter J;

    2006-01-01

    Epstein-Barr virus (EBV)-induced receptor 2 (EBI2) is an orphan seven-transmembrane (7TM) receptor originally identified as the most up-regulated gene (>200-fold) in EBV-infected cells. Here we show that EBI2 signals with constitutive activity through Galpha(i) as determined by a receptor-mediate...

  16. Developmental stability of taurine's activation on glycine receptors in cultured neurons of rat auditory cortex.

    Tang, Zheng-Quan; Lu, Yun-Gang; Chen, Lin

    2008-01-03

    Taurine is an endogenous amino acid that can activate glycine and/or gamma-aminobutyric acid type A (GABA(A)) receptors in the central nervous system. During natural development, taurine's receptor target undergoes a shift from glycine receptors to GABA(A) receptors in cortical neurons. Here, we demonstrate that taurine's receptor target in cortical neurons remains stable during in vitro development. With whole-cell patch-clamp recordings, we found that taurine always activated glycine receptors, rather than GABA(A) receptors, in neurons of rat auditory cortex cultured for 5-22 days. Our results suggest that the functional sensitivity of glycine and GABA(A) receptors to taurine is critically regulated by their developmental environments.

  17. Thrombin-Mediated Direct Activation of Proteinase-Activated Receptor-2: Another Target for Thrombin Signaling.

    Mihara, Koichiro; Ramachandran, Rithwik; Saifeddine, Mahmoud; Hansen, Kristina K; Renaux, Bernard; Polley, Danny; Gibson, Stacy; Vanderboor, Christina; Hollenberg, Morley D

    2016-05-01

    Thrombin is known to signal to cells by cleaving/activating a G-protein-coupled family of proteinase-activated receptors (PARs). The signaling mechanism involves the proteolytic unmasking of an N-terminal receptor sequence that acts as a tethered receptor-activating ligand. To date, the recognized targets of thrombin cleavage and activation for signaling are PAR1 and PAR4, in which thrombin cleaves at a conserved target arginine to reveal a tethered ligand. PAR2, which like PAR1 is also cleaved at an N-terminal arginine to unmask its tethered ligand, is generally regarded as a target for trypsin but not for thrombin signaling. We now show that thrombin, at concentrations that can be achieved at sites of acute injury or in a tumor microenvironment, can directly activate PAR2 vasorelaxation and signaling, stimulating calcium and mitogen-activated protein kinase responses along with triggeringβ-arrestin recruitment. Thus, PAR2 can be added alongside PAR1 and PAR4 to the targets, whereby thrombin can affect tissue function.

  18. High-affinity benzodiazepine receptor ligands among benzodiazepines and betacarbolines with different intrinsic activity

    Yliniemelae, A.; Gynther, J. (Univ. of Kuopio (Finland)); Konschin, H.; Tylli, H. (Univ. of Helsinki (Finland)); Rouvinen, J. (Univ. of Joensuu (Finland))

    1989-01-01

    Structural and electrostatic features of diazepam, flumazenil, and methyl betacarboline-3-carboxylate (BCCM) have been investigated using the molecular superimposition method. These high-affinity benzodiazepine (BZ) receptor ligands are structurally unrelated and they have different intrinsic activity. These ligands are superimposed in such a way that common structural and electrostatic features essential for the high receptor binding affinity overlap. In addition to this binding pharmacophore, there are roughly three separate binding zones in the BZ receptor, one for each class of ligands. The intrinsic activity of BZ receptor ligands depends on the molecular structures and the way the ligand approaches the receptor.

  19. Family C 7TM receptor dimerization and activation

    Bonde, Marie Mi; Sheikh, Søren P; Hansen, Jakob Lerche

    2006-01-01

    The family C seven transmembrane (7TM) receptors constitutes a small and especially well characterized subfamily of the large 7TM receptor superfamily. Approximately 50% of current prescription drugs target 7TM receptors, this biologically important family represents the largest class of drug-tar...

  20. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1....... Canofins mimic the cognate ligand interaction with the receptor and preserve the neuritogenic and neuroprotective properties of FGF2. Canofins were shown by surface plasmon resonance analysis to bind to FGFR1 and promote receptor activation. However, FGF2-induced receptor phosphorylation was inhibited...

  1. Mu-opioid receptor knockout mice show diminished food-anticipatory activity

    Kas, Martien J H; van den Bos, Ruud; Baars, Annemarie M; Lubbers, Marianne; Lesscher, Heidi M B; Hillebrand, Jacquelien J G; Schuller, Alwin G; Pintar, John E; Spruijt, Berry M

    2004-01-01

    We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, mu-opioid receptors are activated. To test this hypothesis schedule induced food-anticipatory activity in mu-opioid receptor knockout mice was measured using running wheels. We hypothesized

  2. Recovery of network-driven glutamatergic activity in rat hippocampal neurons during chronic glutamate receptor blockade.

    Leininger, Eric; Belousov, Andrei B

    2009-01-28

    Previous studies indicated that a long-term decrease in the activity of ionotropic glutamate receptors induces cholinergic activity in rat and mouse hypothalamic neuronal cultures. Here we studied whether a prolonged inactivation of ionotropic glutamate receptors also induces cholinergic activity in hippocampal neurons. Receptor activity was chronically suppressed in rat hippocampal primary neuronal cultures with two proportionally increasing sets of concentrations of NMDA plus non-NMDA receptor antagonists: 100 microM/10 microM AP5/CNQX (1X cultures) and 200 microM/20 microM AP5/CNQX (2X cultures). Using calcium imaging we demonstrate that cholinergic activity does not develop in these cultures. Instead, network-driven glutamate-dependent activity, that normally is detected in hyper-excitable conditions, reappears in each culture group in the presence of these antagonists and can be reversibly suppressed by higher concentrations of AP5/CNQX. This activity is mediated by non-NMDA receptors and is modulated by NMDA receptors. Further, non-NMDA receptors, the general level of glutamate receptor activity and CaMK-dependent signaling are critical for development of this network-driven glutamatergic activity in the presence of receptor antagonists. Using electrophysiology, western blotting and calcium imaging we show that some neuronal parameters are either reduced or not affected by chronic glutamate receptor blockade. However, other parameters (including neuronal excitability, mEPSC frequency, and expression of GluR1, NR1 and betaCaMKII) become up-regulated and, in some cases, proportionally between the non-treated, 1X and 2X cultures. Our data suggest recovery of the network-driven glutamatergic activity after chronic glutamate receptor blockade. This recovery may represent a form of neuronal plasticity that compensates for the prolonged suppression of the activity of glutamate receptors.

  3. Activation of EphA receptors mediates the recruitment of the adaptor protein Slap, contributing to the downregulation of N-methyl-D-aspartate receptors.

    Semerdjieva, Sophia; Abdul-Razak, Hayder H; Salim, Sharifah S; Yáñez-Muñoz, Rafael J; Chen, Philip E; Tarabykin, Victor; Alifragis, Pavlos

    2013-04-01

    Regulation of the activity of N-methyl-d-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

  4. Calcium is the switch in the moonlighting dual function of the ligand-activated receptor kinase phytosulfokine receptor 1

    Muleya, Victor

    2014-09-23

    Background: A number of receptor kinases contain guanylate cyclase (GC) catalytic centres encapsulated in the cytosolic kinase domain. A prototypical example is the phytosulfokine receptor 1 (PSKR1) that is involved in regulating growth responses in plants. PSKR1 contains both kinase and GC activities however the underlying mechanisms regulating the dual functions have remained elusive. Findings: Here, we confirm the dual activity of the cytoplasmic domain of the PSKR1 receptor. We show that mutations within the guanylate cyclase centre modulate the GC activity while not affecting the kinase catalytic activity. Using physiologically relevant Ca2+ levels, we demonstrate that its GC activity is enhanced over two-fold by Ca2+ in a concentration-dependent manner. Conversely, increasing Ca2+ levels inhibits kinase activity up to 500-fold at 100 nM Ca2+. Conclusions: Changes in calcium at physiological levels can regulate the kinase and GC activities of PSKR1. We therefore propose a functional model of how calcium acts as a bimodal switch between kinase and GC activity in PSKR1 that could be relevant to other members of this novel class of ligand-activated receptor kinases.

  5. Repressive effects of resveratrol on androgen receptor transcriptional activity.

    Wen-feng Shi

    Full Text Available BACKGROUND: The chemopreventive effects of resveratrol (RSV on prostate cancer have been well established; the androgen receptor (AR plays pivotal roles in prostatic tumorigenesis. However, the exact underlying molecular mechanisms about the effects of RSV on AR have not been fully elucidated. A model system is needed to determine whether and how RSV represses AR transcriptional activity. METHODOLOGY: The AR cDNA was first cloned into the retroviral vector pOZ-N and then integrated into the genome of AR-negative HeLa cells to generate the AR(+ cells. The constitutively expressed AR was characterized by monitoring hormone-stimulated nuclear translocation, DNA binding, and transcriptional activation, with the AR(- cells serving as controls. AR(+ cells were treated with RSV, and both AR protein levels and AR transcriptional activity were measured simultaneously. Chromatin immunoprecipitation (ChIP assays were used to detect the effects of RSV on the recruitment of AR to its cognate element (ARE. RESULTS: AR in the AR (+ stable cell line functions in a manner similar to that of endogenously expressed AR. Using this model system we clearly demonstrated that RSV represses AR transcriptional activity independently of any effects on AR protein levels. However, neither the hormone-mediated nucleus translocation nor the AR/ARE interaction was affected by RSV treatment. CONCLUSION: We demonstrated unambiguously that RSV regulates AR target gene expression, at least in part, by repressing AR transcriptional activity. Repressive effects of RSV on AR activity result from mechanisms other than the affects of AR nuclear translocation or DNA binding.

  6. Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta

    Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li

    2007-01-01

    Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow...... or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p...

  7. Immunomodulator CD200 promotes neurotrophic activity by interacting with and activating the fibroblast growth factor receptor

    Pankratova, Stanislava; Bjornsdottir, Halla; Christensen, Claus;

    2016-01-01

    in the suppression of microglia activation. We for the first time demonstrated that CD200 can interact with and transduce signaling through activation of the fibroblast growth factor receptor (FGFR), thereby inducing neuritogenesis and promoting neuronal survival in primary neurons. CD200-induced FGFR...... phosphorylation was abrogated by CD200R, whereas FGF2-induced FGFR activation was inhibited by CD200. We also identified a sequence motif located in the first Ig-like module of CD200, likely representing the minimal CD200 binding site for FGFR. The FGFR binding motif overlaps with the CD200R binding site......, suggesting that they can compete for CD200 binding in cells that express both receptors. We propose that CD200 in neurons functions as a ligand of FGFR....

  8. Significance of AT1 receptor independent activation of mineralocorticoid receptor in murine diabetic cardiomyopathy.

    Yuji Nagatomo

    Full Text Available BACKGROUND: Diabetes mellitus (DM has deleterious influence on cardiac performance independent of coronary artery disease and hypertension. The objective of the present study was to investigate the role of the renin-angiotensin-aldosterone system, especially angiotensin II type 1a receptor (AT1aR and mineralocorticoid receptor (MR signaling, in left ventricular (LV dysfunction induced by diabetes mellitus (DM. METHODS AND RESULTS: DM was induced by intraperitoneal injection of streptozotocin (200 mg/kg BW in wild-type (WT or AT1aR knockout (KO male mice, and they were bred during 6 or 12 weeks. Some KO mice were administered the MR antagonist eplerenone (100 mg/kg body weight. At 6 weeks, LV diastolic function was impaired in WT-DM, but preserved in KO-DM. At that time point MR mRNA expression was upregulated, NADPH oxidase subunit (p47phox and glutathione peroxidase (GPx1 mRNA expression were upregulated, the staining intensities of LV tissue for 4-hydroxy-2-nonenal was stronger in immunohistochemistry, the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL positive cells was increased, Bcl-2 protein expression was significantly downregulated, and the expression of SERCA2a and phosphorylated phospholamban was depressed in WT-DM, while these changes were not seen in KO-DM. At 12 weeks, however, these changes were also noted in KO-DM. Eplerenone arrested those changes. The plasma aldosterone concentration was elevated in WT-DM but not in KO-DM at 6 weeks. It showed 3.7-fold elevation at 12 weeks even in KO-DM, which suggests "aldosterone breakthrough" phenomenon. However, the aldosterone content in LV tissue was unchanged in KO-DM. CONCLUSIONS: DM induced diastolic dysfunction was observed even in KO at 12 weeks, which was ameliorated by minelarocorticoid receptor antagonist, eplerenone. AT1-independent MR activation in the LV might be responsible for the pathogenesis of diabetic cardiomyopathy.

  9. Activation of transient receptor potential ankyrin 1 by eugenol.

    Chung, G; Im, S T; Kim, Y H; Jung, S J; Rhyu, M-R; Oh, S B

    2014-03-07

    Eugenol is a bioactive plant extract used as an analgesic agent in dentistry. The structural similarity of eugenol to cinnamaldehyde, an active ligand for transient receptor potential ankyrin 1 (TRPA1), suggests that eugenol might produce its effect via TRPA1, in addition to TRPV1 as we reported previously. In this study, we investigated the effect of eugenol on TRPA1, by fura-2-based calcium imaging and patch clamp recording in trigeminal ganglion neurons and in a heterologous expression system. As the result, eugenol induced robust calcium responses in rat trigeminal ganglion neurons that responded to a specific TRPA1 agonist, allyl isothiocyanate (AITC), and not to capsaicin. Capsazepine, a TRPV1 antagonist failed to inhibit eugenol-induced calcium responses in AITC-responding neurons. In addition, eugenol response was observed in trigeminal ganglion neurons from TRPV1 knockout mice and human embryonic kidney 293 cell lines that express human TRPA1, which was inhibited by TRPA1-specific antagonist HC-030031. Eugenol-evoked TRPA1 single channel activity and eugenol-induced TRPA1 currents were dose-dependent with EC50 of 261.5μM. In summary, these results demonstrate that the activation of TRPA1 might account for another molecular mechanism underlying the pharmacological action of eugenol.

  10. HIV-1 activates macrophages independent of Toll-like receptors.

    Joseph N Brown

    Full Text Available BACKGROUND: Macrophages provide an interface between innate and adaptive immunity and are important long-lived reservoirs for Human Immunodeficiency Virus Type-1 (HIV-1. Multiple genetic networks involved in regulating signal transduction cascades and immune responses in macrophages are coordinately modulated by HIV-1 infection. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate complex interrelated processes and to assemble an integrated view of activated signaling networks, a systems biology strategy was applied to genomic and proteomic responses by primary human macrophages over the course of HIV-1 infection. Macrophage responses, including cell cycle, calcium, apoptosis, mitogen-activated protein kinases (MAPK, and cytokines/chemokines, to HIV-1 were temporally regulated, in the absence of cell proliferation. In contrast, Toll-like receptor (TLR pathways remained unaltered by HIV-1, although TLRs 3, 4, 7, and 8 were expressed and responded to ligand stimulation in macrophages. HIV-1 failed to activate phosphorylation of IRAK-1 or IRF-3, modulate intracellular protein levels of Mx1, an interferon-stimulated gene, or stimulate secretion of TNF, IL-1beta, or IL-6. Activation of pathways other than TLR was inadequate to stimulate, via cross-talk mechanisms through molecular hubs, the production of proinflammatory cytokines typical of a TLR response. HIV-1 sensitized macrophage responses to TLR ligands, and the magnitude of viral priming was related to virus replication. CONCLUSIONS/SIGNIFICANCE: HIV-1 induced a primed, proinflammatory state, M1(HIV, which increased the responsiveness of macrophages to TLR ligands. HIV-1 might passively evade pattern recognition, actively inhibit or suppress recognition and signaling, or require dynamic interactions between macrophages and other cells, such as lymphocytes or endothelial cells. HIV-1 evasion of TLR recognition and simultaneous priming of macrophages may represent a strategy for viral survival, contribute

  11. Oleamide activates peroxisome proliferator-activated receptor gamma (PPARγ in vitro

    Dionisi Mauro

    2012-05-01

    Full Text Available Abstract Background Oleamide (ODA is a fatty acid primary amide first identified in the cerebrospinal fluid of sleep-deprived cats, which exerts effects on vascular and neuronal tissues, with a variety of molecular targets including cannabinoid receptors and gap junctions. It has recently been reported to exert a hypolipidemic effect in hamsters. Here, we have investigated the nuclear receptor family of peroxisome proliferator-activated receptors (PPARs as potential targets for ODA action. Results Activation of PPARα, PPARβ and PPARγ was assessed using recombinant expression in Chinese hamster ovary cells with a luciferase reporter gene assay. Direct binding of ODA to the ligand binding domain of each of the three PPARs was monitored in a cell-free fluorescent ligand competition assay. A well-established assay of PPARγ activity, the differentiation of 3T3-L1 murine fibroblasts into adipocytes, was assessed using an Oil Red O uptake-based assay. ODA, at 10 and 50 μM, was able to transactivate PPARα, PPARβ and PPARγ receptors. ODA bound to the ligand binding domain of all three PPARs, although complete displacement of fluorescent ligand was only evident for PPARγ, at which an IC50 value of 38 μM was estimated. In 3T3-L1 cells, ODA, at 10 and 20 μM, induced adipogenesis. Conclusions We have, therefore, identified a novel site of action of ODA through PPAR nuclear receptors and shown how ODA should be considered as a weak PPARγ ligand in vitro.

  12. Differential effects of exercise on brain opioid receptor binding and activation in rats.

    Arida, Ricardo Mario; Gomes da Silva, Sérgio; de Almeida, Alexandre Aparecido; Cavalheiro, Esper Abrão; Zavala-Tecuapetla, Cecilia; Brand, Serge; Rocha, Luisa

    2015-01-01

    Physical exercise stimulates the release of endogenous opioid peptides supposed to be responsible for changes in mood, anxiety, and performance. Exercise alters sensitivity to these effects that modify the efficacy at the opioid receptor. Although there is evidence that relates exercise to neuropeptide expression in the brain, the effects of exercise on opioid receptor binding and signal transduction mechanisms downstream of these receptors have not been explored. Here, we characterized the binding and G protein activation of mu opioid receptor, kappa opioid receptor or delta opioid receptor in several brain regions following acute (7 days) and chronic (30 days) exercise. As regards short- (acute) or long-term effects (chronic) of exercise, overall, higher opioid receptor binding was observed in acute-exercise animals and the opposite was found in the chronic-exercise animals. The binding of [(35) S]GTPγS under basal conditions (absence of agonists) was elevated in sensorimotor cortex and hippocampus, an effect more evident after chronic exercise. Divergence of findings was observed for mu opioid receptor, kappa opioid receptor, and delta opioid receptor receptor activation in our study. Our results support existing evidence of opioid receptor binding and G protein activation occurring differentially in brain regions in response to diverse exercise stimuli. We characterized the binding and G protein activation of mu, kappa, and delta opioid receptors in several brain regions following acute (7 days) and chronic (30 days) exercise. Higher opioid receptor binding was observed in the acute exercise animal group and opposite findings in the chronic exercise group. Higher G protein activation under basal conditions was noted in rats submitted to chronic exercise, as visible in the depicted pseudo-color autoradiograms.

  13. Triclocarban mediates induction of xenobiotic metabolism through activation of the constitutive androstane receptor and the estrogen receptor alpha.

    Yueh, Mei-Fei; Li, Tao; Evans, Ronald M; Hammock, Bruce; Tukey, Robert H

    2012-01-01

    Triclocarban (3,4,4'-trichlorocarbanilide, TCC) is used as a broad-based antimicrobial agent that is commonly added to personal hygiene products. Because of its extensive use in the health care industry and resistance to degradation in sewage treatment processes, TCC has become a significant waste product that is found in numerous environmental compartments where humans and wildlife can be exposed. While TCC has been linked to a range of health and environmental effects, few studies have been conducted linking exposure to TCC and induction of xenobiotic metabolism through regulation by environmental sensors such as the nuclear xenobiotic receptors (XenoRs). To identify the ability of TCC to activate xenobiotic sensors, we monitored XenoR activities in response to TCC treatment using luciferase-based reporter assays. Among the XenoRs in the reporter screening assay, TCC promotes both constitutive androstane receptor (CAR) and estrogen receptor alpha (ERα) activities. TCC treatment to hUGT1 mice resulted in induction of the UGT1A genes in liver. This induction was dependent upon the constitutive active/androstane receptor (CAR) because no induction occurred in hUGT1Car(-/-) mice. Induction of the UGT1A genes by TCC corresponded with induction of Cyp2b10, another CAR target gene. TCC was demonstrated to be a phenobarbital-like activator of CAR in receptor-based assays. While it has been suggested that TCC be classified as an endocrine disruptor, it activates ERα leading to induction of Cyp1b1 in female ovaries as well as in promoter activity. Activation of ERα by TCC in receptor-based assays also promotes induction of human CYP2B6. These observations demonstrate that TCC activates nuclear xenobiotic receptors CAR and ERα both in vivo and in vitro and might have the potential to alter normal physiological homeostasis. Activation of these xenobiotic-sensing receptors amplifies gene expression profiles that might represent a mechanistic base for potential human

  14. GabaB receptors activation in the NTS blocks the glycemic responses induced by carotid body receptor stimulation.

    Lemus, Mónica; Montero, Sergio; Cadenas, José Luis; Lara, José Jesús; Tejeda-Chávez, Héctor Rafael; Alvarez-Buylla, Ramón; de Alvarez-Buylla, Elena Roces

    2008-08-18

    The carotid body receptors participate in glucose regulation sensing glucose levels in blood entering the cephalic circulation. The carotid body receptors information, is initially processed within the nucleus tractus solitarius (NTS) and elicits changes in circulating glucose and brain glucose uptake. Previous work has shown that gamma-aminobutyric acid (GABA) in NTS modulates respiratory reflexes, but the role of GABA within NTS in glucose regulation remains unknown. Here we show that GABA(B) receptor agonist (baclofen) or antagonists (phaclofen and CGP55845A) locally injected into NTS modified arterial glucose levels and brain glucose retention. Control injections outside NTS did not elicit these responses. In contrast, GABA(A) agonist and antagonist (muscimol or bicuculline) produced no significant changes in blood glucose levels. When these GABAergic drugs were applied before carotid body receptors stimulation, again, only GABA(B) agonist or antagonist significantly affected glycemic responses; baclofen microinjection significantly reduced the hyperglycemic response and brain glucose retention observed after carotid body receptors stimulation, while phaclofen produced the opposite effect, increasing significantly hyperglycemia and brain glucose retention. These results indicate that activation of GABA(B), but not GABA(A), receptors in the NTS modulates the glycemic responses after anoxic stimulation of the carotid body receptors, and suggest the presence of a tonic inhibitory mechanism in the NTS to avoid hyperglycemia.

  15. Tumorigenesis induced by the HHV8-encoded chemokine receptor requires ligand modulation of high constitutive activity

    Holst, P J; Rosenkilde, M M; Manfra, D;

    2001-01-01

    ORF74 (or KSHV-vGPCR) is a highly constitutively active G protein-coupled receptor encoded by HHV8 that is regulated both positively and negatively by endogenous chemokines. When expressed in transgenic mice, this chemokine receptor induces an angioproliferative disease closely resembling Kaposi...... sarcoma (KS). Here we demonstrate that several lines of mice carrying mutated receptors deficient in either constitutive activity or chemokine regulation fail to develop KS-like disease. In addition, animals expressing a receptor that preserves chemokine binding and constitutive activity but that does...

  16. Biased signaling by peptide agonists of protease activated receptor 2.

    Jiang, Yuhong; Yau, Mei-Kwan; Kok, W Mei; Lim, Junxian; Wu, Kai-Chen; Liu, Ligong; Hill, Timothy A; Suen, Jacky Y; Fairlie, David P

    2017-02-07

    Protease activated receptor 2 (PAR2) is associated with metabolism, obesity, inflammatory, respiratory and gastrointestinal disorders, pain, cancer and other diseases. The extracellular N-terminus of PAR2 is a common target for multiple proteases, which cleave it at different sites to generate different N-termini that activate different PAR2-mediated intracellular signaling pathways. There are no synthetic PAR2 ligands that reproduce the same signaling profiles and potencies as proteases. Structure-activity relationships here for 26 compounds spanned a signaling bias over 3 log units, culminating in three small ligands as biased agonist tools for interrogating PAR2 functions. DF253 (2f-LAAAAI-NH2) triggered PAR2-mediated calcium release (EC50 2 μM) but not ERK1/2 phosphorylation (EC50 > 100 μM) in CHO cells transfected with hPAR2. AY77 (Isox-Cha-Chg-NH2) was a more potent calcium-biased agonist (EC50 40 nM, Ca2+; EC50 2 μM, ERK1/2), while its analogue AY254 (Isox-Cha-Chg-A-R-NH2) was an ERK-biased agonist (EC50 2 nM, ERK1/2; EC50 80 nM, Ca2+). Signaling bias led to different functional responses in human colorectal carcinoma cells (HT29). AY254, but not AY77 or DF253, attenuated cytokine-induced caspase 3/8 activation, promoted scratch-wound healing and induced IL-8 secretion, all via PAR2-ERK1/2 signaling. Different ligand components were responsible for different PAR2 signaling and functions, clues that can potentially lead to drugs that modulate different pathway-selective cellular and physiological responses.

  17. Reconstituted B cell receptor signaling reveals carbohydrate-dependent mode of activation

    2016-01-01

    Activation of immune cells (but not B cells) with lectins is widely known. We used the structurally defined interaction between influenza hemagglutinin (HA) and its cell surface receptor sialic acid (SA) to identify a B cell receptor (BCR) activation modality that proceeded through non-cognate interactions with antigen. Using a new approach to reconstitute antigen-receptor interactions in a human reporter B cell line, we found that sequence-defined BCRs from the human germline repertoire coul...

  18. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric carcinomas from high- and low-risk countries

    Alpizar Alpizar, Warner Enrique; Nielsen, Boye Schnack; Sierra, Rafaela;

    2010-01-01

    examined the nonneoplastic mucosa and found that uPAR was more frequently seen in epithelial cells located at the luminal edge of the crypts in cases with Helicobacter pylori infection than in similar epithelial cells in noninfected mucosa (p = 0.033; chi(2) = 4.54). In conclusion, the expression of u...

  19. Activation of Cannabinoid Receptor 2 Enhances Osteogenic Differentiation of Bone Marrow Derived Mesenchymal Stem Cells

    Yong-Xin Sun

    2015-01-01

    Full Text Available Bone marrow derived mesenchymal stem cells (BM-MSCs are considered as the most promising cells source for bone engineering. Cannabinoid (CB receptors play important roles in bone mass turnover. The aim of this study is to test if activation of CB2 receptor by chemical agonist could enhance the osteogenic differentiation and mineralization in bone BM-MSCs. Alkaline phosphatase (ALP activity staining and real time PCR were performed to test the osteogenic differentiation. Alizarin red staining was carried out to examine the mineralization. Small interference RNA (siRNA was used to study the role of CB2 receptor in osteogenic differentiation. Results showed activation of CB2 receptor increased ALP activity, promoted expression of osteogenic genes, and enhanced deposition of calcium in extracellular matrix. Knockdown of CB2 receptor by siRNA inhibited ALP activity and mineralization. Results of immunofluorescent staining showed that phosphorylation of p38 MAP kinase is reduced by knocking down of CB2 receptor. Finally, bone marrow samples demonstrated that expression of CB2 receptor is much lower in osteoporotic patients than in healthy donors. Taken together, data from this study suggested that activation of CB2 receptor plays important role in osteogenic differentiation of BM-MSCs. Lack of CB2 receptor may be related to osteoporosis.

  20. [5-HT1A/5-HT7 receptor interplay: Chronic activation of 5-HT7 receptors decreases the functional activity of 5-HT1A receptor and its сontent in the mouse brain].

    Kondaurova, E M; Bazovkina, D V; Naumenko, V S

    2017-01-01

    Serotonin receptors 5-HT1A and 5-HT7 are involved in the development of various psychopathologies. Some data indicate that there is an interplay between 5-HT1A 5-HT7 receptors that could be implicated in the regulation of their function. This work analyzed the effects of chronic 5-HT7 activation on the functional activity of 5-HT7 and 5-HT1A receptors, on the corresponding protein levels, and on the expression of genes encoding 5-HT7 and 5-HT1A receptors in the mouse brain. Chronic administration of the 5-HT7 selective agonist LP44 (20.5 nmol, i.c.v., 14 days) produced considerable desensitization of both 5-HT7 and 5-HT1A receptors. In LP44-treated mice, the hypothermic responses mediated by both 5-HT7 and 5-HT1A receptors were attenuated. Moreover, the levels of 5-HT1A receptor protein in the midbrain and the frontal cortex of LP44-treated mice were significantly decreased. However, the brain levels of 5-HT7 receptor protein did not differ between LP44-treated and control mice. Chronic LP44 treatment did not alter the expression of the 5-HT7 and 5-HT1A receptor genes in all investigated brain structure. These data suggest that 5-HT7 receptors participate in the posttranscriptional regulation of the 5-HT1A receptors functioning.

  1. Stereostructure-activity studies on agonists at the AMPA and kainate subtypes of ionotropic glutamate receptors

    Johansen, Tommy N; Greenwood, Jeremy R; Frydenvang, Karla Andrea;

    2003-01-01

    -methyl-4-isoxazolyl)propionic acid (AMPA) receptor subtype of ionotropic Glu receptors in the presence or absence of an agonist has provided important information about ligand-receptor interaction mechanisms. The availability of these binding domain crystal structures has formed the basis for rational...... design of ligands, especially for the AMPA and kainate subtypes of ionotropic Glu receptors. This mini-review will focus on structure-activity relationships on AMPA and kainate receptor agonists with special emphasis on stereochemical and three-dimensional aspects....

  2. NMDA receptor activation regulates sociability by its effect on mTOR signaling activity.

    Burket, Jessica A; Benson, Andrew D; Tang, Amy H; Deutsch, Stephen I

    2015-07-01

    Tuberous Sclerosis Complex is one example of a syndromic form of autism spectrum disorder associated with disinhibited activity of mTORC1 in neurons (e.g., cerebellar Purkinje cells). mTORC1 is a complex protein possessing serine/threonine kinase activity and a key downstream molecule in a signaling cascade beginning at the cell surface with the transduction of neurotransmitters (e.g., glutamate and acetylcholine) and nerve growth factors (e.g., Brain-Derived Neurotrophic Factor). Interestingly, the severity of the intellectual disability in Tuberous Sclerosis Complex may relate more to this metabolic disturbance (i.e., overactivity of mTOR signaling) than the density of cortical tubers. Several recent reports showed that rapamycin, an inhibitor of mTORC1, improved sociability and other symptoms in mouse models of Tuberous Sclerosis Complex and autism spectrum disorder, consistent with mTORC1 overactivity playing an important pathogenic role. NMDA receptor activation may also dampen mTORC1 activity by at least two possible mechanisms: regulating intraneuronal accumulation of arginine and the phosphorylation status of a specific extracellular signal regulating kinase (i.e., ERK1/2), both of which are "drivers" of mTORC1 activity. Conceivably, the prosocial effects of targeting the NMDA receptor with agonists in mouse models of autism spectrum disorders result from their ability to dampen mTORC1 activity in neurons. Strategies for dampening mTORC1 overactivity by NMDA receptor activation may be preferred to its direct inhibition in chronic neurodevelopmental disorders, such as autism spectrum disorders.

  3. Activation of toll-like receptors and dendritic cells by a broad range of bacterial molecules

    Boele, L.C.L.; Bajramovic, J.J.; Vries, A.M.M.B.C. de; Voskamp-Visser, I.A.I.; Kaman, W.E.; Kleij, D. van der

    2009-01-01

    Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, the

  4. Plasma soluble urokinase plasminogen activator receptor in children with urinary tract infection

    Wittenhagen, Per; Andersen, Jesper Brandt; Hansen, Anita

    2011-01-01

    In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection.......In this prospective study we investigated the role of plasma levels of soluble urokinase plasminogen activator receptor (suPAR) in children with urinary tract infection....

  5. Peroxisome proliferators-activated receptor (PPAR) regulation in cardiac metabolism and disease

    el Azzouzi, H.

    2009-01-01

    Peroxisome proliferators-activated receptors (PPARs) are members of the nuclear receptor family of ligand activated transcription factors and consist of the three isoforms, PPAR, PPAR/ and PPAR. Considerable evidence has established the importance of PPARs in myocardial lipid homeostasis and car

  6. Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen;

    2015-01-01

    densities in mitochondria. Activation of the cell membrane AT2 receptors by a concomitant treatment with angiotensin II and the AT1 receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT2 receptor...... of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT2 receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

  7. Agonist-biased signaling via proteinase activated receptor-2: differential activation of calcium and mitogen-activated protein kinase pathways.

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; Defea, Kathryn; Hollenberg, Morley D

    2009-10-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR(2)) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR(2) and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR(2)-Leu(37)Ser(38), rPAR(2)-Ala(37-38), and rPAR(2)-Ala(39-42) were compared with the trypsin-revealed wild-type rPAR(2) TL sequence, S(37)LIGRL(42)-. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR(2) and rPAR(2)-Ala(39-42) triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR(2)-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR(2)-Ala(37-38) nor rPAR(2)-Leu(37)Ser(38) constructs recruited beta-arrestins-1 or -2 in response to trypsin stimulation, whereas both beta-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered beta-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Galpha(i) (pertussis toxin), Galpha(q) [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3'-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the

  8. Phytoceramide and sphingoid bases derived from brewer's yeast Saccharomyces pastorianus activate peroxisome proliferator-activated receptors

    Mitsutake Susumu

    2011-08-01

    Full Text Available Abstract Background Peroxisome proliferator-activated receptors (PPARs are ligand-activated transcription factors that regulate lipid and glucose metabolism. PPARα is highly expressed in the liver and controls genes involved in lipid catabolism. We previously reported that synthetic sphingolipid analogs, part of which contains shorter-length fatty acid chains than natural sphingolipids, stimulated the transcriptional activities of PPARs. Sphingosine and dihydrosphingosine (DHS are abundant sphingoid bases, and ceramide and dihydroceramide are major ceramide species in mammals. In contrast, phytosphingosine (PHS and DHS are the main sphingoid bases in fungi. PHS and phytoceramide exist in particular tissues such as the epidermis in mammals, and involvement of ceramide species in PPARβ activation in cultured keratinocytes has been reported. The purpose of the present study is to investigate whether natural sphingolipids with C18 fatty acid and yeast-derived sphingoid bases activate PPARs as PPAR agonists. Method Lipids of brewer's yeast contain PHS- and DHS-based sphingolipids. To obtain the sphingoid bases, lipids were extracted from brewer's yeast and acid-hydrolyzed. The sphingoid base fraction was purified and quantified. To assess the effects of sphingolipids on PPAR activation, luciferase reporter assay was carried out. NIH/3T3 and human hepatoma (HepG2 cells were transfected with expression vectors for PPARs and retinoid × receptors, and PPAR responsive element reporter vector. When indicated, the PPAR/Gal4 chimera system was performed to enhance the credibility of experiments. Sphingolipids were added to the cells and the dual luciferase reporter assay was performed to determine the transcriptional activity of PPARs. Results We observed that phytoceramide increased the transcriptional activities of PPARs significantly, whereas ceramide and dihydroceramide did not change PPAR activities. Phytoceramide also increased transactivation of

  9. Five layers of receptor signalling in γδ T cell differentiation and activation

    Sérgio T. Ribeiro

    2015-01-01

    Full Text Available The contributions of gamma-delta T cells to immunity to infection or tumours critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate gamma-delta T cell physiology. The understanding of how environmental signals are integrated by gamma-delta T cells is critical for their manipulation in clinical settings. Here we discuss how different classes of surface receptors impact on human and murine gamma-delta T cell differentiation, activation and expansion. In particular, we review the role of five receptor types: the T cell receptor (TCR, costimulatory receptors, cytokine receptors, NK receptors and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of gamma-delta T cells; the cytokine receptors IL-2R, IL-7R and IL-15R, which drive functional differentiation and expansion of gamma-delta T cells; the NK receptor NKG2D and its contribution to gamma-delta T cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control gamma-delta T cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signalling in gamma-delta T cell differentiation and activation, and discuss its implications for the manipulation of gamma-delta T cells in immunotherapy.

  10. Troglitazone inhibits cell proliferation by attenuation of epidermal growth factor receptor signaling independent of peroxisome proliferator-activated receptor γ

    Xiaoqi Li; Xuanming Yang; Youli Xu; Xuejun Jiang; Xin Li; Fajun Nan; Hong Tang

    2009-01-01

    Peroxisome proliferator-activated receptors (PPAR) belong to the nuclear hormone receptor superfamily of ligand-dependent transcription factors. Recent results have shown that agonists of PPARy, such as troglitazone (TGZ), can inhibit cell proliferation and promote cell differentiation independent of PPARγ. In the present study, we provide evidence that TGZ may bind directly to EGFR and trigger its signaling and internalization independent of PPARγ. Detailed studies revealed that prolonged incubation with TGZ effectively attenuated EGFR signaling by target-ing the receptor to the endo-lysosomal degradation machinery. Although the extracellular signal-regulated kinase-signaling pathway was transiently activated by TGZ in EGFR overexpressing cancer cells, inhibition of EGF-induced Akt phosphorylation most likely accounted for the growth arrest of tumor cells caused by TGZ at pharmacologically achievable concentrations. Therefore, we have provided a new line of evidence indicating that TGZ inhibits cell pro-liferation by promoting EGFR degradation and attenuating Akt phosphorylation.

  11. Selective glucocorticoid receptor-activating adjuvant therapy in cancer treatments

    Sundahl, Nora; Clarisse, Dorien; Bracke, Marc; Offner, Fritz; Berghe, Wim Vanden; Beck, Ilse M.

    2016-01-01

    Although adverse effects and glucocorticoid resistance cripple their chronic use, glucocorticoids form the mainstay therapy for acute and chronic inflammatory disorders, and play an important role in treatment protocols of both lymphoid malignancies and as adjuvant to stimulate therapy tolerability in various solid tumors. Glucocorticoid binding to their designate glucocorticoid receptor (GR), sets off a plethora of cell-specific events including therapeutically desirable effects, such as cell death, as well as undesirable effects, including chemotherapy resistance, systemic side effects and glucocorticoid resistance. In this context, selective GR agonists and modulators (SEGRAMs) with a more restricted GR activity profile have been developed, holding promise for further clinical development in anti-inflammatory and potentially in cancer therapies. Thus far, the research into the prospective benefits of selective GR modulators in cancer therapy limped behind. Our review discusses how selective GR agonists and modulators could improve the therapy regimens for lymphoid malignancies, prostate or breast cancer. We summarize our current knowledge and look forward to where the field should move to in the future. Altogether, our review clarifies novel therapeutic perspectives in cancer modulation via selective GR targeting.

  12. Estrogen Receptor β Activation Rapidly Modulates Male Sexual Motivation through the Transactivation of Metabotropic Glutamate Receptor 1a.

    Seredynski, Aurore L; Balthazart, Jacques; Ball, Gregory F; Cornil, Charlotte A

    2015-09-23

    In addition to the transcriptional activity of their liganded nuclear receptors, estrogens, such as estradiol (E2), modulate cell functions, and consequently physiology and behavior, within minutes through membrane-initiated events. The membrane-associated receptors (mERs) underlying the acute effects of estrogens on behavior have mostly been documented in females where active estrogens are thought to be of ovarian origin. We determined here, by acute intracerebroventricular injections of specific agonists and antagonists, the type(s) of mERs that modulate rapid effects of brain-derived estrogens on sexual motivation in male Japanese quail. Brain aromatase blockade acutely inhibited sexual motivation. Diarylpropionitrile (DPN), an estrogen receptor β (ERβ)-specific agonist, and to a lesser extent 17α-estradiol, possibly acting through ER-X, prevented this effect. In contrast, drugs targeting ERα (PPT and MPP), GPR30 (G1 and G15), and the Gq-mER (STX) did not affect sexual motivation. The mGluR1a antagonist LY367385 significantly inhibited sexual motivation but mGluR2/3 and mGluR5 antagonists were ineffective. LY367385 also blocked the behavioral restoration induced by E2 or DPN, providing functional evidence that ERβ interacts with metabotropic glutamate receptor 1a (mGluR1a) signaling to acutely regulate male sexual motivation. Together these results show that ERβ plays a key role in sexual behavior regulation and the recently uncovered cooperation between mERs and mGluRs is functional in males where it mediates the acute effects of estrogens produced centrally in response to social stimuli. The presence of an ER-mGluR interaction in birds suggests that this mechanism emerged relatively early in vertebrate history and is well conserved. Significance statement: The membrane-associated receptors underlying the acute effects of estrogens on behavior have mostly been documented in females, where active estrogens are thought to be of ovarian origin. Using acute

  13. Molecular photoacoustic tomography of breast cancer using receptor targeted magnetic iron oxide nanoparticles as contrast agents.

    Xi, Lei; Grobmyer, Stephen R; Zhou, Guangyin; Qian, Weiping; Yang, Lily; Jiang, Huabei

    2014-06-01

    In this report, we present a breast imaging technique combining high-resolution near-infrared (NIR) light induced photoacoustic tomography (PAT) with NIR dye-labeled amino-terminal fragments of urokinase plasminogen activator receptor (uPAR) targeted magnetic iron oxide nanoparticles (NIR830-ATF-IONP) for breast cancer imaging using an orthotopic mouse mammary tumor model. We show that accumulation of the targeted nanoparticles in the tumor led to photoacoustic contrast enhancement due to the high absorption of iron oxide nanoparticles (IONP). NIR fluorescence images were used to validate specific delivery of NIR830-ATF-IONP to mouse mammary tumors. We found that systemic delivery of the targeted IONP produced 4- and 10-fold enhancement in photoacoustic signals in the tumor, compared to the tumor of the mice that received non-targeted IONP or control mice. The use of targeted nanoparticles allowed imaging of tumors located as deep as 3.1 cm beneath the normal tissues. Our study indicates the potential of the combination of photoacoustic tomography and receptor-targeted NIR830-ATF-IONP as a clinical tool that can provide improved specificity and sensitivity for breast cancer detection.

  14. Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

    Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1992-01-01

    Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

  15. Sulindac metabolites inhibit epidermal growth factor receptor activation and expression

    Pangburn Heather A

    2005-09-01

    Full Text Available Abstract Background Regular use of nonsteroidal anti-inflammatory drugs (NSAIDs is associated with a decreased mortality from colorectal cancer (CRC. NSAIDs induce apoptotic cell death in colon cancer cells in vitro and inhibit growth of neoplastic colonic mucosa in vivo however, the biochemical mechanisms required for these growth inhibitory effects are not well defined. We previously reported that metabolites of the NSAID sulindac downregulate extracellular-signal regulated kinase 1/2 (ERK1/2 signaling and that this effect is both necessary and sufficient for the apoptotic effects of these drugs. The goal of this project was to specifically test the hypothesis that sulindac metabolites block activation and/or expression of the epidermal growth factor (EGF receptor (EGFR. Methods HT29 human colon cancer cells were treated with EGF, alone, or in the presence of sulindac sulfide or sulindac sulfone. Cells lysates were assayed by immunoblotting for phosphorylated EGFR (pEGFR, pY1068, total EGFR, phosphorylated ERK1/2 (pERK1/2, total ERK1/2, activated caspase-3, and α-tubulin. Results EGF treatment rapidly induced phosphorylation of both EGFR and ERK1/2 in HT29 colon cancer cells. Pretreatment with sulindac metabolites for 24 h blocked EGF-induced phosphorylation of both EGFR and ERK1/2 and decreased total EGFR protein expression. Under basal conditions, downregulation of pEGFR and total EGFR was detected as early as 12 h following sulindac sulfide treatment and persisted through at least 48 h. Sulindac sulfone induced downregulation of pEGFR and total EGFR was detected as early as 1 h and 24 h, respectively, following drug treatment, and persisted through at least 72 h. EGFR downregulation by sulindac metabolites was observed in three different CRC cell lines, occurred prior to the observed downregulation of pERK1/2 and induction of apoptosis by these drugs, and was not dependent of caspase activation. Conclusion These results suggest that

  16. Activation of NTS A2a adenosine receptors differentially resets baroreflex control of renal vs. adrenal sympathetic nerve activity.

    Ichinose, Tomoko K; O'Leary, Donal S; Scislo, Tadeusz J

    2009-04-01

    The role of nucleus of solitary tract (NTS) A(2a) adenosine receptors in baroreflex mechanisms is controversial. Stimulation of these receptors releases glutamate within the NTS and elicits baroreflex-like decreases in mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA), whereas inhibition of these receptors attenuates HR baroreflex responses. In contrast, stimulation of NTS A(2a) adenosine receptors increases preganglionic adrenal sympathetic nerve activity (pre-ASNA), and the depressor and sympathoinhibitory responses are not markedly affected by sinoaortic denervation and blockade of NTS glutamatergic transmission. To elucidate the role of NTS A(2a) adenosine receptors in baroreflex function, we compared full baroreflex stimulus-response curves for HR, RSNA, and pre-ASNA (intravenous nitroprusside/phenylephrine) before and after bilateral NTS microinjections of selective adenosine A(2a) receptor agonist (CGS-21680; 2.0, 20 pmol/50 nl), selective A(2a) receptor antagonist (ZM-241385; 40 pmol/100 nl), and nonselective A(1) + A(2a) receptor antagonist (8-SPT; 1 nmol/100 nl) in urethane/alpha-chloralose anesthetized rats. Activation of A(2a) receptors decreased the range, upper plateau, and gain of baroreflex-response curves for RSNA, whereas these parameters all increased for pre-ASNA, consistent with direct effects of the agonist on regional sympathetic activity. However, no resetting of baroreflex-response curves along the MAP axis occurred despite the marked decreases in baseline MAP. The antagonists had no marked effects on baseline variables or baroreflex-response functions. We conclude that the activation of NTS A(2a) adenosine receptors differentially alters baroreflex control of HR, RSNA, and pre-ASNA mostly via non-baroreflex mechanism(s), and these receptors have virtually no tonic action on baroreflex control of these sympathetic outputs.

  17. Protease-Activated Receptors and other G-Protein-Coupled Receptors: the Melanoma Connection

    Rosero, Rebecca A.; Villares, Gabriel J.; Bar-Eli, Menashe

    2016-01-01

    The vast array of G-protein-coupled receptors (GPCRs) play crucial roles in both physiological and pathological processes, including vision, coagulation, inflammation, autophagy, and cell proliferation. GPCRs also affect processes that augment cell proliferation and metastases in many cancers including melanoma. Melanoma is the deadliest form of skin cancer, yet limited therapeutic modalities are available to patients with metastatic melanoma. Studies have found that both chemokine receptors ...

  18. 5-HT1A receptors modulate small-conductance Ca2+-activated K+ channels

    Grunnet, Morten; Jespersen, Thomas; Perrier, Jean-François

    2004-01-01

    Small-conductance calcium-activated potassium channels (SK) are responsible for the medium afterhyperpolarisation (mAHP) following action potentials in neurons. Here we tested the ability of serotonin (5-HT) to modulate the activity of SK channels by coexpressing 5-HT1A receptors with different...... subtypes of SK channels (SK1, SK2, and SK3) in Xenopus laevis oocytes. SK channels were activated by intracellular injection of Cd2+. Subsequent activation of 5-HT1A receptors by 8-OH-DPAT always produced an inhibition of the SK current, showing the existence of a specific pathway between the receptor...

  19. Pharmacological activation of 5-HT7 receptors reduces nerve injury-induced mechanical and thermal hypersensitivity.

    Brenchat, Alex; Nadal, Xavier; Romero, Luz; Ovalle, Sergio; Muro, Asunción; Sánchez-Arroyos, Ricard; Portillo-Salido, Enrique; Pujol, Marta; Montero, Ana; Codony, Xavier; Burgueño, Javier; Zamanillo, Daniel; Hamon, Michel; Maldonado, Rafael; Vela, José Miguel

    2010-06-01

    The involvement of the 5-HT(7) receptor in nociception and pain, particularly chronic pain (i.e., neuropathic pain), has been poorly investigated. In the present study, we examined whether the 5-HT(7) receptor participates in some modulatory control of nerve injury-evoked mechanical hypersensitivity and thermal (heat) hyperalgesia in mice. Activation of 5-HT(7) receptors by systemic administration of the selective 5-HT(7) receptor agonist AS-19 (1 and 10mg/kg) exerted a clear-cut reduction of mechanical and thermal hypersensitivities that were reversed by co-administering the selective 5-HT(7) receptor antagonist SB-258719. Interestingly, blocking of 5-HT(7) receptors with SB-258719 (2.5 and 10mg/kg) enhanced mechanical (but not thermal) hypersensitivity in nerve-injured mice and induced mechanical hypersensitivity in sham-operated mice. Effectiveness of the treatment with a 5-HT(7) receptor agonist was maintained after repeated systemic administration: no tolerance to the antiallodynic and antihyperalgesic effects was developed following treatment with the selective 5-HT(7) receptor agonist E-57431 (10mg/kg) twice daily for 11 days. The 5-HT(7) receptor co-localized with GABAergic cells in the dorsal horn of the spinal cord, suggesting that the activation of spinal inhibitory GABAergic interneurons could contribute to the analgesic effects of 5-HT(7) receptor agonists. In addition, a significant increase of 5-HT(7) receptors was found by immunohistochemistry in the ipsilateral dorsal horn of the spinal cord after nerve injury, suggesting a "pain"-triggered regulation of receptor expression. These results support the idea that the 5-HT(7) receptor subtype is involved in the control of pain and point to a new potential use of 5-HT(7) receptor agonists for the treatment of neuropathic pain.

  20. Comparative study on transcriptional activity of 17 parabens mediated by estrogen receptor α and β and androgen receptor.

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-07-01

    The structure-activity relationships of parabens which are widely used as preservatives for transcriptional activities mediated by human estrogen receptor α (hERα), hERβ and androgen receptor (hAR) were investigated. Fourteen of 17 parabens exhibited hERα and/or hERβ agonistic activity at concentrations of ≤ 1 × 10(-5)M, whereas none of the 17 parabens showed AR agonistic or antagonistic activity. Among 12 parabens with linear alkyl chains ranging in length from C₁ to C₁₂, heptylparaben (C₇) and pentylparaben (C₅) showed the most potent ERα and ERβ agonistic activity in the order of 10(-7)M and 10(-8)M, respectively, and the activities decreased in a stepwise manner as the alkyl chain was shortened to C₁ or lengthened to C₁₂. Most parabens showing estrogenic activity exhibited ERβ-agonistic activity at lower concentrations than those inducing ERα-agonistic activity. The estrogenic activity of butylparaben was markedly decreased by incubation with rat liver microsomes, and the decrease of activity was blocked by a carboxylesterase inhibitor. These results indicate that parabens are selective agonists for ERβ over ERα; their interactions with ERα/β are dependent on the size and bulkiness of the alkyl groups; and they are metabolized by carboxylesterases, leading to attenuation of their estrogenic activity.

  1. Quantitative impedimetric NPY-receptor activation monitoring and signal pathway profiling in living cells.

    te Kamp, Verena; Lindner, Ricco; Jahnke, Heinz-Georg; Krinke, Dana; Kostelnik, Katja B; Beck-Sickinger, Annette G; Robitzki, Andrea A

    2015-05-15

    Label-free and non-invasive monitoring of receptor activation and identification of the involved signal pathways in living cells is an ongoing analytic challenge and a great opportunity for biosensoric systems. In this context, we developed an impedance spectroscopy-based system for the activation monitoring of NPY-receptors in living cells. Using an optimized interdigital electrode array for sensitive detection of cellular alterations, we were able for the first time to quantitatively detect the NPY-receptor activation directly without a secondary or enhancer reaction like cAMP-stimulation by forskolin. More strikingly, we could show that the impedimetric based NPY-receptor activation monitoring is not restricted to the Y1-receptor but also possible for the Y2- and Y5-receptor. Furthermore, we could monitor the NPY-receptor activation in different cell lines that natively express NPY-receptors and proof the specificity of the observed impedimetric effect by agonist/antagonist studies in recombinant NPY-receptor expressing cell lines. To clarify the nature of the observed impedimetric effect we performed an equivalent circuit analysis as well as analyzed the role of cell morphology and receptor internalization. Finally, an antagonist based extensive molecular signal pathway analysis revealed small alterations of the actin cytoskeleton as well as the inhibition of at least L-type calcium channels as major reasons for the observed NPY-induced impedance increase. Taken together, our novel impedance spectroscopy based NPY-receptor activation monitoring system offers the opportunity to identify signal pathways as well as for novel versatile agonist/antagonist screening systems for identification of novel therapeutics in the field of obesity and cancer.

  2. Novel indole and azaindole (pyrrolopyridine) cannabinoid (CB) receptor agonists: design, synthesis, structure-activity relationships, physicochemical properties and biological activity

    Blaazer, A.R.; Lange, J.H.M.; van der Neut, M.A.W.; Mulder, A.; den Boon, F.S.; Werkman, T.R.; Kruse, C.G.; Wadman, W.J.

    2011-01-01

    The discovery, synthesis and structure-activity relationship (SAR) of a novel series of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands are reported. Based on the aminoalkylindole class of cannabinoid receptor agonists, a biphenyl moiety was introduced as novel lipophilic indole 3-acyl

  3. Src Family Kinases and Receptors: Analysis of Three Activation Mechanisms by Dynamic Systems Modeling

    Fuß, Hendrik; Dubitzky, Werner; Downes, C. Stephen; Kurth, Mary Jo

    2007-01-01

    Src family kinases (SFKs) interact with a number of cellular receptors. They participate in diverse signaling pathways and cellular functions. Most of the receptors involved in SFK signaling are characterized by similar modes of regulation. This computational study discusses a general kinetic model of SFK-receptor interaction. The analysis of the model reveals three major ways of SFK activation: release of inhibition by C-terminal Src kinase, weakening of the inhibitory intramolecular phospho...

  4. Mechanisms involved in VPAC receptors activation and regulation: lessons from pharmacological and mutagenesis studies.

    Ingrid eLanger

    2012-10-01

    Full Text Available VIP plays diverse and important role in human physiology and physiopathology and their receptors constitute potential targets for the treatment of several diseases such as neurodegenerative disorder, asthma, diabetes and inflammatory diseases. This article reviews the current knowledge regarding the two VIP receptors, VPAC1 and VPAC2, with respect to mechanisms involved in receptor activation, G protein coupling, signaling, regulation and oligomerization.

  5. Activity of protease-activated receptors in primary cultured human myenteric neurons

    Eva Maria Kugler

    2012-09-01

    Full Text Available Activity of the four known protease-activated receptors (PARs has been well studied in rodent enteric nervous system and results in animal models established an important role for neuronal PAR2. We recently demonstrated that, unlike in rodents, PAR1 is the dominant neuronal protease receptor in the human submucous plexus. With this study we investigated whether this also applies to the human myenteric plexus. We used voltage sensitive dye recordings to detect action potential discharge in primary cultures of human myenteric neurons in response to PAR activating peptides (AP. Application of the PAR1-AP (TFLLR or PAR4-AP (GYPGQV evoked spike discharge in 79% or 23% of myenteric neurons, respectively. The PAR1-AP response was mimicked by the endogenous PAR1 activator thrombin and blocked by the PAR1 antagonists SCH79797. Human myenteric neurons did not respond to PAR2-AP. This was not due to culture conditions because all three PAR-APs evoked action potentials in cultured guinea pig myenteric neurons. Consecutive application of PAR-APs revealed coexpression (relative to the population responding to PAR-APs of PAR1/PAR2 in 51%, PAR1/PAR4 in 43% and of PAR2/PAR4 in 29% of guinea pig myenteric neurons. Our study provided further evidence for the prominent role of neuronal PAR1 in the human enteric nervous system.

  6. Adenosine A3 receptor activation is neuroprotective against retinal neurodegeneration.

    Galvao, Joana; Elvas, Filipe; Martins, Tiago; Cordeiro, M Francesca; Ambrósio, António Francisco; Santiago, Ana Raquel

    2015-11-01

    Death of retinal neural cells, namely retinal ganglion cells (RGCs), is a characteristic of several retinal neurodegenerative diseases. Although the role of adenosine A3 receptor (A3R) in neuroprotection is controversial, A3R activation has been reported to afford protection against several brain insults, with few studies in the retina. In vitro models (retinal neural and organotypic cultures) and animal models [ischemia-reperfusion (I-R) and partial optic nerve transection (pONT)] were used to study the neuroprotective properties of A3R activation against retinal neurodegeneration. The A3R selective agonist (2-Cl-IB-MECA, 1 μM) prevented apoptosis (TUNEL(+)-cells) induced by kainate and cyclothiazide (KA + CTZ) in retinal neural cultures (86.5 ± 7.4 and 37.2 ± 6.1 TUNEL(+)-cells/field, in KA + CTZ and KA + CTZ + 2-Cl-IB-MECA, respectively). In retinal organotypic cultures, 2-Cl-IB-MECA attenuated NMDA-induced cell death, assessed by TUNEL (17.3 ± 2.3 and 8.3 ± 1.2 TUNEL(+)-cells/mm(2) in NMDA and NMDA+2-Cl-IB-MECA, respectively) and PI incorporation (ratio DIV4/DIV2 3.3 ± 0.3 and 1.3 ± 0.1 in NMDA and NMDA+2-Cl-IB-MECA, respectively) assays. Intravitreal 2-Cl-IB-MECA administration afforded protection against I-R injury decreasing the number of TUNEL(+) cells by 72%, and increased RGC survival by 57%. Also, intravitreal administration of 2-Cl-IB-MECA inhibited apoptosis (from 449.4 ± 37.8 to 207.6 ± 48.9 annexin-V(+)-cells) and RGC loss (from 1.2 ± 0.6 to 8.1 ± 1.7 cells/mm) induced by pONT. This study demonstrates that 2-Cl-IB-MECA is neuroprotective to the retina, both in vitro and in vivo. Activation of A3R may have great potential in the management of retinal neurodegenerative diseases characterized by RGC death, as glaucoma and diabetic retinopathy, and ischemic diseases.

  7. Pharmacology and toxicology of fibrates as hypolipidemic drugs mediated by nuclear receptor peroxisome proliferator—activated receptor

    SugaT

    2002-01-01

    PPAR(peroxisome proliferator-activated receptor) is a family of nuclear receptor.In recent years,it has been focused for the discovery and development of new drugs which are mediated by PPARs.Fibrate hypolipidemic drugs are the specific and potent ligands to PPAR alpha and have been widely used for the treatment of hyperlipidemia.But these drugs induce hepatocarcinogenesis in rodent animals after the long-term administration.However,there are species differences on these phenomena which are not seen in mammals ioncluding human.To clarify the mechanism of carcinogenesis by these drugs in important for the evaluation of safety of these drugs in human.

  8. Xenobiotic-induced hepatocyte proliferation associated with constitutive active/androstane receptor (CAR or peroxisome proliferator-activated receptor α (PPARα is enhanced by pregnane X receptor (PXR activation in mice.

    Ryota Shizu

    Full Text Available Xenobiotic-responsive nuclear receptors pregnane X receptor (PXR, constitutive active/androstane receptor (CAR and peroxisome proliferator-activated receptor α (PPARα play pivotal roles in the metabolic functions of the liver such as xenobiotics detoxification and energy metabolism. While CAR or PPARα activation induces hepatocyte proliferation and hepatocarcinogenesis in rodent models, it remains unclear whether PXR activation also shows such effects. In the present study, we have investigated the role of PXR in the xenobiotic-induced hepatocyte proliferation with or without CAR activation by 1,4-bis[2-(3,5-dichloropyridyloxy]benzene (TCPOBOP and phenobarbital, or PPARα activation by Wy-14643 in mice. Treatment with TCPOBOP or phenobarbital increased the percentage of Ki-67-positive nuclei as well as mRNA levels of cell proliferation-related genes in livers as expected. On the other hand, treatment with the PXR activator pregnenolone 16α-carbonitrile (PCN alone showed no such effects. Surprisingly, PCN co-treatment significantly augmented the hepatocyte proliferation induced by CAR activation with TCPOBOP or phenobarbital in wild-type mice but not in PXR-deficient mice. Intriguingly, PXR activation also augmented the hepatocyte proliferation induced by Wy-14643 treatment. Moreover, PCN treatment increased the RNA content of hepatocytes, suggesting the induction of G0/G1 transition, and reduced mRNA levels of Cdkn1b and Rbl2, encoding suppressors of cell cycle initiation. Our present findings indicate that xenobiotic-induced hepatocyte proliferation mediated by CAR or PPARα is enhanced by PXR co-activation despite that PXR activation alone does not cause the cell proliferation in mouse livers. Thus PXR may play a novel and unique role in the hepatocyte/liver hyperplasia upon exposure to xenobiotics.

  9. Cellular phosphatases facilitate combinatorial processing of receptor-activated signals

    Siddiqui Zaved

    2008-09-01

    Full Text Available Abstract Background Although reciprocal regulation of protein phosphorylation represents a key aspect of signal transduction, a larger perspective on how these various interactions integrate to contribute towards signal processing is presently unclear. For example, a key unanswered question is that of how phosphatase-mediated regulation of phosphorylation at the individual nodes of the signaling network translates into modulation of the net signal output and, thereby, the cellular phenotypic response. Results To address the above question we, in the present study, examined the dynamics of signaling from the B cell antigen receptor (BCR under conditions where individual cellular phosphatases were selectively depleted by siRNA. Results from such experiments revealed a highly enmeshed structure for the signaling network where each signaling node was linked to multiple phosphatases on the one hand, and each phosphatase to several nodes on the other. This resulted in a configuration where individual signaling intermediates could be influenced by a spectrum of regulatory phosphatases, but with the composition of the spectrum differing from one intermediate to another. Consequently, each node differentially experienced perturbations in phosphatase activity, yielding a unique fingerprint of nodal signals characteristic to that perturbation. This heterogeneity in nodal experiences, to a given perturbation, led to combinatorial manipulation of the corresponding signaling axes for the downstream transcription factors. Conclusion Our cumulative results reveal that it is the tight integration of phosphatases into the signaling network that provides the plasticity by which perturbation-specific information can be transmitted in the form of a multivariate output to the downstream transcription factor network. This output in turn specifies a context-defined response, when translated into the resulting gene expression profile.

  10. The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis

    Jansen Sandra

    2011-02-01

    Full Text Available Abstract Background Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure mediates activation of the immune response in bacterial infection of the central nervous system (CNS. The chemotactic G-protein-coupled receptor (GPCR formyl-peptide-receptor like-1 (FPRL1 plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD. Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear. Methods Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP and Neisseria meningitides (NM. Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2 phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene expression and signal transduction were determined. Results We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between

  11. Identification of chemical modulators of the constitutive activated receptor (CAR) in a gene expression compendium

    Oshida, Keiyu; Vasani, Naresh; Jones, Carlton; Moore, Tanya; Hester, Susan; Nesnow, Stephen; Auerbach, Scott; Geter, David R.; Aleksunes, Lauren M; Thomas, Russell S.; Applegate, Dawn; Klaassen, Curtis D.; Corton, J. Christopher

    2015-01-01

    The nuclear receptor family member constitutive activated receptor (CAR) is activated by structurally diverse drugs and environmentally-relevant chemicals leading to transcriptional regulation of genes involved in xenobiotic metabolism and transport. Chronic activation of CAR increases liver cancer incidence in rodents, whereas suppression of CAR can lead to steatosis and insulin insensitivity. Here, analytical methods were developed to screen for chemical treatments in a gene expression comp...

  12. The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection

    Kawabata, Atsufumi; Kinoshita, Mitsuhiro; Nishikawa, Hiroyuki; Kuroda, Ryotaro; Nishida, Minoru; Araki, Hiromasa; Arizono, Naoki; Oda, Yasuo; Kakehi, Kazuaki

    2001-01-01

    Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2–activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsai...

  13. Analysis of the epidermal growth factor receptor specific transcriptome: effect of receptor expression level and an activating mutation

    Pedersen, Mikkel W; Pedersen, Nina; Damstrup, Lars;

    2005-01-01

    moderately expressed or overexpressed at an in-itself transforming level. These changes were compared to those induced by the naturally occurring constitutively active variant EGFRvIII. This study provides novel insight on the activities and mechanisms of EGFRvIII and EGFR mediated transformation, as genes...... by interferons. Expression of this module was absent in the EGFRvIII-expressing cell line and the parental cell line. Treatment with the specific EGFR inhibitor AG1478 indicated that the regulations were primary, receptor-mediated events. Furthermore, activation of this module correlated with activation of STAT1...

  14. Tumor-Suppressive Activity of Lunatic Fringe in Prostate through Differential Modulation of Notch Receptor Activation

    Shubing Zhang

    2014-02-01

    Full Text Available Elevated Notch ligand and receptor expression has been associated with aggressive forms of prostate cancer, suggesting a role for Notch signaling in regulation of prostate tumor initiation and progression. Here, we report a critical role for Lunatic Fringe (Lfng, which encodes an O-fucosylpeptide 3-ß-N-acetylglucosaminyltransferase known to modify epidermal growth factor repeats of Notch receptor proteins, in regulation of prostate epithelial differentiation and proliferation, as well as in prostate tumor suppression. Deletion of Lfng in mice caused altered Notch activation in the prostate, associated with elevated accumulation of Notch1, Notch2, and Notch4 intracellular domains, decreased levels of the putative Notch3 intracellular fragment, as well as increased expression of Hes1, Hes5, and Hey2. Loss of Lfng resulted in expansion of the basal layer, increased proliferation of both luminal and basal cells, and ultimately, prostatic intraepithelial neoplasia. The Lfng-null prostate showed down-regulation of prostatic tumor suppressor gene NKX3.1 and increased androgen receptor expression. Interestingly, expression of LFNG and NKX3.1 were positively correlated in publically available human prostate cancer data sets. Knockdown of LFNG in DU-145 prostate cancer cells led to expansion of CD44+CD24− and CD49f+CD24− stem/progenitor-like cell population associated with enhanced prostatosphere-forming capacity. Taken together, these data revealed a tumor-suppressive role for Lfng in the prostate through differential regulation of Notch signaling.

  15. An angiotensin II type 1 receptor activation switch patch revealed through evolutionary trace analysis

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong;

    2010-01-01

    in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model...... for 7TM receptor activation and signaling. Six mutations: F66A, L67R, L70R, L119R, D125A, and I245F were targeted to the putative switch and assayed for changes in activation state by their ligand binding, signaling, and trafficking properties. All but one receptor mutant (that was not expressed well...

  16. A2A adenosine receptor antagonism enhances synaptic and motor effects of cocaine via CB1 cannabinoid receptor activation.

    Alessandro Tozzi

    Full Text Available BACKGROUND: Cocaine increases the level of endogenous dopamine (DA in the striatum by blocking the DA transporter. Endogenous DA modulates glutamatergic inputs to striatal neurons and this modulation influences motor activity. Since D2 DA and A2A-adenosine receptors (A2A-Rs have antagonistic effects on striatal neurons, drugs targeting adenosine receptors such as caffeine-like compounds, could enhance psychomotor stimulant effects of cocaine. In this study, we analyzed the electrophysiological effects of cocaine and A2A-Rs antagonists in striatal slices and the motor effects produced by this pharmacological modulation in rodents. PRINCIPAL FINDINGS: Concomitant administration of cocaine and A2A-Rs antagonists reduced glutamatergic synaptic transmission in striatal spiny neurons while these drugs failed to produce this effect when given in isolation. This inhibitory effect was dependent on the activation of D2-like receptors and the release of endocannabinoids since it was prevented by L-sulpiride and reduced by a CB1 receptor antagonist. Combined application of cocaine and A2A-R antagonists also reduced the firing frequency of striatal cholinergic interneurons suggesting that changes in cholinergic tone might contribute to this synaptic modulation. Finally, A2A-Rs antagonists, in the presence of a sub-threshold dose of cocaine, enhanced locomotion and, in line with the electrophysiological experiments, this enhanced activity required activation of D2-like and CB1 receptors. CONCLUSIONS: The present study provides a possible synaptic mechanism explaining how caffeine-like compounds could enhance psychomotor stimulant effects of cocaine.

  17. Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction.

    Varty, LoriAnn M; Gustafson, Eric; Laverty, Maureen; Hey, John A

    2004-01-19

    The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa. Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from

  18. Activation of peroxisome proliferator-activated receptor alpha in rat spinal cord after peripheral noxious stimulation.

    Benani, A; Heurtaux, T; Netter, P; Minn, A

    2004-10-07

    Following recurrent noxious stimulation, both functional modification and structural reorganization such as activation of the arachidonate cascade or axon sprouting occur in the central nervous system (CNS). It has been recently proposed that these alterations observed during chronic pain state were supported by an intensification of the lipid metabolism. In this regard, it has been shown that mRNA coding for several fatty acid metabolizing enzymes are up-regulated in the rat lumbar spinal cord in response to persistent nociception induced by a peripheral inflammation. As peroxisome proliferators-activated receptor (PPAR) could mediate such effects, we therefore investigated the activation of this transcription factor in the rat spinal cord following subcutaneous injection of complete Freund's adjuvant (CFA) into a hind paw. In this study, we compared the DNA-binding activity of nuclear proteins extracted from healthy and inflamed rats toward a PPAR response element. Using electrophoretic mobility-shift assay (EMSA), we found that only the PPARalpha isoform was activated in the rat spinal cord after CFA injection. This activation occurred rapidly, as early as 30 min post-CFA injection, and was persistent up to 10 h, reaching a maximum at 6h after CFA injection. In view of the consequences of PPARalpha activation in other tissues, these results suggest that fatty acid utilization is enhanced in the CNS during chronic pain state. Although the physiopathological relevance of PPARalpha activation during hyperalgesia needs further investigation, we provided here a new player in the molecular modeling of pain pathways.

  19. Different efficacy of adenosine and NECA derivatives at the human A3 adenosine receptor: insight into the receptor activation switch.

    Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Kachler, Sonja; Falgner, Nico; Marucci, Gabriella; Thomas, Ajiroghene; Cristalli, Gloria; Volpini, Rosaria; Klotz, Karl-Norbert

    2014-01-15

    A3 Adenosine receptors are promising drug targets for a number of diseases and intense efforts are dedicated to develop selective agonists and antagonists of these receptors. A series of adenosine derivatives with 2-(ar)-alkynyl chains, with high affinity and different degrees of selectivity for human A3 adenosine receptors was tested for the ability to inhibit forskolin-stimulated adenylyl cyclase. All these derivatives are partial agonists at A3 adenosine receptors; their efficacy is not significantly modified by the introduction of small alkyl substituents in the N(6)-position. In contrast, the adenosine-5'-N-ethyluronamide (NECA) analogs of 2-(ar)-alkynyladenosine derivatives are full A3 agonists. Molecular modeling analyses were performed considering both the conformational behavior of the ligands and the impact of 2- and 5'-substituents on ligand-target interaction. The results suggest an explanation for the different agonistic behavior of adenosine and NECA derivatives, respectively. A sub-pocket of the binding site was analyzed as a crucial interaction domain for receptor activation.

  20. Enhancement of CA3 hippocampal network activity by activation of group II metabotropic glutamate receptors.

    Ster, Jeanne; Mateos, José María; Grewe, Benjamin Friedrich; Coiret, Guyllaume; Corti, Corrado; Corsi, Mauro; Helmchen, Fritjof; Gerber, Urs

    2011-06-14

    Impaired function or expression of group II metabotropic glutamate receptors (mGluRIIs) is observed in brain disorders such as schizophrenia. This class of receptor is thought to modulate activity of neuronal circuits primarily by inhibiting neurotransmitter release. Here, we characterize a postsynaptic excitatory response mediated by somato-dendritic mGluRIIs in hippocampal CA3 pyramidal cells and in stratum oriens interneurons. The specific mGluRII agonists DCG-IV or LCCG-1 induced an inward current blocked by the mGluRII antagonist LY341495. Experiments with transgenic mice revealed a significant reduction of the inward current in mGluR3(-/-) but not in mGluR2(-/-) mice. The excitatory response was associated with periods of synchronized activity at theta frequency. Furthermore, cholinergically induced network oscillations exhibited decreased frequency when mGluRIIs were blocked. Thus, our data indicate that hippocampal responses are modulated not only by presynaptic mGluRIIs that reduce glutamate release but also by postsynaptic mGluRIIs that depolarize neurons and enhance CA3 network activity.

  1. Kaempferol inhibits cancer cell growth by antagonizing estrogen-related receptor α and γ activities.

    Wang, Haibin; Gao, Minghui; Wang, Junjian

    2013-11-01

    Kaempferol is a dietary flavonoid that can function as a selective estrogen receptor modulator (SERM). Estrogen-related receptors alpha and gamma (ERRα and ERRγ) are orphan nuclear receptors that play important roles in mitochondrial biogenesis and cancer development. We have shown that kaempferol can functionally antagonize the activities of ERRs based on both response element reporter systems and target gene analysis. Kaempferol modulation of mitochondrial function and suppression cancer cell growth has been confirmed. These findings suggest that kaempferol may exert their anti-cancer activities through antagonizing ERRs activities.

  2. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D;

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...

  3. Allosteric regulation of G protein-coupled receptor activity by phospholipids.

    Dawaliby, Rosie; Trubbia, Cataldo; Delporte, Cédric; Masureel, Matthieu; Van Antwerpen, Pierre; Kobilka, Brian K; Govaerts, Cédric

    2016-01-01

    Lipids are emerging as key regulators of membrane protein structure and activity. These effects can be attributed either to the modification of bilayer properties (thickness, curvature and surface tension) or to the binding of specific lipids to the protein surface. For G protein-coupled receptors (GPCRs), the effects of phospholipids on receptor structure and activity remain poorly understood. Here we reconstituted purified β2-adrenergic receptor (β2R) in high-density lipoparticles to systematically characterize the effect of biologically relevant phospholipids on receptor activity. We observed that the lipid headgroup type affected ligand binding (agonist and antagonist) and receptor activation. Specifically, phosphatidylgycerol markedly favored agonist binding and facilitated receptor activation, whereas phosphatidylethanolamine favored antagonist binding and stabilized the inactive state of the receptor. We then showed that these effects could be recapitulated with detergent-solubilized lipids, demonstrating that the functional modulation occurred in the absence of a bilayer. Our data suggest that phospholipids act as direct allosteric modulators of GPCR activity.

  4. DMPD: Proximal effects of Toll-like receptor activation in dendritic cells. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 17142025 Proximal effects of Toll-like receptor activation in dendritic cells. Watt...) (.svg) (.html) (.csml) Show Proximal effects of Toll-like receptor activation in dendritic cells. PubmedID... 17142025 Title Proximal effects of Toll-like receptor activation in dendritic ce

  5. Discoidin domain receptor 1 is activated independently of beta(1) integrin

    Vogel, W; Brakebusch, C; Fässler, R

    2000-01-01

    Various types of collagen have been identified as potential ligands for the two mammalian discoidin domain receptor (DDR) tyrosine kinases, DDR1 and DDR2. It is presently unclear whether collagen-induced DDR receptor activation, which occurs with very slow kinetics, involves additional proteins...... with kinase activity or membrane-anchored proteins serving as coreceptors. In particular, the role of the collagen-binding integrins alpha(1)beta(1) or alpha(2)beta(1) in the DDR activation process is undefined. Here, we provide three lines of evidence suggesting that DDR1 signaling is distinct from integrin...... activation. First we demonstrate that the enzymatic activity of DDR1 is essential for receptor tyrosine phosphorylation. Collagen-induced DDR receptor autophosphorylation can be blocked either by a dominant negative mutant or by a preparation of recombinant extracellular domain. Second, we show DDR1 signals...

  6. Allosteric activation of membrane-bound glutamate receptors using coordination chemistry within living cells

    Kiyonaka, Shigeki; Kubota, Ryou; Michibata, Yukiko; Sakakura, Masayoshi; Takahashi, Hideo; Numata, Tomohiro; Inoue, Ryuji; Yuzaki, Michisuke; Hamachi, Itaru

    2016-10-01

    The controlled activation of proteins in living cells is an important goal in protein-design research, but to introduce an artificial activation switch into membrane proteins through rational design is a significant challenge because of the structural and functional complexity of such proteins. Here we report the allosteric activation of two types of membrane-bound neurotransmitter receptors, the ion-channel type and the G-protein-coupled glutamate receptors, using coordination chemistry in living cells. The high programmability of coordination chemistry enabled two His mutations, which act as an artificial allosteric site, to be semirationally incorporated in the vicinity of the ligand-binding pockets. Binding of Pd(2,2‧-bipyridine) at the allosteric site enabled the active conformations of the glutamate receptors to be stabilized. Using this approach, we were able to activate selectively a mutant glutamate receptor in live neurons, which initiated a subsequent signal-transduction pathway.

  7. Transcriptional integration of metabolism by the nuclear sterol-activated receptors LXR and FXR.

    Calkin, Anna C; Tontonoz, Peter

    2012-03-14

    Nuclear receptors are integrators of hormonal and nutritional signals, mediating changes to metabolic pathways within the body. Given that modulation of lipid and glucose metabolism has been linked to diseases including type 2 diabetes, obesity and atherosclerosis, a greater understanding of pathways that regulate metabolism in physiology and disease is crucial. The liver X receptors (LXRs) and the farnesoid X receptors (FXRs) are activated by oxysterols and bile acids, respectively. Mounting evidence indicates that these nuclear receptors have essential roles, not only in the regulation of cholesterol and bile acid metabolism but also in the integration of sterol, fatty acid and glucose metabolism.

  8. Maturational alterations in constitutive activity of medial prefrontal cortex kappa-opioid receptors in Wistar rats.

    Sirohi, Sunil; Walker, Brendan M

    2015-11-01

    Opioid receptors can display spontaneous agonist-independent G-protein signaling (basal signaling/constitutive activity). While constitutive κ-opioid receptor (KOR) activity has been documented in vitro, it remains unknown if KORs are constitutively active in native systems. Using [(35) S] guanosine 5'-O-[gamma-thio] triphosphate coupling assay that measures receptor functional state, we identified the presence of medial prefrontal cortex KOR constitutive activity in young rats that declined with age. Furthermore, basal signaling showed an age-related decline and was insensitive to neutral opioid antagonist challenge. Collectively, the present data are first to demonstrate age-dependent alterations in the medial prefrontal cortex KOR constitutive activity in rats and changes in the constitutive activity of KORs can differentially impact KOR ligand efficacy. These data provide novel insights into the functional properties of the KOR system and warrant further consideration of KOR constitutive activity in normal and pathophysiological behavior. Opioid receptors exhibit agonist-independent constitutive activity; however, kappa-opioid receptor (KOR) constitutive activity has not been demonstrated in native systems. Our results confirm KOR constitutive activity in the medial prefrontal cortex (mPFC) that declines with age. With the ability to presynaptically inhibit multiple neurotransmitter systems in the mPFC, maturational or patho-logical alterations in constitutive activity could disrupt corticofugal glutamatergic pyramidal projection neurons mediating executive function. Regulation of KOR constitutive activity could serve as a therapeutic target to treat compromised executive function.

  9. Activation, internalization, and recycling of the serotonin 2A receptor by dopamine

    Bhattacharyya, Samarjit; Raote, Ishier; Bhattacharya, Aditi; Miledi, Ricardo; Panicker, Mitradas M.

    2006-01-01

    Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or “primed” by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations ≈10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders. PMID:17005723

  10. Activated receptors for peroxisomic proliferators. Its role in the atherosclerosis, obesity and high blood pressure.

    Mikhail Benet Rodríguez

    2004-08-01

    Full Text Available The receptors activated by peroxisome proliferators are a family of factors of transcription that belong to the superfamily of the steroid receptors and include tree subtypes which are PPARá, PPAR©¬ and PPAR ã. These receptors join to direct hexameric repetitions in the form of heterodimers with the retinoid receptor. PPAR receptors regulate the expressions of a great variety of genes that codify the proteins that are implied in the lipid metabolism, the energetic homeostasis, the cellular differentiation and the formation of tumours. This review describes the features, regulation and target genes of the PPAR receptor and the physiopathological and pharmacological implications of the regulation of the lipid and glucose metabolism, the energetic homeostasis ,hypertension and endothelial dysfunction.

  11. In vitro translation of androgen receptor cRNA results in an activated androgen receptor protein

    G.G.J.M. Kuiper (George); P.E. de Ruiter (Petra); J. Trapman (Jan); G.W. Jenster (Guido); A.O. Brinkmann (Albert)

    1993-01-01

    textabstractTranslation of androgen receptor (AR) cRNA in a reticulocyte lysate and subsequent analysis of the translation products by SDS/PAGE showed a protein with an apparent molecular mass of 108 kDa. Scatchard-plot analysis revealed a single binding component with

  12. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels;

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  13. Transcriptional activation by the thyroid hormone receptor through ligand-dependent receptor recruitment and chromatin remodelling

    Grøntved, Lars; Waterfall, Joshua J; Kim, Dong Wook;

    2015-01-01

    A bimodal switch model is widely used to describe transcriptional regulation by the thyroid hormone receptor (TR). In this model, the unliganded TR forms stable, chromatin-bound complexes with transcriptional co-repressors to repress transcription. Binding of hormone dissociates co-repressors and...

  14. Research Resource: Androgen Receptor Activity Is Regulated Through the Mobilization of Cell Surface Receptor Networks.

    Hsiao, Jordy J; Ng, Brandon H; Smits, Melinda M; Martinez, Harryl D; Jasavala, Rohini J; Hinkson, Izumi V; Fermin, Damian; Eng, Jimmy K; Nesvizhskii, Alexey I; Wright, Michael E

    2015-08-01

    The aberrant expression of androgen receptor (AR)-dependent transcriptional programs is a defining pathology of the development and progression of prostate cancers. Transcriptional cofactors that bind AR are critical determinants of prostate tumorigenesis. To gain a deeper understanding of the proteins linked to AR-dependent gene transcription, we performed a DNA-affinity chromatography-based proteomic screen designed to identify proteins involved in AR-mediated gene transcription in prostate tumor cells. Functional experiments validated the coregulator roles of known AR-binding proteins in AR-mediated transcription in prostate tumor cells. More importantly, novel coregulatory functions were detected in components of well-established cell surface receptor-dependent signal transduction pathways. Further experimentation demonstrated that components of the TNF, TGF-β, IL receptor, and epidermal growth factor signaling pathways modulated AR-dependent gene transcription and androgen-dependent proliferation in prostate tumor cells. Collectively, our proteomic dataset demonstrates that the cell surface receptor- and AR-dependent pathways are highly integrated, and provides a molecular framework for understanding how disparate signal-transduction pathways can influence AR-dependent transcriptional programs linked to the development and progression of human prostate cancers.

  15. Activity-induced and developmental downregulation of the Nogo receptor

    Josephson, Anna; Trifunovski, Alexandra; Schéele, Camilla;

    2003-01-01

    The three axon growth inhibitory proteins, myelin associated glycoprotein, oligodendrocyte-myelin glycoprotein and Nogo-A, can all bind to the Nogo-66 receptor (NgR). This receptor is expressed by neurons with high amounts in regions of high plasticity where Nogo expression is also high. We...... hypothesized that simultaneous presence of high levels of Nogo and its receptor in neurons confers a locked state to hippocampal and cortical microcircuitry and that one or both of these proteins must be effectively and temporarily downregulated to permit plastic structural changes underlying formation of long...... that NgR levels in developing and adult neurons are regulated in vivo under different conditions. Strong, rapid and transient downregulation of NgR mRNA in response to kainic acid and after wheel running in cortex and hippocampus suggests a role for NgR and Nogo-A in plasticity, learning and memory....

  16. GABA-B receptor activation and conflict behavior

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.; Bruinvels, J.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render it unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.

  17. G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?

    Roberts, David J; Waelbroeck, Magali

    2004-09-01

    G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymatic model of G protein activation and compares its predictions with the ternary complex model and with observed receptor phenomenon. This alternative model does not merely provide a new set of formulae but leads to a new philosophical outlook and more readily accommodates experimental observations. The ternary complex model implies that, HRG being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).

  18. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways.

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P; Monczor, Federico

    2015-12-04

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration.

  19. Effects of histamine H1 receptor signaling on glucocorticoid receptor activity. Role of canonical and non-canonical pathways

    Zappia, Carlos Daniel; Granja-Galeano, Gina; Fernández, Natalia; Shayo, Carina; Davio, Carlos; Fitzsimons, Carlos P.; Monczor, Federico

    2015-01-01

    Histamine H1 receptor (H1R) antagonists and glucocorticoid receptor (GR) agonists are used to treat inflammatory conditions such as allergic rhinitis, atopic dermatitis and asthma. Consistent with the high morbidity levels of such inflammatory conditions, these receptors are the targets of a vast number of approved drugs, and in many situations their ligands are co-administered. However, this drug association has no clear rationale and has arisen from clinical practice. We hypothesized that H1R signaling could affect GR-mediated activity, impacting on its transcriptional outcome. Indeed, our results show a dual regulation of GR activity by the H1R: a potentiation mediated by G-protein βγ subunits and a parallel inhibitory effect mediated by Gαq-PLC pathway. Activation of the H1R by its full agonists resulted in a composite potentiating effect. Intriguingly, inactivation of the Gαq-PLC pathway by H1R inverse agonists resulted also in a potentiation of GR activity. Moreover, histamine and clinically relevant antihistamines synergized with the GR agonist dexamethasone to induce gene transactivation and transrepression in a gene-specific manner. Our work provides a delineation of molecular mechanisms underlying the widespread clinical association of antihistamines and GR agonists, which may contribute to future dosage optimization and reduction of well-described side effects associated with glucocorticoid administration. PMID:26635083

  20. Peripheral Sensitization Increases Opioid Receptor Expression and Activation by Crotalphine in Rats

    Zambelli, Vanessa Olzon; Fernandes, Ana Carolina de Oliveira; Gutierrez, Vanessa Pacciari; Ferreira, Julio Cesar Batista; Parada, Carlos Amilcar; Mochly-Rosen, Daria; Cury, Yara

    2014-01-01

    Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids. PMID:24594607

  1. Effects related to gene-gene interactions of peroxisome proliferator-activated receptor on essential hypertension

    俞浩

    2013-01-01

    Objective To explore the impact of the gene-gene interaction among the single nucleotide polymorphisms(SNPs) of peroxisome proliferator-activated receptorα/δ/γ on essential hypertension(EH).Methods

  2. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  3. An Angiotensin II type 1 receptor activation switch patch revealed through Evolutionary Trace analysis

    Bonde, Marie Mi; Yao, Rong; Ma, Jian-Nong

    2010-01-01

    in the cytoplasmic parts of TM2, TM3, and TM6 to form an activation switch that is common to all family A 7TM receptors. We tested this hypothesis in the rat Angiotensin II (Ang II) type 1a (AT1a) receptor. The receptor has important roles in the cardiovascular system, but has also frequently been applied as a model......) displayed phenotypes associated with changed activation state, such as increased agonist affinity or basal activity, promiscuous activation, or constitutive internalization highlighting the importance of testing different signaling pathways. We conclude that this evolutionary important patch mediates...... to be completely resolved. Evolutionary Trace (ET) analysis is a computational method, which identifies clusters of functionally important residues by integrating information on evolutionary important residue variations with receptor structure. Combined with known mutational data, ET predicted a patch of residues...

  4. Altered activity profile of a tertiary silanol analog of multi-targeting nuclear receptor modulator T0901317.

    Toyama, Hirozumi; Sato, Shoko; Shirakawa, Hitoshi; Komai, Michio; Hashimoto, Yuichi; Fujii, Shinya

    2016-04-01

    We report the design, synthesis, and physicochemical/biological evaluation of novel silanol derivative 6 (sila-T) as a silanol analog of multi-target nuclear receptor modulator T0901317 (5). Compound 6 showed intermediate hydrophobicity between the corresponding alcohol 13 and perfluoroalcohol 5. While 5 exhibited potent activities toward liver X receptor α and β, farnesoid X receptor, pregnane X receptor (PXR) and retinoic acid receptor-related orphan receptor (ROR)γ, silanol 6 exhibited activity only toward PXR and RORs. Incorporation of silanol instead of perfluoroalcohol is a promising option for developing novel target-selective, biologically active compounds.

  5. Identification of (beta-carboxyethyl)-rhodanine derivatives exhibiting peroxisome proliferator-activated receptor gamma activity.

    Choi, Jiwon; Ko, Yoonae; Lee, Hui Sun; Park, Yun Sun; Yang, Young; Yoon, Sukjoon

    2010-01-01

    We applied an improved virtual screening scheme combining ligand-centric and receptor-centric methods for the identification of a new series of PPARgamma agonists known as (beta-carboxyethyl)-rhodanine derivatives which include a thiazolidin-based core structure, 2-thioxo-thiazolidine-4-one. An in vitro assay confirmed the nanomolar binding affinity in one of the (beta-carboxyethyl)-rhodanine derivatives, SP1818. It showed a PPARgamma agonistic activity similar to that of a known PPARgamma drug, pioglitazone, in a cell-based transactivation assay. Furthermore, the structure-activity relationships of the rhodanine derivatives were investigated through comparative molecular field analysis. We also characterized the inconsistency between the in vitro binding affinity and cell-based transactivation ability by using a set of property-based molecular descriptors. The binding mode analysis provided new insight concerning their agonistic effect on PPARgamma.

  6. Structural and functional characterization of alternative transmembrane domain conformations in VEGF receptor 2 activation.

    Manni, Sandro; Mineev, Konstantin S; Usmanova, Dinara; Lyukmanova, Ekaterina N; Shulepko, Mikhail A; Kirpichnikov, Mikhail P; Winter, Jonas; Matkovic, Milos; Deupi, Xavier; Arseniev, Alexander S; Ballmer-Hofer, Kurt

    2014-08-05

    Transmembrane signaling by receptor tyrosine kinases (RTKs) entails ligand-mediated dimerization and structural rearrangement of the extracellular domains. RTK activation also depends on the specific orientation of the transmembrane domain (TMD) helices, as suggested by pathogenic, constitutively active RTK mutants. Such mutant TMDs carry polar amino acids promoting stable transmembrane helix dimerization, which is essential for kinase activation. We investigated the effect of polar amino acids introduced into the TMD of vascular endothelial growth factor receptor 2, regulating blood vessel homeostasis. Two mutants showed constitutive kinase activity, suggesting that precise TMD orientation is mandatory for kinase activation. Nuclear magnetic resonance spectroscopy revealed that TMD helices in activated constructs were rotated by 180° relative to the interface of the wild-type conformation, confirming that ligand-mediated receptor activation indeed results from transmembrane helix rearrangement. A molecular dynamics simulation confirmed the transmembrane helix arrangement of wild-type and mutant TMDs revealed by nuclear magnetic resonance spectroscopy.

  7. The phosphoproteome of toll-like receptor-activated macrophages

    Weintz, Gabriele; Olsen, Jesper Velgaard; Frühauf, Katja;

    2010-01-01

    Recognition of microbial danger signals by toll-like receptors (TLR) causes re-programming of macrophages. To investigate kinase cascades triggered by the TLR4 ligand lipopolysaccharide (LPS) on systems level, we performed a global, quantitative and kinetic analysis of the phosphoproteome...

  8. Environmental polycyclic aromatic hydrocarbons affect androgen receptor activation in vitro

    Vinggaard, Anne Marie; Hnida, Christina; Larsen, John Christian

    2000-01-01

    Nine structurally different polycyclic aromatic hydrocarbons (PAHs) were tested for their ability to either agonize or antagonize the human androgen receptor (hAR) in a sensitive reporter gene assay based on CHO cells transiently cotransfected with a hAR vector and an MMTV-LUC vector. Benz...

  9. Multiple activities of insect repellents on odorant receptors in mosquitoes

    Several lines of evidence suggest that insect repellent molecules reduce mosquito-host contacts by interacting with odorants and odorant receptors (ORs) ultimately affecting olfactory-driven behaviors. We describe the molecular effects of ten insect repellents and a pyrethroid insecticide with known...

  10. Obstructing Androgen Receptor Activation in Prostate Cancer Cells Through Post-translational Modification by NEDD8

    2012-11-01

    Nonidet P - 40 ). Pre-cleared with 25 µl protein-A/G agarose beads, 700 µg lysate was subjected to immunoprecipitation with 20 µl anti-FLAG antibody...cell growth. REPORTABLE OUTCOMES: 1. Chang, K. H., Hsiao, P .-W. and Chen, J. D. Modulation of Androgen Receptor Activity by Reversible NEDD8...Hsiao, P .-W. and Chen, J. D. Modulation of Androgen Receptor Activity by Reversible NEDD8 Modification. (under revision) 1 Modulation of

  11. Estradiol increases the anorexia associated with increased 5-HT2C receptor activation in ovariectomized rats

    Rivera, Heidi M.; Santollo, Jessica; Nikonova, Larissa V.; Eckel, Lisa A.

    2011-01-01

    Estradiol’s inhibitory effect on food intake is mediated, in part, by its ability to increase the activity of meal-related signals, including serotonin (5-HT), which hasten satiation. The important role that postsynaptic 5-HT2C receptors play in mediating 5-HT’s anorexigenic effect prompted us to investigate whether a regimen of acute estradiol treatment increases the anorexia associated with increased 5-HT2C receptor activation in ovariectomized (OVX) rats. We demonstrated that intraperitone...

  12. Regulation of breast cancer stem cell activity by signaling through the Notch4 receptor

    Harrison, Hannah; Farnie, Gillian; Howell, Sacha J.; Rock, Rebecca E; Stylianou, Spyros; Brennan, Keith R.; Bundred, Nigel J; Clarke, Robert B.

    2010-01-01

    Notch receptor signaling pathways play an important role not only in normal breast development but also in breast cancer development and progression. We assessed the role of Notch receptors in stem cell activity in breast cancer cell lines and nine primary human tumor samples. Stem cells were enriched by selection of anoikis-resistant cells or cells expressing the membrane phenotype ESA+/CD44+/CD24low. Using these breast cancer stem cell populations, we compared the activation status of Notch...

  13. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2014-01-15

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

  14. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

    2003-07-01

    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  15. EPO-independent functional EPO receptor in breast cancer enhances estrogen receptor activity and promotes cell proliferation

    Reinbothe, Susann; Larsson, Anna-Maria; Vaapil, Marica; Wigerup, Caroline [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Sun, Jianmin [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Jögi, Annika [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Neumann, Drorit [Department of Cell and Developmental Biology, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Rönnstrand, Lars [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); Påhlman, Sven, E-mail: sven.pahlman@med.lu.se [Department of Laboratory Medicine, Translational Cancer Research, Medicon Village, Lund University, SE-223 81 Lund (Sweden); CREATE Health, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-02-28

    Highlights: • New anti-human EPOR antibody confirms full-length EPOR expression in breast cancer cells. • Proliferation of breast cancer cells is not affected by rhEPO treatment in vitro. • EPOR knockdown impairs proliferation of ERa positive breast cancer cells. • EPOR knockdown reduces AKT phosphorylation and ERa activity. - Abstract: The main function of Erythropoietin (EPO) and its receptor (EPOR) is the stimulation of erythropoiesis. Recombinant human EPO (rhEPO) is therefore used to treat anemia in cancer patients. However, clinical trials have indicated that rhEPO treatment might promote tumor progression and has a negative effect on patient survival. In addition, EPOR expression has been detected in several cancer forms. Using a newly produced anti-EPOR antibody that reliably detects the full-length isoform of the EPOR we show that breast cancer tissue and cells express the EPOR protein. rhEPO stimulation of cultured EPOR expressing breast cancer cells did not result in increased proliferation, overt activation of EPOR (receptor phosphorylation) or a consistent activation of canonical EPOR signaling pathway mediators such as JAK2, STAT3, STAT5, or AKT. However, EPOR knockdown experiments suggested functional EPO receptors in estrogen receptor positive (ERα{sup +}) breast cancer cells, as reduced EPOR expression resulted in decreased proliferation. This effect on proliferation was not seen in ERα negative cells. EPOR knockdown decreased ERα activity further supports a mechanism by which EPOR affects proliferation via ERα-mediated mechanisms. We show that EPOR protein is expressed in breast cancer cells, where it appears to promote proliferation by an EPO-independent mechanism in ERα expressing breast cancer cells.

  16. Soluble Urokinase Receptors in Focal Segmental Glomerulosclerosis: A Review on the Scientific Point of View

    Andreas Kronbichler

    2016-01-01

    Full Text Available Focal segmental glomerulosclerosis (FSGS is one of the primary glomerular disorders in both children and adults which can progress to end-stage renal failure. Although there are genetic and secondary causes, circulating factors have also been regarded as an important factor in the pathogenesis of FSGS, because about 40% of the patients with FSGS have recurrence after renal transplantation. Soluble urokinase-type plasminogen activator receptor (suPAR is a soluble form of uPAR, which is a membrane-bound protein linked to GPI in various immunologically active cells, including podocytes. It has recently been suggested as a potential circulating factor in FSGS by in vitro podocyte experiments, in vivo mice models, and human studies. However, there have also been controversies on this issue, because subsequent studies showed conflicting results. suPAR levels were also increased in patients with other glomerular diseases and were inversely correlated with estimated glomerular filtration rate. Nevertheless, there has been no balanced review on this issue. In this review, we compare the conflicting data on the involvement of suPAR in the pathogenesis of FSGS and shed light on interpretation by taking into account many points and the potential variables and confounders influencing serum suPAR levels.

  17. In silico analysis of the histaprodifen induced activation pathway of the guinea-pig histamine H1-receptor

    Straßer, Andrea; Wittmann, Hans-Joachim

    2010-09-01

    The binding of (partial) agonists in the binding pocket of biogenic amine receptors induces a conformational change from the inactive to the active state of the receptors. There is only little knowledge about the binding pathways of ligands into binding pocket on molecular level. So far, it was not possible with molecular dynamic simulations to observe the ligand binding and receptor activation. Furthermore, there is nearly nothing known, in which state of ligand binding, the receptor gets activated. The aim of this study was to get more detailed insight into the process of ligand binding and receptor activation. With the recently developed LigPath algorithm, we scanned the potential energy surface of the binding process of dimeric histaprodifen, a partial agonist at the histamine H1-receptor, into the guinea pig histamine H1-receptor, taking also into account the receptor activation. The calculations exhibited large conformational changes of Trp6.48 and Phe6.55 during ligand binding and receptor activation. Additionally, conformational changes were also observed for Phe6.52, Tyr6.51 and Phe6.44. Conformational changes of Trp6.48 and Phe6.52 are discussed in literature as rotamer toggle switch in context with receptor activation. Additionally, the calculations indicate that the binding of dimeric histaprodifen, accompanied by receptor activation is energetically preferred. In general, this study gives new, theoretical insights onto ligand binding and receptor activation on molecular level.

  18. [Dependence of EGF receptor and STAT factor activation on redox of A431 cells].

    Gonchar, I V; Burova, E B; Dorosh, V N; Gamaleĭ, I A; Nikol'skiĭ, N N

    2003-01-01

    Reactive oxygen species (ROS) were established to play an important role in cellular signaling as second messengers by integrating different pathways. Recently, we showed that EGF initiated a rapid tyrosine phosphorylation of both EGF-receptor and STAT factors with simultaneous increase in the intracellular ROS level. Now, we have investigated the effect of intracellular red-ox state on EGF- and H2O2-induced activation of EGF receptor, STAT1 and STAT3. We demonstrated that the pretreatment of A431 cells with antioxidant N-acetyl-L-cysteine (NAC) partly reduced the level of EGF-induced phosphorylation of proteins under investigation. Besides, H2O2-induced activation of EGF receptor, and STAT factors was fully prevented by NAC pretreatment. The inhibition of ROS generation by DPI declined EGF-dependent activation of EGF receptor and STAT factors to basal level. Our results demonstrate the essential role of cellular red-ox status in the modulation of EGF-mediated activation of receptor and STAT factors. We have postulated that EGF-induced ROS generation is a very important initial event promoting physiological activation of EGF receptor and subsequent STAT factor activation.

  19. Peroxisome proliferator-activated receptor subtype- and cell-type-specific activation of genomic target genes upon adenoviral transgene delivery

    Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G

    2006-01-01

    Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral...... delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes...

  20. Metastin receptor is overexpressed in papillary thyroid cancer and activates MAP kinase in thyroid cancer cells.

    Ringel, Matthew D; Hardy, Elena; Bernet, Victor J; Burch, Henry B; Schuppert, Frank; Burman, Kenneth D; Saji, Motoyasu

    2002-05-01

    The development of distant metastasis is the most important predictor of death from thyroid cancer. KiSS-1 is a recently cloned human metastasis suppressor gene whose product, metastin, was recently identified as the endogenous agonist for a novel Gq/11 coupled receptor (metastin receptor). The expression and functional consequences of metastin and the metastin receptor have not been evaluated in thyroid cancer. We measured metastin and metastin receptor mRNA levels in 10 FCs and 13 papillary carcinomas (PCs), 2 benign non-functioning follicular adenomas (FAs), and 11 normal thyroid samples, and evaluated the signaling pathways activated by metastin in ARO thyroid cancer cells that express the metastin receptor endogenously. Paired normal and tumor samples were available for 4 PC and 3 PFC samples. Metastin mRNA was detected in 6/11 normal samples, and 0/2 FA, 2/10 FC, and 9/13 PC samples (p Metastin receptor was not expressed in any normal thyroid or benign FA samples, and was expressed in only a minority (2/10) of FC samples. However, the receptor was expressed in the majority (10/13) of PCs (p = 0.002 for PC vs. normal tissue). Increased levels of metastin receptor were detected in all four PCs compared to adjacent normal tissue. Incubation levels of metastin receptor were detected in all four PCs compared to adjacent normal tissue. Incubation of metastin receptor expressing ARO thyroid cancer cells with metastin resulted in activation of ERK, but not Akt. Taken together, these data suggest a potential role for metastin and/or metastin receptors in modulating the biological behavior of thyroid cancers.

  1. The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, does not require functional leptin receptor, serotonin, and hypothalamic POMC and CART activities in mice.

    Nonogaki, Katsunori; Kaji, Takao

    2016-10-01

    The acute anorexic effect of liraglutide, a GLP-1 receptor agonist, did not require functional leptin receptor, serotonin, and hypothalamic proopiomelanocortin and cocaine amphetamine regulated transcript activities in mice, although decrease in functional hypothalamic orexin activity might be involved in the acute anorexic effect of liraglutide.

  2. Rapid effect of stress concentration corticosterone on glutamate receptor and its subtype NMDA receptor activity in cultured hippocampal neurons

    刘玲; 孙继虎; 王春安

    2003-01-01

    Objective:To study the rapid effect of glucocorticoids(GCs)on NMDA receptor activity in hippocampal neurons in stress and to elucidate its underlying probable membrane mechanisms.Methods:Whole-cell patch-clamp recording was used to assess the effect of stress concentration corticosterone(B)on the responses of cultured hippocampal neurons to glutamate and NMDA(N-methy-D-asparatic acid).To make clear the target of B,intracellular dialysis of B(10 μ mol/L)through patch pipette and extracellular application of bovine serum albumin-conjugated corticosterone(B-BSA,10 μmol/L)were carried out to observe their influence on peak amplitude of NMDA-evoked current.Results:B had a rapid,reversible and inhibitory effect on peak amplitude of GLU- or NMDA-evoked current in cultured hippocampal neurons.Furthermore,B-BSA had the inhibitory effect on INMDA as that of B,but intracellularly dialyzed B had no significant effect on INMDA.Conclusion:These results suggest that under the condition of stress,GCs may rapidly,negatively regulate excitatory synaptic receptors-glutamate receptors(GluRs),especially NMDA receptor(NMDAR)in central nervous system,which is mediated by rapid membrane mechanisms,but not by classical,genomic mechanisms.

  3. Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

    Hikichi, Yukiko; Yamaoka, Masuo; Kusaka, Masami; Hara, Takahito

    2015-10-15

    Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity.

  4. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  5. Impaired effect of activation of rat hippocampal 5-HT7 receptors, induced by treatment with the 5-HT7 receptor antagonist SB 269970.

    Kusek, M; Sowa, J; Tokarski, K; Hess, G

    2015-04-01

    Effects of the 5-HT(7) receptor antagonist SB 269970, administered for 14 days (1.25 mg/kg), were studied in ex vivo slices of rat hippocampus. To activate the 5-HT(7) receptor, 5-carboxamidotryptamine (5-CT, 200 nM) was applied in the presence of WAY 100635 (2 μM), a 5-HT(1A) receptor antagonist. In contrast to control preparations, no 5-HT(7) receptor-mediated increase in excitability nor depolarization and an increase in the input resistance of CA1 and CA3 pyramidal neurons were present in slices prepared from rats treated with SB 269970. The treatment also abolished the stimulatory effect of 5-HT(7) receptor activation on spontaneous excitatory postsynaptic currents recorded from CA1 stratum radiatum/lacunosum-moleculare interneurons. These data demonstrate that repeated administration of SB 269970 impairs the reactivity of the CA1 hippocampal neuronal network to 5-HT(7) receptor activation.

  6. The liberated domain I of urokinase plasminogen activator receptor - a new tumour marker in small cell lung cancer

    Almasi, Charlotte E; Drivsholm, Lars; Pappot, Helle;

    2013-01-01

    measured using time-resolved fluorescence immunoassays (TR-FIA 1-3). Assessment of association of the uPAR forms to overall survival (OS) was done using Cox regression analysis adjusted for clinical covariates [age, gender, stage, lactate dehydrogenase (LDH), WHO performance status (PS)]. Multivariate...... the first tertile. High serum uPAR(I) levels are associated with short OS in SCLC patient, independent of LDH and PS....

  7. Inhibition of migration and invasion of carcinoma cells by urokinase-derived antagonists of alphavbeta5 integrin activation.

    Vocca, Immacolata; Franco, Paola; Alfano, Daniela; Votta, Giuseppina; Carriero, Maria Vincenza; Estrada, Yeriel; Caputi, Mario; Netti, Paolo A; Ossowski, Liliana; Stoppelli, Maria Patrizia

    2009-01-15

    We previously showed that, while binding to urokinase receptor (uPAR) through its growth factor domain (GFD, residues 1-49), urokinase (uPA) can engage alphavbeta5 integrin through an internal domain (CP, residues 132-158). This novel uPA/alphavbeta5 interaction promotes cytoskeletal rearrangements and directional cell migration (Franco et al., J Cell Sci 2006;119:3424-34). We now show that treatment of cells with phosphomimic uPA (uPA138E/303E, serine 138 and 303 substituted with glutamic acid) strongly inhibits matrix-induced cell migration. Unlike uPA, binding of uPA138E/303E to cell surface did not induce F-actin enriched protruding structures and caused a 5-fold reduction in cell translocation speed, as determined by video tracking of living cells. Inhibition of migration was found to be independent of uPAR, since uPA variants lacking the GFD domain, but carrying the relevant Ser to Glu substitutions were as effective inhibitor as uPA138E/303E. Through several independent approaches, we established that the phosphomimics specifically bind to alphavbeta5 integrin through the CP region carrying the S138E mutation. This interaction blocks integrin activation, as determined by a decreased affinity of alphavbeta5 to vitronectin and a reduced association of the beta5 cytoplasmic tail with talin. Finally, stable expression of uPA138E/303E in human squamous carcinoma cells prevented tumor cell invasion in vivo. Thus, when expressed in cancer cells, the inhibitory phosphomimic effect was dominant over the effect of endogenously produced uPA. These results shed light on the regulation of cell migration by uPA phosphorylation and provide a realistic opportunity for a novel antiinvasive/metastatic therapeutic intervention.

  8. Peroxisome Proliferator-Activated Receptor γ Activity is Required for Appropriate Cardiomyocyte Differentiation

    Maryam Peymani

    2016-07-01

    Full Text Available Objective Peroxisome proliferator-activated receptor γ (PPARγ is a member of the PPAR nuclear receptor superfamily. Although PPARγ acts as a master transcription factor in adipocyte differentiation, it is also associated with a variety of cell functions including carbohydrate and lipid metabolism, glucose homeostasis, cell proliferation and cell differentiation. This study aimed to assess the expression level of PPARγ in order to address its role in cardiac cell differentiation of mouse embryonic stem cells (mESCs. Materials and Methods In this an intervening study, mESCs were subjected to cardiac differentiation. Total RNA was extracted from the cells and quantitative real time polymerase chain reaction (qPCR was carried out to estimate level of gene expression. Furthermore, the requirement of PPARγ in cardiac differentiation of mESCs, during cardiac progenitor cells (CPCs formation, was examined by applying the respective agonist and antagonist. Results The obtained data revealed an elevation in the expression level of PPARγ during spontaneous formation of CPCs and cardiomyocytes. Our results indicated that during CPC formation, PPARγ inactivation via treatment with GW9662 (GW reduced expression of CPC and cardiac markers. Conclusion We conclude that PPARγ modulation has an effective role on cardiac differentiation of mESCs at the early stage of cardiomyogenesis.

  9. Pharmacological activation of CB1 receptor modulates long term potentiation by interfering with protein synthesis.

    Navakkode, Sheeja; Korte, Martin

    2014-04-01

    Cognitive impairment is one of the most important side effects associated with cannabis drug abuse, as well as the serious issue concerning the therapeutic use of cannabinoids. Cognitive impairments and neuropsychiatric symptoms are caused by early synaptic dysfunctions, such as loss of synaptic connections in different brain structures including the hippocampus, a region that is believed to play an important role in certain forms of learning and memory. We report here that metaplastic priming of synapses with a cannabinoid type 1 receptor (CB1 receptor) agonist, WIN55,212-2 (WIN55), significantly impaired long-term potentiation in the apical dendrites of CA1 pyramidal neurons. Interestingly, the CB1 receptor exerts its effect by altering the balance of protein synthesis machinery towards higher protein production. Therefore the activation of CB1 receptor, prior to strong tetanization, increased the propensity to produce new proteins. In addition, WIN55 priming resulted in the expression of late-LTP in a synaptic input that would have normally expressed early-LTP, thus confirming that WIN55 priming of LTP induces new synthesis of plasticity-related proteins. Furthermore, in addition to the effects on protein translation, WIN55 also induced synaptic deficits due to the ability of CB1 receptors to inhibit the release of acetylcholine, mediated by both muscarinic and nicotinic acetylcholine receptors. Taken together this supports the notion that the modulation of cholinergic activity by CB1 receptor activation is one mechanism that regulates the synthesis of plasticity-related proteins.

  10. Receptor-binding domain of ephrin-A1: production in bacterial expression system and activity.

    Nekrasova, O V; Sharonov, G V; Tikhonov, R V; Kolosov, P M; Astapova, M V; Yakimov, S A; Tagvey, A I; Korchagina, A A; Bocharova, O V; Wulfson, A N; Feofanov, A V; Kirpichnikov, M P

    2012-12-01

    Eph receptor tyrosine kinases and their ligands, the ephrins, perform an important regulatory function in tissue organization, as well as participate in malignant transformation of cells. Ephrin-A1, a ligand of A class Eph receptors, is a modulator of tumor growth and progression, and the mechanism of its action needs detailed investigation. Here we report on the development of a system for bacterial expression of an ephrin-A1 receptor-binding domain (eA1), a procedure for its purification, and its renaturation with final yield of 50 mg/liter of culture. Functional activity of eA1 was confirmed by immunoblotting, laser scanning confocal microscopy, and flow cytometry. It is shown that monomeric non-glycosylated receptor-binding domain of ephrin-A1 is able to activate cellular EphA2 receptors, stimulating their phosphorylation. Ligand eA1 can be used to study the features of ephrin-A1 interactions with different A class Eph receptors. The created expression cassette is suitable for the development of ligands with increased activity and selectivity and experimental systems for the delivery of cytotoxins into tumor cells that overexpress EphA2 or other class A Eph receptors.

  11. Molecular Imaging Provides Novel Insights on Estrogen Receptor Activity in Mouse Brain

    Alessia Stell

    2008-11-01

    Full Text Available Estrogen receptors have long been known to be expressed in several brain areas in addition to those directly involved in the control of reproductive functions. Investigations in humans and in animal models suggest a strong influence of estrogens on limbic and motor functions, yet the complexity and heterogeneity of neural tissue have limited our approaches to the full understanding of estrogen activity in the central nervous system. The aim of this study was to examine the transcriptional activity of estrogen receptors in the brain of male and female mice. Exploiting the ERE-Luc reporter mouse, we set up a novel, bioluminescence-based technique to study brain estrogen receptor transcriptional activity. Here we show, for the first time, that estrogen receptors are similarly active in male and female brains and that the estrous cycle affects estrogen receptor activity in regions of the central nervous system not known to be associated with reproductive functions. Because of its reproducibility and sensitivity, this novel bioluminescence application stands as a candidate as an innovative methodology for the study and development of drugs targeting brain estrogen receptors.

  12. Attenuation of eph receptor kinase activation in cancer cells by coexpressed ephrin ligands.

    Giulia Falivelli

    Full Text Available The Eph receptor tyrosine kinases mediate juxtacrine signals by interacting "in trans" with ligands anchored to the surface of neighboring cells via a GPI-anchor (ephrin-As or a transmembrane segment (ephrin-Bs, which leads to receptor clustering and increased kinase activity. Additionally, soluble forms of the ephrin-A ligands released from the cell surface by matrix metalloproteases can also activate EphA receptor signaling. Besides these trans interactions, recent studies have revealed that Eph receptors and ephrins coexpressed in neurons can also engage in lateral "cis" associations that attenuate receptor activation by ephrins in trans with critical functional consequences. Despite the importance of the Eph/ephrin system in tumorigenesis, Eph receptor-ephrin cis interactions have not been previously investigated in cancer cells. Here we show that in cancer cells, coexpressed ephrin-A3 can inhibit the ability of EphA2 and EphA3 to bind ephrins in trans and become activated, while ephrin-B2 can inhibit not only EphB4 but also EphA3. The cis inhibition of EphA3 by ephrin-B2 implies that in some cases ephrins that cannot activate a particular Eph receptor in trans can nevertheless inhibit its signaling ability through cis association. We also found that an EphA3 mutation identified in lung cancer enhances cis interaction with ephrin-A3. These results suggest a novel mechanism that may contribute to cancer pathogenesis by attenuating the tumor suppressing effects of Eph receptor signaling pathways activated by ephrins in trans.

  13. Soluble TL1A is sufficient for activation of death receptor 3.

    Bittner, Sebastian; Knoll, Gertrud; Füllsack, Simone; Kurz, Maria; Wajant, Harald; Ehrenschwender, Martin

    2016-01-01

    Death receptor 3 (DR3) is a typical member of the tumor necrosis factor receptor family, and was initially identified as a T-cell co-stimulatory molecule. However, further studies revealed a more complex and partly dichotomous role for DR3 and its ligand TL1A under (patho)physiological conditions. TL1A and DR3 are not only a driving force in the development of autoimmune and inflammatory diseases, but also play an important role in counteracting these processes through an increase in the number of regulatory T cells. Ligands of the tumor necrosis factor family typically occur in two forms, membrane-bound and soluble, that can differ strikingly with respect to their efficacy in activating their corresponding receptor(s). Ligand-based approaches to activate the TL1A-DR3 pathway therefore require understanding of the molecular prerequisites of TL1A-based DR3 activation. To date, this has not been addressed. Here, we show that recombinant soluble trimeric TL1A is fully sufficient to strongly activate DR3-associated pro- and anti-apoptotic signaling pathways. In contrast to the TRAIL death receptors, which are much better activated by soluble TRAIL upon secondary ligand oligomerization, but similarly to the death receptor tumor necrosis factor receptor 1, DR3 is efficiently activated by soluble TL1A trimers. Additionally, we have measured the affinity of TL1A-DR3 interaction in a cell-based system, and demonstrated TL1A-induced DR3 internalization. Identification of DR3 as a tumor necrosis factor receptor that responds to soluble ligand trimers without further oligomerization provides a basis for therapeutic exploitation of the TL1A-DR3 pathway.

  14. Molecular mechanisms of action of the soy isoflavones includes activation of promiscuous nuclear receptors. A review.

    Ricketts, Marie-Louise; Moore, David D; Banz, William J; Mezei, Orsolya; Shay, Neil F

    2005-06-01

    Consumption of soy has been demonstrated to reduce circulating cholesterol levels, most notably reducing low-density lipoprotein (LDL) cholesterol levels in hypercholesterolemic individuals. The component or components that might be responsible for this effect is still a matter of debate or controversy among many researchers. Candidate agents include an activity of soy protein itself, bioactive peptides produced during the digestive process, or the soy isoflavones. Although soy intake may provide other health benefits including preventative or remediative effects on cancer, osteoporosis and symptoms of menopause, this review will focus on isoflavones as agents affecting lipid metabolism. Isoflavones were first discovered as a bioactive agent disrupting estrogen action in female sheep, thereby earning the often-used term 'phytoestrogens'. Subsequent work confirmed the ability of isoflavones to bind to estrogen receptors. Along with the cholesterol-lowering effect of soy intake, research that is more recent has pointed to a beneficial antidiabetic effect of soy intake, perhaps mediated by soy isoflavones. The two common categories of antidiabetic drugs acting on nuclear receptors known as peroxisome proliferator activated receptors (PPARs) are the fibrates and glitazones. We and others have recently asked the research question 'do the soy isoflavones have activities as either "phytofibrates" or "phytoglitazones"?' Such an activity should be able to be confirmed both in vivo and in vitro. In both the in vivo and in vitro cases, this action has indeed been confirmed. Further work suggests a possible action of isoflavones similar to the nonestrogenic ligands that bind the estrogen-related receptors (ERRs). Recently, these receptors have been demonstrated to contribute to lipolytic processes. Finally, evaluation of receptor activation studies suggests that thyroid receptor activation may provide additional clues explaining the metabolic action of isoflavones. The recent

  15. Progression of Osteosarcoma from a Non-Metastatic to a Metastatic Phenotype Is Causally Associated with Activation of an Autocrine and Paracrine uPA Axis.

    Liliana Endo-Munoz

    Full Text Available Pulmonary metastasis is the major untreatable complication of osteosarcoma (OS resulting in 10-20% long-term survival. The factors and pathways regulating these processes remain unclear, yet their identification is crucial in order to find new therapeutic targets. In this study we used a multi-omics approach to identify molecules in metastatic and non-metastatic OS cells that may contribute to OS metastasis, followed by validation in vitro and in vivo. We found elevated levels of the urokinase plasminogen activator (uPA and of the uPA receptor (uPAR exclusively in metastatic OS cells. uPA was secreted in soluble form and as part of the protein cargo of OS-secreted extracellular vesicles, including exosomes. In addition, in the tumour microenvironment, uPA was expressed and secreted by bone marrow cells (BMC, and OS- and BMC-derived uPA significantly and specifically stimulated migration of metastatic OS cells via uPA-dependent signaling pathways. Silencing of uPAR in metastatic OS cells abrogated the migratory response to uPA in vitro and decreased metastasis in vivo. Finally, a novel small-molecule inhibitor of uPA significantly (P = 0.0004 inhibited metastasis in an orthotopic mouse model of OS. Thus, we show for the first time that malignant conversion of OS cells to a metastatic phenotype is defined by activation of the uPA/uPAR axis in both an autocrine and paracrine fashion. Furthermore, metastasis is driven by changes in OS cells as well as in the microenvironment. Finally, our data show that pharmacological inhibition of the uPA/uPAR axis with a novel small-molecule inhibitor can prevent the emergence of metastatic foci.

  16. Activation of intracellular angiotensin AT₂ receptors induces rapid cell death in human uterine leiomyosarcoma cells.

    Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen; Zuhayra, Maaz; Schütze, Stefan; Steckelings, Ulrike M; Recanti, Chiara; Namsoleck, Pawel; Unger, Thomas; Culman, Juraj

    2015-05-01

    The presence of angiotensin type 2 (AT₂) receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT₂ receptors including their presence in mitochondria and their role in the induction of apoptosis and cell death in cultured human uterine leiomyosarcoma (SK-UT-1) cells and control human uterine smooth muscle cells (HutSMC). The intracellular levels of the AT₂ receptor are low in proliferating SK-UT-1 cells but the receptor is substantially up-regulated in quiescent SK-UT-1 cells with high densities in mitochondria. Activation of the cell membrane AT₂ receptors by a concomitant treatment with angiotensin II and the AT₁ receptor antagonist, losartan, induces apoptosis but does not affect the rate of cell death. We demonstrate for the first time that the high-affinity, non-peptide AT₂ receptor agonist, Compound 21 (C21), penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT₂ receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped cell death, displayed activation of the mitochondrial apoptotic pathway, i.e. down-regulation of the Bcl-2 protein, induction of the Bax protein and activation of caspase-3. All quiescent SK-UT-1 cells died within 5 days after treatment with a single dose of C21. C21 was devoid of cytotoxic effects in proliferating SK-UT-1 cells and in quiescent HutSMC. Our results point to a new, unique approach enabling the elimination non-cycling uterine leiomyosarcoma cells providing that they over-express the AT₂ receptor.

  17. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Park, Mi Kyung; Cho, Min Kyoung; Kang, Shin Ae; Park, Hye-Kyung; Kim, Dong-Hee; Yu, Hak Sun

    2014-01-01

    Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES) proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25) in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF) inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  18. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  19. Angiotensin type 2-receptor (AT2R) activation induces hypotension in apolipoprotein E-deficient mice by activating peroxisome proliferator-activated receptor

    Li, Ming; Tejada, Thor; Lambert, Jonathan P; Nicholson, Chad K; Yahiro, Eiji; Ambai, Vats T; Ali, Syeda F; Bradley, Eddie W; Graham, Robert M; Dell’Italia, Louis J; Calvert, John W; Naqvi, Nawazish

    2016-01-01

    Angiotensin II (Ang II) modulates blood pressure and atherosclerosis development through its vascular type-1 (AT1R) and type-2 (AT2R) receptors, which have opposing effects. AT2R activation produces hypotension, and is anti-atherogenic. Targeted overexpression of AT2Rs in vascular smooth muscle cells (VSMCs) indicates that these effects are due to increased nitric oxide (NO) generation. However, the role of endogenous VSMC AT2Rs in these events is unknown. Effect of 7-day low-dose Ang II-infusion (12 µg/kg/hr) on blood pressure was tested in 9-week-old apoE(-/-) mice fed a low or high cholesterol diet (LCD or HCD, respectively). Cardiac output was measured by echocardiography. Immunohistochemistry was performed to localize and quantify AT2Rs and p-Ser1177-endothelial nitric oxide synthase (eNOS) levels in the aortic arch. PD123319 and GW-9662 were used to selectively block the AT2R and peroxisome proliferator-activated receptor-γ (PPAR-γ), respectively. Ang II infusion decreased blood pressure by 12 mmHg (P LCD/apoE(-/-) mice without altering cardiac output; a response blocked by PD123319. Although, AT2R stimulation neither activated eNOS (p-Ser1177-eNOS) nor changed plasma NO metabolites, it caused an ~6-fold increase in VSMC PPAR-γ levels (P < 0.001) and the AT2R-mediated hypotension was abolished by GW-9662. AT2R-mediated hypotension was also inhibited by HCD, which selectively decreased VSMC AT2R expression by ~6-fold (P < 0.01). These findings suggest a novel pathway for the Ang II/AT2R-mediated hypotensive response that involves PPAR-γ, and is down regulated by a HCD. PMID:27679746

  20. Activation of peroxisome proliferator-activated receptor α in human endothelial cells increases plasminogen activator inhibitor type-1 expression

    叶平; 胡晓晖; 刘永学; 赵亚力

    2003-01-01

    Objective To investigate the effect of peroxisome proliferator-activated receptors (PPARs) activators on plasminogen activator inhibitor 1 (PAI-1) expression in human umbilical vein endothelial cells and elucidate a possible mechanism.Methods Human umbilical vein endothelial cells (HUVECs) were obtained from normal fetus, and cultured conventionally. Then the HUVEC were exposed to fatty acids and prostaglandin J2 in varying concentrations with fresh media. RT-PCR and ELISA were used to determine the expression of PPAR and PAI-1 in HUVECs. Transient co-transfection of PAI-1 promoter and PPARα gene or PPARγ gene to ECV304 was performed.Results PPARα, PPARδ and PPARγ mRNA in HUVECs were detected by RT-PCR. Treatment of HUVECs with PPARα and PPARγ activators-linolenic acid, linoleic acid, oleic acid and prostaglandin J2, but not with stearic acid could augment PAI-I mRNA expression and protein secretion in a concentration-dependent manner. Proportional induction of PAI-1 promoter activity was observed through increasing amounts of PPARα DNA in HUVECs through a transient gene transfection assay, although the mRNA expression of the 3 subtypes of PPAR with their activators were not changed compared with controls.Conclusions HUVECs express PPARs. PPARs activators may increase PAI-1 expression in endothelial cells (EC). Although PPARs expression was not enhanced after being stimulated by their activators in EC, the functionally active PPARα is probably involved in regulating PAI-1 expression in EC.

  1. Molecular mechanism of 7TM receptor activation--a global toggle switch model

    Schwartz, Thue W; Frimurer, Thomas M; Holst, Birgitte;

    2006-01-01

    The multitude of chemically highly different agonists for 7TM receptors apparently do not share a common binding mode or active site but nevertheless act through induction of a common molecular activation mechanism. A global toggle switch model is proposed for this activation mechanism to reconcile...... to this model, a vertical see-saw movement of TM-VI-and to some degree TM-VII-around a pivot corresponding to the highly conserved prolines will occur during receptor activation, which may involve the outer segment of TM-V in an as yet unclear fashion. Small-molecule agonists can stabilize such a proposed...

  2. Pharmacology of the hypothermic response to 5-HT1A receptor activation in humans.

    Lesch, K P; Poten, B; Söhnle, K; Schulte, H M

    1990-01-01

    The selective 5-HT1A receptor ligand ipsapirone (IPS) caused dose-related hypothermia in humans. The response was attenuated by the nonselective 5-HT1/2 receptor antagonist metergoline and was completely antagonized by the nonselective beta-adrenoceptor antagonist pindolol, which interacts stereoselectively with the 5-HT1A receptor. The selective beta 1-adrenergic antagonist betaxolol had no effect. The findings indicate that IPS-induced hypothermia specifically involves activation of (presynaptic) 5-HT1A receptors. Therefore, the hypothermic response to IPS may provide a convenient in vivo paradigma to assess the function of the presynaptic 5-HT receptor in affective disorders and its involvement in the effects of psychotropic drugs.

  3. Transition of arrestin into the active receptor-binding state requires an extended interdomain hinge.

    Vishnivetskiy, Sergey A; Hirsch, Joel A; Velez, Maria-Gabriela; Gurevich, Yulia V; Gurevich, Vsevolod V

    2002-11-15

    Arrestins selectively bind to the phosphorylated activated form of G protein-coupled receptors, thereby blocking further G protein activation. Structurally, arrestins consist of two domains topologically connected by a 12-residue long loop, which we term the "hinge" region. Both domains contain receptor-binding elements. The relative size and shape of arrestin and rhodopsin suggest that dramatic changes in arrestin conformation are required to bring all of its receptor-binding elements in contact with the cytoplasmic surface of the receptor. Here we use the visual arrestin/rhodopsin system to test the hypothesis that the transition of arrestin into its active receptor-binding state involves a movement of the two domains relative to each other that might be limited by the length of the hinge. We have introduced three insertions and 24 deletions in the hinge region and measured the binding of all of these mutants to light-activated phosphorylated (P-Rh*), dark phosphorylated (P-Rh), dark unphosphorylated (Rh), and light-activated unphosphorylated rhodopsin (Rh*). The addition of 1-3 extra residues to the hinge has no effect on arrestin function. In contrast, sequential elimination of 1-8 residues results in a progressive decrease in P-Rh* binding without changing arrestin selectivity for P-Rh*. These results suggest that there is a minimum length of the hinge region necessary for high affinity binding, consistent with the idea that the two domains move relative to each other in the process of arrestin transition into its active receptor-binding state. The same length of the hinge is also necessary for the binding of "constitutively active" arrestin mutants to P-Rh*, dark P-Rh, and Rh*, suggesting that the active (receptor-bound) arrestin conformation is essentially the same in both wild type and mutant forms.

  4. Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

    Silva, Jose Alberto Alves da

    2009-07-01

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  5. Activation of muscarinic receptors in porcine airway smooth muscle elicits a transient increase in phospholipase D activity.

    Mamoon, A M; Smith, J; Baker, R C; Farley, J M

    1999-01-01

    Phospholipase D (PLD) is a phosphodiesterase that catalyses hydrolysis of phosphatidylcholine to produce phosphatidic acid and choline. In the presence of ethanol, PLD also catalyses the formation of phosphatidylethanol, which is a unique characteristic of this enzyme. Muscarinic receptor-induced changes in the activity of PLD were investigated in porcine tracheal smooth muscle by measuring the formation of [3H]phosphatidic acid ([3H]PA) and [3H]phosphatidylethanol ([3H]PEth) after labeling the muscle strips with [3H]palmitic acid. The cholinergic receptor agonist acetylcholine (Ach) significantly but transiently increased formation of both [3H]PA and [3H]PEth in a concentration-dependent manner (>105-400% vs. controls in the presence of 10(-6) to 10(-4) M Ach) when pretreated with 100 mM ethanol. The Ach receptor-mediated increase in PLD activity was inhibited by atropine (10(-6) M), indicating that activation of PLD occurred via muscarinic receptors. Activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) increased PLD activity that was effectively blocked by the PKC inhibitors calphostin C (10(-8) to 10(-6) M) and GFX (10(-8) to 10(-6) M). Ach-induced increases in PLD activity were also significantly, but incompletely, inhibited by both GFX and calphostin C. From the present data, we conclude that in tracheal smooth muscle, muscarinic acetylcholine receptor-induced PLD activation is transient in nature and coupled to these receptors via PKC. However, PKC activation is not solely responsible for Ach-induced activation of PLD in porcine tracheal smooth muscle.

  6. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor

    Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-01-01

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered “off-target” effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo. PMID:27036020

  7. Research on drug-receptor interactions and prediction of drug activity via oriented immobilized receptor capillary electrophoresis.

    Liu, Chunye; Zhang, Xuejiao; Jing, Hui; Miao, Yanqing; Zhao, Lingzhi; Han, Yan; Cui, Cuixia

    2015-10-01

    Oriented covalent immobilized β2 -adrenergic receptor (β2 -AR) CE (OIRCE) was developed to determine the interactions between a set of natural extracts of Radix Paeoniae Rubra (NERPR) and β2 -AR, and to predict the activity of NERPR. The inner capillary surface is chemically bonded with stable β2 -AR coating via microwave-assisted technical synthesis. The modified capillaries were characterized via infrared spectroscopy and fluorescence microscopy. Furthermore, the bonding amounts of β2 -AR were first obtained via fluorescence spectroscopy method. In determining the amount of bonded β2 -AR, the regression equation A  =  576 707C + 35.449 and the correlation coefficient 0.9995 were obtained. This result revealed an excellent linear relationship in the range of 2 × 10(-4)  mg/mL to 1 × 10(-3)  mg/mL. The normalized capacity factor (KRCE ) was obtained using OIRCE in evaluating drug-receptor interactions. Related theories and equations were used to calculate KRCE values from apparent migration times of a solute and EOF. The order of KRCE and the binding constant (Kb ) values between drugs and β2 -AR was well consistent. The results confirmed that the OIRCE and KRCE values can be effectually used to investigate drug-receptor interactions, and OIRCE has the potential to predict drug activity and to select leading compounds from natural chemicals.

  8. A high-content EMT screen identifies multiple receptor tyrosine kinase inhibitors with activity on TGFβ receptor.

    Lotz-Jenne, Carina; Lüthi, Urs; Ackerknecht, Sabine; Lehembre, François; Fink, Tobias; Stritt, Manuel; Wirth, Matthias; Pavan, Simona; Bill, Ruben; Regenass, Urs; Christofori, Gerhard; Meyer-Schaller, Nathalie

    2016-05-03

    An epithelial to mesenchymal transition (EMT) enables epithelial tumor cells to break out of the primary tumor mass and to metastasize. Understanding the molecular mechanisms driving EMT in more detail will provide important tools to interfere with the metastatic process. To identify pharmacological modulators and druggable targets of EMT, we have established a novel multi-parameter, high-content, microscopy-based assay and screened chemical compounds with activities against known targets. Out of 3423 compounds, we have identified 19 drugs that block transforming growth factor beta (TGFβ)-induced EMT in normal murine mammary gland epithelial cells (NMuMG). The active compounds include inhibitors against TGFβ receptors (TGFBR), Rho-associated protein kinases (ROCK), myosin II, SRC kinase and uridine analogues. Among the EMT-repressing compounds, we identified a group of inhibitors targeting multiple receptor tyrosine kinases, and biochemical profiling of these multi-kinase inhibitors reveals TGFBR as a thus far unknown target of their inhibitory spectrum. These findings demonstrate the feasibility of a multi-parameter, high-content microscopy screen to identify modulators and druggable targets of EMT. Moreover, the newly discovered "off-target" effects of several receptor tyrosine kinase inhibitors have important consequences for in vitro and in vivo studies and might beneficially contribute to the therapeutic effects observed in vivo.

  9. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    Takemura, Kenichi [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan); Suzuki, Hiroshi [National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Hokkaido (Japan); Kodama, Tatsuhiko [Department of Molecular Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo (Japan); Mizuta, Hiroshi [Department of Orthopaedic and Neuro-Musculoskeletal Surgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto (Japan); Takeya, Motohiro, E-mail: takeya@kumamoto-u.ac.jp [Department of Cell Pathology, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556 (Japan)

    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. 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.

  10. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    Vondrácek, Jan; Chramostová, Katerina; Plísková, Martina; Bláha, Ludek; Brack, Werner; Kozubík, Alois; Machala, Miroslav

    2004-09-01

    A group of heterocyclic aromatic compounds, dinaphthofurans (DNFs), recently have been identified as potentially significant contaminants in freshwater sediments. In the present study, a battery of in vitro assays was used for detection of toxic effects of DNFs that are potentially associated with endocrine disruption and tumor promotion. Dinaphthofurans were found to act as relatively potent inducers of aryl hydrocarbon receptor (AhR)-mediated activity in the chemical-activated luciferase reporter gene expression DR-CALUX assay. The relative AhR-inducing potencies of DNFs were similar or even higher than relative potencies of unsubstituted polycyclic aromatic hydrocarbons (PAHs), with dinaphtho[1,2-b;2'3'-d]furan being the most potent AhR agonist. Two compounds, dinaphtho[2,1-b;2'3'-d]furan and dinaphtho[1,2-b;1'2'-d]furan, induced estrogen receptor (ER)-mediated activity in the estrogen receptor-mediated CALUX (the ER-CALUX) assay. Two types of potential tumor-promoting effects of DNFs were investigated, using in vitro bioassays for detection of inhibition of gap-junctional intercellular communication and detection of a release from contact inhibition. Although the acute inhibition of gap-junctional intercellular communication was not observed, all six tested DNFs were able to release rat liver epithelial WB-F344 cells from contact inhibition at concentrations as low as 100 nM. In summary, the present study indicated that DNFs can exert multiple biological effects in vitro, including induction of the AhR-mediated activity, release of cells from contact inhibition, and induction of ER-mediated activity.

  11. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition.

    Alari-Pahissa, Elisenda; Grandclément, Camille; Jeevan-Raj, Beena; Leclercq, Georges; Veillette, André; Held, Werner

    2016-01-01

    Natural Killer (NK) cells attack normal hematopoietic cells that do not express inhibitory MHC class I (MHC-I) molecules, but the ligands that activate NK cells remain incompletely defined. Here we show that the expression of the Signaling Lymphocyte Activation Molecule (SLAM) family members CD48 and Ly9 (CD229) by MHC-I-deficient tumor cells significantly contributes to NK cell activation. When NK cells develop in the presence of T cells or B cells that lack inhibitory MHC-I but express activating CD48 and Ly9 ligands, the NK cells' ability to respond to MHC-I-deficient tumor cells is severely compromised. In this situation, NK cells express normal levels of the corresponding activation receptors 2B4 (CD244) and Ly9 but these receptors are non-functional. This provides a partial explanation for the tolerance of NK cells to MHC-I-deficient cells in vivo. Activating signaling via 2B4 is restored when MHC-I-deficient T cells are removed, indicating that interactions with MHC-I-deficient T cells dominantly, but not permanently, impair the function of the 2B4 NK cell activation receptor. These data identify an important role of SLAM family receptors for NK cell mediated "missing-self" reactivity and suggest that NK cell tolerance in MHC-I mosaic mice is in part explained by an acquired dysfunction of SLAM family receptors.

  12. Involvement of Ca2+-activated K+ Channels in Receptor-Regulated Sperm Motility in Rats

    2002-01-01

    Previous voltage-clamp studies have demonstrated the modulation of sperm Ca2+-activated K+ (KCa) channels expressed in Xenopus oocytes by angiotensin Ⅱ (Ang Ⅱ)and extracellular ATP via AT1 receptor and P2U receptor, respectively. In the presentstudy, we investigated the involvement of KCa channels in receptor-regulated spermmotility of the rat using a computer-aided sperm analysis system, HTM-IVOS, in con-junction with Ca2+-mobilizing agents, receptor agonists/antagonists and KCa channelsblockers.The percentage of motile sperm was increased by ionomycin (0. 5 μmol/L), whichcould be inhibited by K+ channel blockers, tetraethylammonium (TEA 1 μmol/L ) orcharybdotoxin (ChTX, 300 nmol/L) indicating the presence of KCa channels. AngⅡ, at low concentration, 10 nmol/L, was found to increase motility, however, athigher concentration, 1 μmol/L, percentage of motility was found to be suppressed.Both stimulatory and inhibitory effects of Ang Ⅱ could be reversed by losartan, aspecific antagonist of AT 1 receptors, but not AT 2 antagonist PD123177, indicating theinvolvement of AT1 but not AT2 receptor in mediating both effects. ChTX also abol-ished both stimulatory and inhibitory effects of Ang H, suggesting the involvement ofKCa channels. The percentage of motility was also enhanced by extracellular ATP, afactor known to be involved in sperm activation. The ATP-enhanced sperm motilitywas mimicked by UTP , and inhibited by ChTX and reactive blue, an antagonist of P2receptor, indicating the involvement of both P2U and KCa channels. RT-PCR studywas also conducted to confirm the expression of KCa channels, AT1 receptors and P2Ureceptor, but not AT2 receptor, in rat caudal epididymal sperm. The present findingssuggest an important role of KCa channels in the regulation of sperm motility by AT1and P 2U receptors.

  13. Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

    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.

  14. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner.

    Sjöqvist, Marika; Antfolk, Daniel; Ferraris, Saima; Rraklli, Vilma; Haga, Cecilia; Antila, Christian; Mutvei, Anders; Imanishi, Susumu Y; Holmberg, Johan; Jin, Shaobo; Eriksson, John E; Lendahl, Urban; Sahlgren, Cecilia

    2014-04-01

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick central nervous system and in cultured myoblasts, Notch-stimulated cells were allowed to undergo differentiation. PKCζ phosphorylates membrane-tethered forms of Notch and regulates two distinct routing steps, depending on the Notch activation state. When Notch is activated, PKCζ promotes re-localization of Notch from late endosomes to the nucleus and enhances production of the Notch intracellular domain, which leads to increased Notch activity. In the non-activated state, PKCζ instead facilitates Notch receptor internalization, accompanied with increased ubiquitylation and interaction with the endosomal sorting protein Hrs. Collectively, these data identify PKCζ as a key regulator of Notch trafficking and demonstrate that distinct steps in intracellular routing are differentially modulated depending on Notch signaling status.

  15. Mechanism for Src activation by the CCK2 receptor: Patho-physiological functions of this receptor in pancreas

    Audrey Ferrand; Sebastien Vatinel; Aline Kowalski-Chauvel; Claudine Bertrand; Chantal Escrieut; Daniel Fourmy; Marlene Dufresne; Catherine Seva

    2006-01-01

    AIM: To investigate in vivo, whether CCK2 receptors (CCK2R) regulate proteins known to play a crucial role in cell proliferation and cancer development and analyse in vitro the molecular mechanisms that lead to Src activation; in particular, to identify the domains within the CCK2R sequence that are implicated in this activation.METHODS: The expression and activation of Src and ERK were studied in vivo using immunofluorescence and western-blot techniques. We used pancreatic tissues derived from wild type or ElasCCK2 mice that expressed the CCK2R in pancreatic acini, displayed an increased pancreatic growth and developed preneoplastic lesions. The pancreatic tumor cell line AR4-2J expressing the endogenous CCK2R or COS-7 cells transiently transfected with wild type or mutant CCK2R were used as in vitro models to study the mechanism of Src activation.Src activation was measured by in vitro kinase assays, ERK activation by western blot using antiphospho-ERK antibodies and the involvement of Src in gastrin-induced cell proliferation by MTT test.RESULTS: We showed in vivo that the targeted CCK2R expression in the pancreas of Elas-CCK2 mice, led to the activation of Src and the ERK pathway. Src was activated upstream of the ERK pathway by the CCK2R in pancreatic tumoral cells and contributed to the proliferative effects mediated by this receptor. In vitro results demonstrated that activation of the Src/ERK pathway by the CCK2R required the NPXXY motif, located within the CCK2R sequence at the end of the 7th transmembrane domain,and suggested the putative role of Gq in this mechanism.CONCLUSION: Deregulation of the Src/ERK pathway by the CCK2R might represent an early step that contributes to cell proliferation, formation of preneoplastic lesions and pancreatic tumor development.

  16. Receptor density is key to the alpha2/beta interferon differential activities.

    Moraga, Ignacio; Harari, Daniel; Schreiber, Gideon; Uzé, Gilles; Pellegrini, Sandra

    2009-09-01

    Multiple type I interferons (IFN-alpha/beta) elicit Jak/Stat activation, rapid gene induction, and pleiotropic effects, such as differentiation, antiviral protection, and blocks in proliferation, which are dependent on the IFN subtype and the cellular context. To date, ligand- and receptor-specific molecular determinants underlying IFN-alpha/beta differential activities or potencies have been well characterized. To analyze cellular determinants that impact subtype-specific potency, human fibrosarcoma U5A-derived clones, exhibiting a gradient of IFN sensitivity by virtue of increasing receptor levels, were monitored for Jak/Stat signaling, gene induction, cell cycle lengthening, and apoptosis. In cells with scarce receptors, IFN-beta was more potent than IFN-alpha2 in antiproliferative activity, while the two subtypes were equipotent in all other readouts. Conversely, in cells with abundant receptors, IFN-alpha2 matched or even surpassed IFN-beta in all readouts tested. Our results suggest that the differential activities of the IFN subtypes are dictated not only by the intrinsic ligand/receptor binding kinetics but also by the density of cell surface receptor components.

  17. Ligand Perception, Activation, and Early Signaling of Plant Steroid Receptor Brassinosteroid Insensitive 1

    Jianjun Jiang; Chi Zhang; Xuelu Wang

    2013-01-01

    Leucine-rich repeat receptor-like kinases (LRR-RLKs) belong to a large group of cell surface proteins involved in many aspects of plant development and environmental responses in both monocots and dicots. Brassinosteroid insensitive 1 (BRI1), a member of the LRR X subfamily, was first identified through several forward genetic screenings for mutants insensitive to brassinosteroids (BRs), which are a class of plant-specific steroid hormones. Since its identification, BRI1 and its homologs had been proved as receptors perceiving BRs and initiating BR signaling. The co-receptor BRI1-associated kinase 1 and its homologs, and other BRI1 interacting proteins such as its inhibitor BRI1 kinase inhibitor 1 (BKI1) were identified by genetic and biochemical approaches. The detailed mechanisms of BR perception by BRI1 and the activation of BRI1 receptor complex have also been elucidated. Moreover, several mechanisms for termination of the activated BRI1 signaling were also discovered. In this review, we will focus on the recent advances on the mechanism of BRI1 phosphorylation and activation, the regulation of its receptor complex, the structure basis of BRI1 ectodomain and BR recognition, its direct substrates, and the termination of the activated BRI1 receptor complex.

  18. Activity-dependent upregulation of presynaptic kainate receptors at immature CA3-CA1 synapses.

    Clarke, Vernon R J; Molchanova, Svetlana M; Hirvonen, Teemu; Taira, Tomi; Lauri, Sari E

    2014-12-10

    Presynaptic kainate-type glutamate receptors (KARs) regulate glutamate release probability and short-term plasticity in various areas of the brain. Here we show that long-term depression (LTD) in the area CA1 of neonatal rodent hippocampus is associated with an upregulation of tonic inhibitory KAR activity, which contributes to synaptic depression and causes a pronounced increase in short-term facilitation of transmission. This increased KAR function was mediated by high-affinity receptors and required activation of NMDA receptors, nitric oxide (NO) synthetase, and postsynaptic calcium signaling. In contrast, KAR activity was irreversibly downregulated in response to induction of long-term potentiation in a manner that depended on activation of the TrkB-receptor of BDNF. Both tonic KAR activity and its plasticity were restricted to early stages of synapse development and were lost in parallel with maturation of the network due to ongoing BDNF-TrkB signaling. These data show that presynaptic KARs are targets for activity-dependent modulation via diffusible messengers NO and BDNF, which enhance and depress tonic KAR activity at immature synapses, respectively. The plasticity of presynaptic KARs in the developing network allows nascent synapses to shape their response to incoming activity. In particular, upregulation of KAR function after LTD allows the synapse to preferentially pass high-frequency afferent activity. This can provide a potential rescue from synapse elimination by uncorrelated activity and also increase the computational dynamics of the developing CA3-CA1 circuitry.

  19. P2X receptors mediate ATP-induced primary nociceptive neurone activation.

    Bland-Ward, P A; Humphrey, P P

    2000-07-01

    ATP-gated P2X ion-channel receptors are localised throughout the mammalian nervous system and have been identified on neurones which participate in conduction of nociceptive information from the periphery to, and within, the CNS. This article briefly reviews recently published research describing the role that ATP and P2X receptors may play in pain perception, highlighting the importance of the P2X(3) receptor in this process. The P2X(3) receptor subunit is almost exclusively expressed on a subset of small and medium diameter sensory neurones innervating cutaneous and visceral tissue. Activation of P2X receptors present on the peripheral terminals of primary afferents results in neuronal depolarisation and, in conscious animals, leads to the manifestation of acute nociceptive behaviour. Recent animal studies have also shown that P2X(3) receptor expression is increased in sensory ganglia following acute neuronal injury, hinting that similar plasticity in the expression of this receptor subtype could underlie the mechanisms involved in a range of conditions characterised by sensory hypersensitivity in man. It is apparent from the evidence available that functional antagonists at specific P2X receptor subtypes could represent an important class of novel analgesic agents.

  20. Dimerization by a cytokine receptor is necessary for constitutive activation of JAK2V617F.

    Lu, Xiaohui; Huang, Lily Jun-Shen; Lodish, Harvey F

    2008-02-29

    The majority of the BCR-ABL-negative myeloproliferative disorders express the mutant JAK2, JAK2V617F. Previously we showed that constitutive activation of this oncogenic JAK2 mutant in Ba/F3 or 32D cells requires coexpression of a cognate homodimeric cytokine receptor, such as the EpoR. However, overexpression of JAK2V617F in Ba/F3 cells renders them cytokine-independent for growth in the absence of an exogenous cytokine receptor. Here, we demonstrated that JAK2V617F domains required for receptor association are essential for cytokine-independent growth by overexpressed JAK2V617F, suggesting JAK2V617F is binding to an unknown endogenous cytokine receptor(s) for its activation. We further showed that disruption of EpoR dimerization by coexpressing a truncated EpoR disrupted JAK2V617F-mediated transformation, indicating that EpoR dimerization plays an essential role in the activation of JAK2V617F. Interestingly, coexpression of JAK2V617F with EpoR mutants that retain JAK2 binding but are defective in mediating Epo-dependent JAK2 activation due to mutations in a conserved juxtamembrane motif does lead to cytokine-independent activation of JAK2V617F. Overall, these findings confirm that JAK2V617F requires binding to a dimerized cytokine receptor for its activation, and that the key EpoR juxtamembrane regulatory motif essential for Epo-dependent JAK2 activation is not essential for the activation of JAK2V617F. The structure of the activated JAK2V617F is thus likely to be different from that of the activated wild-type JAK2, raising the possibility of developing a specifically targeted therapy for myeloproliferative disorders.

  1. Role of NK1 and NK2 receptors in mouse gastric mechanical activity.

    Mulè, Flavia; Amato, Antonella; Vannucchi, Maria Giuliana; Faussone-Pellegrini, Maria Simonetta; Serio, Rosa

    2006-02-01

    1. The aim of the present study was to examine the role of NK1 and NK2 receptors in the control of mechanical activity of mouse stomach. In this view, the motor effects induced by NK1 and NK2 receptor agonists and antagonists were analyzed, measuring motility as intraluminal pressure changes in mouse-isolated stomach preparations. In parallel, immunohistochemical studies were performed to identify the location of NK1 and NK2 receptors on myenteric neurons and smooth muscle cells. 2. Substance P (SP) induced biphasic effects: a contraction followed by relaxation; neurokinin A (NKA) and [beta-Ala8]-NKA(4-10), selective agonist of NK2 receptors, evoked concentration-dependent contractions, whereas [Sar9, Met(O2)11]-SP, selective agonist of NK1 receptors, induced concentration-dependent relaxation. 3. SR48968, NK2 receptor antagonist, did not modify the spontaneous activity and reduced the contractile effects induced by tachykinins without affecting the relaxation. SR140333, NK1 receptor antagonist, did not modify the spontaneous activity and antagonized the relaxant response to tachykinins, failing to affect the contractile effects. 4. The relaxation to SP or to [Sar9, Met(O2)11]-SP was abolished by tetrodotoxin (TTX) and significantly reduced by N(omega)-nitro-L-arginine methyl ester (L-NAME). 5. NK2-immunoreactivity (NK2-IR) was seen at the level of the smooth muscle cells of both circular and longitudinal muscle layers. NK1-immunoreactive (NK1-IR) neurons were seen in the myenteric ganglia and NK1/nNOS double labeling revealed that some neurons were both NK1-IR and nNOS-IR. 6. These results suggest that, in mouse stomach, NK1 receptors, causing relaxant responses, are present on nitrergic inhibitory myenteric neurons, whereas NK2 receptors, mediating contractile responses, are present at muscular level.

  2. Dosage-dependent effect of dopamine D2 receptor activation on motor cortex plasticity in humans.

    Fresnoza, Shane; Stiksrud, Elisabeth; Klinker, Florian; Liebetanz, David; Paulus, Walter; Kuo, Min-Fang; Nitsche, Michael A

    2014-08-06

    The neuromodulator dopamine plays an important role in synaptic plasticity. The effects depend on receptor subtypes, affinity, concentration level, and the kind of neuroplasticity induced. In animal experiments, dopamine D2-like receptor stimulation revealed partially antagonistic effects on plasticity, which might be explained by dosage dependency. In humans, D2 receptor block abolishes plasticity, and the D2/D3, but predominantly D3, receptor agonist ropinirol has a dosage-dependent nonlinear affect on plasticity. Here we aimed to determine the specific affect of D2 receptor activation on neuroplasticity in humans, because physiological effects of D2 and D3 receptors might differ. Therefore, we combined application of the selective D2 receptor agonist bromocriptine (2.5, 10, and 20 mg or placebo medication) with anodal and cathodal transcranial direct current stimulation (tDCS), which induces nonfocal plasticity, and with paired associative stimulation (PAS) generating a more focal kind of plasticity in the motor cortex of healthy humans. Plasticity was monitored by transcranial magnetic stimulation-induced motor-evoked potential amplitudes. For facilitatory tDCS, bromocriptine prevented plasticity induction independent from drug dosage. However, its application resulted in an inverted U-shaped dose-response curve on inhibitory tDCS, excitability-diminishing PAS, and to a minor degree on excitability-enhancing PAS. These data support the assumption that modulation of D2-like receptor activity exerts a nonlinear dose-dependent effect on neuroplasticity in the human motor cortex that differs from predominantly D3 receptor activation and that the kind of plasticity-induction procedure is relevant for its specific impact.

  3. Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

    Patricia Dillenburg-Pilla

    Full Text Available Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

  4. Analgesic tone conferred by constitutively active mu opioid receptors in mice lacking β-arrestin 2

    Hales Tim G

    2011-04-01

    Full Text Available Abstract Hedonic reward, dependence and addiction are unwanted effects of opioid analgesics, linked to the phasic cycle of μ opioid receptor activation, tolerance and withdrawal. In vitro studies of recombinant G protein coupled receptors (GPCRs over expressed in cell lines reveal an alternative tonic signaling mechanism that is independent of agonist. Such studies demonstrate that constitutive GPCR signaling can be inhibited by inverse agonists but not by neutral antagonists. However, ligand-independent activity has been difficult to examine in vivo, at the systems level, due to relatively low levels of constitutive activity of most GPCRs including μ receptors, often necessitating mutagenesis or pharmacological manipulation to enhance basal signaling. We previously demonstrated that the absence of β-arrestin 2 (β-arr2 augments the constitutive coupling of μ receptors to voltage-activated Ca2+ channels in primary afferent dorsal root ganglion neurons from β-arr2-/- mice. We used this in vitro approach to characterize neutral competitive antagonists and inverse agonists of the constitutively active wild type μ receptors in neurons. We administered these agents to β-arr2-/- mice to explore the role of constitutive μ receptor activity in nociception and hedonic tone. This study demonstrates that the induction of constitutive μ receptor activity in vivo in β-arr2-/- mice prolongs tail withdrawal from noxious heat, a phenomenon that was reversed by inverse agonists, but not by antagonists that lack negative efficacy. By contrast, the aversive effects of inverse agonists were similar in β-arr2-/- and β-arr2+/+ mice, suggesting that hedonic tone was unaffected.

  5. Soluble urokinase plasminogen activator receptor is a marker of dysmetabolism in HIV-infected patients receiving highly active antiretroviral therapy

    Andersen, Ove; Eugen-Olsen, Jesper; Kofoed, Kristian;

    2008-01-01

    Circulating soluble urokinase plasminogen activator receptor (suPAR) reflects the immune and pro-inflammatory status of the HIV-infected patient. Highly active antiretroviral therapy (HAART) suppresses suPAR. Independent of the immune response to HAART, suPAR remains elevated in some HIV-infected...

  6. Protease-Activated Receptor-2 Activation Contributes to House Dust Mite-Induced IgE Responses in Mice

    Post, Sijranke; Heijink, Irene; Petersen, A H; de Bruin, Harold G.; van Oosterhout, Antoon J. M.; Nawijn, Martijn C.

    2014-01-01

    Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium,

  7. Protease-activated receptor-2 activation contributes to house dust mite-induced IgE responses in mice

    Post, Sijranke; Heijink, Irene H; Petersen, Arjen H; de Bruin, Harold G; van Oosterhout, Antoon J M; Nawijn, Martijn C

    2014-01-01

    Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium,

  8. Cytisine induces autonomic cardiovascular responses via activations of different nicotinic receptors.

    Li, Yi-Fan; Lacroix, Carly; Freeling, Jessica

    2010-04-19

    Nicotinic cholinergic receptors mediate autonomic transmission at ganglia. However, whether different subtypes of nicotinic cholinergic receptors expressed in autonomic ganglia elicit distinct roles in mediating sympathetic and parasympathetic regulations remain to be defined. In this study, we observed that different subtypes of nicotinic receptors were responsible for the sympathetic and parasympathetic cardiovascular responses. In urethane anesthetized mice, intravenous injection with cytisine, a non-selective nicotinic agonist, induced a brief but pronounced decrease in heart rate, followed by increases in heart rate and arterial blood pressure. The bradycardic response was blocked by atropine, and the pressor response was blocked by prazosin, confirming that these responses were parasympathetic and sympathetic activities, respectively. Hexamethonium, a ganglionic blocker, blocked both sympathetic and parasympathetic responses. Pretreatment with methyllycaconitine citrate, a selective alpha7 nicotinic receptor antagonist, significantly attenuated cytisine-induced sympathetic response with little effect on the parasympathetic response. In contrast, pretreatment with dihydro-beta-erythroidine hydrobromide, a selective alpha4beta2 nicotinic receptor antagonist, blocked cytisine-induced parasympathetic response but not the sympathetic response. Pretreatment with dihydro-beta-erythroidine hydrobromide also blocked baroreflex associated parasympathetic bradycardic response. Moreover, treatment with nicotine induced a bradycardic response without a significant pressor response, which was also attenuated by dihydro-beta-erythroidine hydrobromide. Collectively, these data suggest that different nicotinic receptors play distinct roles in sympathetic and parasympathetic ganglia. Specifically, activations of alpha7 and alpha4beta2 nicotinic receptors are involved in cytisine-induced cardiovascular sympathetic and parasympathetic responses, respectively.

  9. A pharmacological analysis of serotonergic receptors: effects of their activation of blockade in learning.

    Meneses, A; Hong, E

    1997-02-01

    1. The authors have tested several 5-HT selective agonists and antagonists (5-HT1A/1B, 5-HT2A/2B/2C, 5-HT3 or 5-HT4), an uptake inhibitor and 5-HT depletors in the autoshaping learning task. 2. The present work deals with the receptors whose stimulation increases or decreases learning. 3. Impaired consolidation of learning was observed after the presynaptic activation of 5-HT1B, 5-HT3 or 5-HT4 or the blockade of postsynaptic 5-HT2C/2B receptors. 4. In contrast, an improvement occurred after the presynaptic activation of 5-HT1A, 5-HT2C, and the blockade of presynaptic 5-HT2A, 5-HT2C and 5-HT3 receptors. 5. The blockade of postsynaptic 5-HT1A, 5-HT1B, 5-HT3 or 5-HT4 receptors and 5-HT inhibition of synthesis and its depletion did no alter learning by themselves. 6. The present data suggest that multiple pre- and postsynaptic serotonergic receptors are involved in the consolidation of learning. 7. Stimulation of most 5-HT receptors increases learning, however, some of 5-HT subtypes seem to limit the data storage. 8. Furthermore, the role of 5-HT receptors in learning seem to require an interaction with glutamatergic, GABAergic and cholinergic neurotransmission systems.

  10. Activation of GLP-1 Receptor Promotes Bone Marrow Stromal Cell Osteogenic Differentiation through β-Catenin

    Jingru Meng

    2016-04-01

    Full Text Available Glucagon-like peptide 1 (GLP-1 plays an important role in regulating bone remodeling, and GLP-1 receptor agonist shows a positive relationship with osteoblast activity. However, GLP-1 receptor is not found in osteoblast, and the mechanism of GLP-1 receptor agonist on regulating bone remodeling is unclear. Here, we show that the GLP-1 receptor agonist exendin-4 (Ex-4 promoted bone formation and increased bone mass and quality in a rat unloading-induced bone loss model. These functions were accompanied by an increase in osteoblast number and serum bone formation markers, while the adipocyte number was decreased. Furthermore, GLP-1 receptor was detected in bone marrow stromal cells (BMSCs, but not in osteoblast. Activation of GLP-1 receptor by Ex-4 promoted the osteogenic differentiation and inhibited BMSC adipogenic differentiation through regulating PKA/β-catenin and PKA/PI3K/AKT/GSK3β signaling. These findings reveal that GLP-1 receptor regulates BMSC osteogenic differentiation and provide a molecular basis for therapeutic potential of GLP-1 against osteoporosis.

  11. Imaging of urokinase-type plasminogen activator receptor expression using a 64Cu-labeled linear peptide antagonist by microPET

    Li, Z.B.; Niu, G.; Wang, H.;

    2008-01-01

    for positron emission tomography (PET) imaging. A linear, high-affinity uPAR-binding peptide antagonist AE105 was conjugated with 1,4,7,10-tetraazadodecane-N,N',N'',N'''-tetraacetic acid (DOTA) and labeled with (64)Cu for microPET imaging of mice bearing U87MG human glioblastoma (uPAR positive) and MDA-MB-435...... human breast cancer (uPAR negative). RESULTS: Surface plasmon resonance measurements show that AE105 with DOTA conjugated at the alpha-amino group (DOTA-AE105) has high affinity toward uPAR. microPET imaging reveals a rapid and high accumulation of (64)Cu-DOTA-AE105 in uPAR-positive U87MG tumors (10...... translation of this class of radiopharmaceuticals for uPAR-positive cancer detection and patient stratification for uPA/uPAR system-based cancer therapy Udgivelsesdato: 2008/8/1...

  12. Design and Synthesis of Norendoxifen Analogues with Dual Aromatase Inhibitory and Estrogen Receptor Modulatory Activities

    Lv, Wei; Liu, Jinzhong; Skaar, Todd C.; Flockhart, David A.; Cushman, Mark

    2015-01-01

    Both selective estrogen receptor modulators and aromatase inhibitors are widely used for the treatment of breast cancer. Compounds with both aromatase inhibitory and estrogen receptor modulatory activities could have special advantages for treatment of breast cancer. Our previous efforts led to the discovery of norendoxifen as the first compound with dual aromatase inhibitory and estrogen receptor binding activities. To optimize its efficacy and aromatase selectivity versus other cytochrome P450 enzymes, a series of structurally related norendoxifen analogues were designed and synthesized. The most potent compound, 4'-hydroxynorendoxifen (10), displayed elevated inhibitory potency against aromatase and enhanced affinity for estrogen receptors when compared to norendoxifen. The selectivity of 10 for aromatase versus other cytochrome P450 enzymes was also superior to norendoxifen. 4'-Hydroxynorendoxifen is therefore an interesting lead for further development to obtain new anticancer agents of potential value for the treatment of breast cancer. PMID:25751283

  13. Conformational variability of the glycine receptor M2 domain in response to activation by different agonists.

    Pless, Stephan A; Dibas, Mohammed I; Lester, Henry A; Lynch, Joseph W

    2007-12-07

    Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric alpha1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19' or 22' positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19'C increased by approximately 20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and beta-alanine were weak partial agonists at the alpha1R19'C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or beta-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22'C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

  14. Activation of NTS A(1) adenosine receptors inhibits regional sympathetic responses evoked by activation of cardiopulmonary chemoreflex.

    Ichinose, Tomoko K; Minic, Zeljka; Li, Cailian; O'Leary, Donal S; Scislo, Tadeusz J

    2012-09-01

    Previously we have shown that adenosine operating via the A(1) receptor subtype may inhibit glutamatergic transmission in the baroreflex arc within the nucleus of the solitary tract (NTS) and differentially increase renal (RSNA), preganglionic adrenal (pre-ASNA), and lumbar (LSNA) sympathetic nerve activity (ASNA>RSNA≥LSNA). Since the cardiopulmonary chemoreflex and the arterial baroreflex are mediated via similar medullary pathways, and glutamate is a primary transmitter in both pathways, it is likely that adenosine operating via A(1) receptors in the NTS may differentially inhibit regional sympathetic responses evoked by activation of cardiopulmonary chemoreceptors. Therefore, in urethane-chloralose-anesthetized rats (n = 37) we compared regional sympathoinhibition evoked by the cardiopulmonary chemoreflex (activated with right atrial injections of serotonin 5HT(3) receptor agonist phenylbiguanide, PBG, 1-8 μg/kg) before and after selective stimulation of NTS A(1) adenosine receptors [microinjections of N(6)-cyclopentyl adenosine (CPA), 0.033-330 pmol/50 nl]. Activation of cardiopulmonary chemoreceptors evoked differential, dose-dependent sympathoinhibition (RSNA>ASNA>LSNA), and decreases in arterial pressure and heart rate. These differential sympathetic responses were uniformly attenuated in dose-dependent manner by microinjections of CPA into the NTS. Volume control (n = 11) and blockade of adenosine receptor subtypes in the NTS via 8-(p-sulfophenyl)theophylline (8-SPT, 1 nmol in 100 nl) (n = 9) did not affect the reflex responses. We conclude that activation of NTS A(1) adenosine receptors uniformly inhibits neural and cardiovascular cardiopulmonary chemoreflex responses. A(1) adenosine receptors have no tonic modulatory effect on this reflex under normal conditions. However, when adenosine is released into the NTS (i.e., during stress or severe hypotension/ischemia), it may serve as negative feedback regulator for depressor and sympathoinhibitory reflexes

  15. Molecular simulation of receptors of physiologically active compounds for purposes of medical chemistry

    Baskin, Igor I.; Palyulin, Vladimir A.; Zefirov, Nikolai S.

    2009-06-01

    The general strategy of the molecular simulation of biological receptors and their interaction with ligands is considered. The procedures for construction of 3D protein models, molecular docking, evaluation of model quality, determination of the free energy of protein binding with ligands are discussed. The methods of molecular design of new medicaments based on molecular models of biological targets: virtual screening and de novo design, are presented. Examples of the above-listed approaches for the simulation of a number of pharmacologically significant receptors, analysis of receptor-ligand interactions and design of new biologically active organic compounds are given.

  16. Molecular simulation of receptors of physiologically active compounds for purposes of medical chemistry

    Baskin, Igor I; Palyulin, Vladimir A; Zefirov, Nikolai S [Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow (Russian Federation)

    2009-06-30

    The general strategy of the molecular simulation of biological receptors and their interaction with ligands is considered. The procedures for construction of 3D protein models, molecular docking, evaluation of model quality, determination of the free energy of protein binding with ligands are discussed. The methods of molecular design of new medicaments based on molecular models of biological targets: virtual screening and de novo design, are presented. Examples of the above-listed approaches for the simulation of a number of pharmacologically significant receptors, analysis of receptor-ligand interactions and design of new biologically active organic compounds are given.

  17. Role of NK1 and NK2 receptors in mouse gastric mechanical activity

    2006-01-01

    The aim of the present study was to examine the role of NK1 and NK2 receptors in the control of mechanical activity of mouse stomach. In this view, the motor effects induced by NK1 and NK2 receptor agonists and antagonists were analyzed, measuring motility as intraluminal pressure changes in mouse-isolated stomach preparations. In parallel, immunohistochemical studies were performed to identify the location of NK1 and NK2 receptors on myenteric neurons and smooth muscle cells.Substance P (SP)...

  18. Acutely increasing δGABAA receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABAA) receptors that contain the δ subunit (δGABAA receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABAA receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABAA receptor–preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enh...

  19. Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion.

    Alex de Voux

    Full Text Available The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·⁴⁹(¹²⁵ and Arg⁶·³²(²²⁵ residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·⁵⁶(⁸², in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr²·⁵⁶(⁸² mutants were fully stabilized in active conformations. The Thr²·⁵⁶(⁸²Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·⁵⁶(⁸²Lys mutation with an Arg⁶·³²(²²⁵Gln mutation partially reversed the decrease in expression. Mutants with Thr²·⁵⁶(⁸²Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·⁶⁵(⁸²Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·⁶⁵(⁸²Lys and Thr²·⁶⁵(⁸²Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82 stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated

  20. Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion.

    de Voux, Alex; Chan, Mei-Chi; Folefoc, Asongna T; Madziva, Michael T; Flanagan, Colleen A

    2013-01-01

    The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp³·⁴⁹(¹²⁵) and Arg⁶·³²(²²⁵) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr²·⁵⁶(⁸²), in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr²·⁵⁶(⁸²) mutants were fully stabilized in active conformations. The Thr²·⁵⁶(⁸²)Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr²·⁵⁶(⁸²)Lys mutation with an Arg⁶·³²(²²⁵)Gln mutation partially reversed the decrease in expression. Mutants with Thr²·⁵⁶(⁸²)Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr²·⁶⁵(⁸²)Pro substitution exhibited full co-receptor function. Our results suggest that the Thr²·⁶⁵(⁸²)Lys and Thr²·⁶⁵(⁸²)Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations

  1. Activation of murine microglial N9 cells is attenuated through cannabinoid receptor CB2 signaling.

    Ma, Lei; Jia, Ji; Liu, Xiangyu; Bai, Fuhai; Wang, Qiang; Xiong, Lize

    2015-02-27

    Inhibition of microglial activation is effective in treating various neurological disorders. Activation of microglial cannabinoid CB2 receptor induces anti-inflammatory effects, and the mechanism, however, is still elusive. Microglia could be activated into the classic activated state (M1 state) or the alternative activated state (M2 state), the former is cytotoxic, and the latter is neurotrophic. In this study, we used lipopolysaccharide (LPS) plus interferon-γ (IFNγ) to activate N9 microglia and hypothesized the pretreatment with cannabinoid CB2 receptor agonist AM1241 attenuates microglial activation by shifting microglial M1 to M2 state. We found that pretreatment with 5 μM AM1241 at 1 h before microglia were exposed to LPS plus IFNγ decreased the expression of inducible nitric oxide synthase (iNOS) and the release of pro-inflammatory factors, increased the expression of arginase 1 (Arg-1) and the release of anti-inflammatory and neurotrophic factors in microglia. However, these effects induced by AM1241 pretreatment were significantly reversed in the presence of 10 μM cannabinoid CB2 receptor antagonist AM630 or 10 μM protein kinase C (PKC) inhibitor chelerythrine. These findings indicated that AM1241 pretreatment attenuates microglial activation by shifting M1 to M2 activated state via CB2 receptor, and the AM1241-induced anti-inflammatory effects may be mediated by PKC.

  2. Pleiotropic β-Agonist–Promoted Receptor Conformations and Signals Independent of Intrinsic Activity

    2006-01-01

    β-Agonists used for treatment of obstructive lung disease have a variety of different structures but are typically classified by their intrinsic activities for stimulation of cAMP, and predictions are made concerning other downstream signals based on such a classification. We generated modified β2-adrenergic receptors with insertions of energy donor and acceptor moieties to monitor agonist-promoted conformational changes of the receptor using intramolecular bioluminescence resonance energy tr...

  3. Kainate-type glutamate receptors modulating network activity in developing hippocampus

    Juuri, Juuso

    2015-01-01

    Kainate-type of ionotropic glutamate (KA) receptors are associated with the modulation of neuronal excitability, synaptic transmission, and activity of neuronal networks. They are believed to have an important role in the development of neuronal connections. In this thesis, the role of KA receptors in the early brain development was assessed by conducting in vitro electrophysiological recordings from individual neurons at CA3 region in acute slices of neonatal rodent hippocampi. It was f...

  4. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer.

  5. T-cell receptor-induced JNK activation requires proteolytic inactivation of CYLD by MALT1.

    Staal, Jens; Driege, Yasmine; Bekaert, Tine; Demeyer, Annelies; Muyllaert, David; Van Damme, Petra; Gevaert, Kris; Beyaert, Rudi

    2011-05-04

    The paracaspase mucosa-associated lymphoid tissue 1 (MALT1) is central to lymphocyte activation and lymphomagenesis. MALT1 mediates antigen receptor signalling to NF-κB by acting as a scaffold protein. Furthermore, MALT1 has proteolytic activity that contributes to optimal NF-κB activation by cleaving the NF-κB inhibitor A20. Whether MALT1 protease activity is involved in other signalling pathways, and the identity of the relevant substrates, is unknown. Here, we show that T-cell receptors (TCR) activation, as well as overexpression of the oncogenic API2-MALT1 fusion protein, results in proteolytic inactivation of CYLD by MALT1, which is specifically required for c-jun N-terminal kinase (JNK) activation and the inducible expression of a subset of genes. These results indicate a novel role for MALT1 proteolytic activity in TCR-induced JNK activation and reveal CYLD cleavage as the underlying mechanism.

  6. Requirement for caspase-8 in NF-kappaB activation by antigen receptor.

    Su, Helen; Bidère, Nicolas; Zheng, Lixin; Cubre, Alan; Sakai, Keiko; Dale, Janet; Salmena, Leonardo; Hakem, Razqallah; Straus, Stephen; Lenardo, Michael

    2005-03-04

    Caspase-8, a proapoptotic protease, has an essential role in lymphocyte activation and protective immunity. We show that caspase-8 deficiency (CED) in humans and mice specifically abolishes activation of the transcription factor nuclear factor kappaB (NF-kappaB) after stimulation through antigen receptors, Fc receptors, or Toll-like receptor 4 in T, B, and natural killer cells. Caspase-8 also causes the alphabeta complex of the inhibitor of NF-kappaB kinase (IKK) to associate with the upstream Bcl10-MALT1 (mucosa-associated lymphatic tissue) adapter complex. Recruitment of the IKKalpha, beta complex, its activation, and the nuclear translocation of NF-kappaB require enzyme activity of full-length caspase-8. These findings thus explain the paradoxical association of defective apoptosis and combined immunodeficiency in human CED.

  7. Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation.

    Sommer, Anselm; Fries, Anja; Cornelsen, Isabell; Speck, Nancy; Koch-Nolte, Friedrich; Gimpl, Gerald; Andrä, Jörg; Bhakdi, Sucharit; Reiss, Karina

    2012-07-06

    Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.

  8. Natural compounds regulate energy metabolism by the modulating the activity of lipid-sensing nuclear receptors.

    Goto, Tsuyoshi; Kim, Young-Il; Takahashi, Nobuyuki; Kawada, Teruo

    2013-01-01

    Obesity causes excess fat accumulation in various tissues, most notoriously in the adipose tissue, along with other insulin-responsive organs such as skeletal muscle and the liver, which predisposes an individual to the development of metabolic abnormalities. The molecular mechanisms underlying obesity-induced metabolic abnormalities have not been completely elucidated; however, in recent years, the search for therapies to prevent the development of obesity and obesity-associated metabolic disorders has increased. It is known that several nuclear receptors, when activated by specific ligands, regulate carbohydrate and lipid metabolism at the transcriptional level. The expression of lipid metabolism-related enzymes is directly regulated by the activity of various nuclear receptors via their interaction with specific response elements in promoters of those genes. Many natural compounds act as ligands of nuclear receptors and regulate carbohydrate and lipid metabolism by regulating the activities of these nuclear receptors. In this review, we describe our current knowledge of obesity, the role of lipid-sensing nuclear receptors in energy metabolism, and several examples of food factors that act as agonists or antagonists of nuclear receptors, which may be useful for the management of obesity and the accompanying energy metabolism abnormalities.

  9. Estimation of the receptor-state affinity constants of ligands in functional studies using wild type and constitutively active mutant receptors: Implications for estimation of agonist bias.

    Ehlert, Frederick J; Stein, Richard S L

    We describe a method for estimating the affinities of ligands for active and inactive states of a G protein-coupled receptor (GPCR). Our protocol involves measuring agonist-induced signaling responses of a wild type GPCR and a constitutively active mutant of it under control conditions and after partial receptor inactivation or reduced receptor expression. Our subsequent analysis is based on the assumption that the activating mutation increases receptor isomerization into the active state without affecting the affinities of ligands for receptor states. A means of confirming this assumption is provided. Global nonlinear regression analysis yields estimates of 1) the active (Kact) and inactive (Kinact) receptor-state affinity constants, 2) the isomerization constant of the unoccupied receptor (Kq-obs), and 3) the sensitivity constant of the signaling pathway (KE-obs). The latter two parameters define the output response of the receptor, and hence, their ratio (Kq-obs/KE) is a useful measure of system bias. If the cellular system is reasonably stable and the Kq-obs and KE-obs values of the signaling pathway are known, the Kact and Kinact values of additional agonists can be estimated in subsequent experiments on cells expressing the wild type receptor. We validated our method through computer simulation, an analytical proof, and analysis of previously published data. Our approach provides 1) a more meaningful analysis of structure-activity relationships, 2) a means of validating in silico docking experiments on active and inactive receptor structures and 3) an absolute, in contrast to relative, measure of agonist bias.

  10. Spontaneous release of GABA activates GABAB receptors and controls network activity in the neonatal rat hippocampus.

    McLean, H A; Caillard, O; Khazipov, R; Ben-Ari, Y; Gaiarsa, J L

    1996-08-01

    giant glutamatergic potentials were observed in simultaneously recorded CA3 pyramidal cells and interneurons. CGP 35348 (0.5 mM) progressively increased the duration of these bicuculline-induced glutamatergic bursts leading to the simultaneous appearance of ictal discharges in both pyramidal cells and interneurons. 6. These results suggest that in the neonatal CA3 hippocampal region, when synchronous giant polysynaptic GABAergic PSPs are present (i.e., under basal, control conditions), spontaneously released GABA reaches a critical level and activates GABAB receptors on both pyramidal cells and interneurons thus regulating the level of glutamatergic and GABAergic activity in the CA3 neuronal network.

  11. A common model for cytokine receptor activation: combined scissor-like rotation and self-rotation of receptor dimer induced by class I cytokine.

    Xiaodong Pang

    Full Text Available The precise mechanism by which the binding of a class I cytokine to the extracellular domain of its corresponding receptor transmits a signal through the cell membrane remains unclear. Receptor activation involves a cytokine-receptor complex with a 1∶2 stoichiometry. Previously we used our transient-complex theory to calculate the rate constant of the initial cytokine-receptor binding to form a 1∶1 complex. Here we computed the binding pathway leading to the 1∶2 activation complex. Three cytokine systems (growth hormone, erythropoietin, and prolactin were studied, and the focus was on the binding of the extracellular domain of the second receptor molecule after forming the 1∶1 complex. According to the transient-complex theory, translational and rotation diffusion of the binding entities bring them together to form a transient complex, which has near-native relative separation and orientation but not the short-range specific native interactions. Subsequently conformational rearrangement leads to the formation of the native complex. We found that the changes in relative orientations between the two receptor molecules from the transient complex to the 1∶2 native complex are similar for the three cytokine-receptor systems. We thus propose a common model for receptor activation by class I cytokines, involving combined scissor-like rotation and self-rotation of the two receptor molecules. Both types of rotations seem essential: the scissor-like rotation separates the intracellular domains of the two receptor molecules to make room for the associated Janus kinase molecules, while the self-rotation allows them to orient properly for transphosphorylation. This activation model explains a host of experimental observations. The transient-complex based approach presented here may provide a strategy for designing antagonists and prove useful for elucidating activation mechanisms of other receptors.

  12. Structural mechanism of ligand activation in human calcium-sensing receptor

    Geng, Yong; Mosyak, Lidia; Kurinov, Igor; Zuo, Hao; Sturchler, Emmanuel; Cheng, Tat Cheung; Subramanyam, Prakash; Brown, Alice P.; Brennan, Sarah C.; Mun, Hee-chang; Bush, Martin; Chen, Yan; Nguyen, Trang X.; Cao, Baohua; Chang, Donald D.; Quick, Matthias; Conigrave, Arthur D.; Colecraft, Henry M.; McDonald, Patricia; Fan, Qing R.

    2016-07-19

    Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+and PO43-ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ions stabilize the active state, PO43-ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits.

  13. Differential activation of nitric oxide synthase through muscarinic acetylcholine receptors in rat salivary glands.

    Leirós, C P; Rosignoli, F; Genaro, A M; Sales, M E; Sterin-Borda, L; Santiago BordaE

    2000-03-15

    Muscarinic receptors play an important role in secretory and vasodilator responses in rat salivary glands. Nitric oxide synthase (NOS) appears to be one of the multiple effectors coupled to muscarinic receptors in both submandibular and sublingual glands although some differences have been found depending on the gland studied. First, submandibular glands had a lower basal activity of nitric oxide synthase than sublingual glands and the concentration-response curve for carbachol was bell-shaped in the former but not in sublingual glands. Second, cGMP levels displayed a similar profile to that observed for NOS activity in both glands. Third, protein kinase C also coupled to muscarinic receptor activation in the glands might have a regulatory effect on nitric oxide production since its activity was higher in basal conditions in submandibular than sublingual glands and it also increased in the presence of the agonist at a concentration that inhibited NOS activity in submandibular glands. The effects appear to be partly related to the expression of a minor population of M(1) receptors in submandibular glands absent in sublingual as determined in binding and signaling experiments with the muscarinic receptor antagonist pirenzepine.

  14. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity.

    Chen, Xingjuan; Egly, Christian; Riley, Ashley M; Li, Wennan; Tewson, Paul; Hughes, Thomas E; Quinn, Anne Marie; Obukhov, Alexander G

    2014-01-01

    Transient receptor potential canonical 6 (TRPC6) is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R), TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG) and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  15. PKC-dependent Phosphorylation of the H1 Histamine Receptor Modulates TRPC6 Activity

    Xingjuan Chen

    2014-04-01

    Full Text Available Transient receptor potential canonical 6 (TRPC6 is a cation selective, DAG-regulated, Ca2+-permeable channel activated by the agonists of Gq-protein-coupled heptahelical receptors. Dysfunctions of TRPC6 are implicated in the pathogenesis of various cardiovascular and kidney conditions such as vasospasm and glomerulosclerosis. When stimulated by agonists of the histamine H1 receptor (H1R, TRPC6 activity decays to the baseline despite the continuous presence of the agonist. In this study, we examined whether H1R desensitization contributes to regulating the decay rate of TRPC6 activity upon receptor stimulation. We employed the HEK expression system and a biosensor allowing us to simultaneously detect the changes in intracellular diacylglycerol (DAG and Ca2+ concentrations. We found that the histamine-induced DAG response was biphasic, in which a transient peak was followed by maintained elevated plateau, suggesting that desensitization of H1R takes place in the presence of histamine. The application of PKC inhibitor Gö6983 slowed the decay rate of intracellular DAG concentration. Activation of the mouse H1R mutant lacking a putative PKC phosphorylation site, Ser399, responsible for the receptor desensitization, resulted in a prolonged intracellular DAG increase and greater Mn2+ influx through the TRPC6 channel. Thus, our data support the hypothesis that PKC-dependent H1R phosphorylation leads to a reduced production of intracellular DAG that contributes to TRPC6 activity regulation.

  16. Expression of the Inhibitory CD200 Receptor Is Associated with Alternative Macrophage Activation

    N. Koning; M. van Eijk; W. Pouwels; M.S.M. Brouwer; D. Voehringer; I. Huitinga; R.M. Hoek; G. Raes; J. Hamann

    2010-01-01

    Classical macrophage activation is inhibited by the CD200 receptor (CD200R). Here, we show that CD200R expression was specifically induced on human in vitro polarized macrophages of the alternatively activated M2a subtype, generated by incubation with IL-4 or IL-13. In mice, peritoneal M2 macrophage

  17. Prediction of in vitro and in vivo oestrogen receptor activity using hierarchical clustering

    In this study, hierarchical clustering classification models were developed to predict in vitro and in vivo oestrogen receptor (ER) activity. Classification models were developed for binding, agonist, and antagonist in vitro ER activity and for mouse in vivo uterotrophic ER bindi...

  18. Eph receptors are involved in the activity-dependent synaptic wiring in the mouse cerebellar cortex.

    Roberta Cesa

    Full Text Available Eph receptor tyrosine kinases are involved in many cellular processes. In the developing brain, they act as migratory and cell adhesive cues while in the adult brain they regulate dendritic spine plasticity. Here we show a new role for Eph receptor signalling in the cerebellar cortex. Cerebellar Purkinje cells are innervated by two different excitatory inputs. The climbing fibres contact the proximal dendritic domain of Purkinje cells, where synapse and spine density is low; the parallel fibres contact the distal dendritic domain, where synapse and spine density is high. Interestingly, Purkinje cells have the intrinsic ability to generate a high number of spines over their entire dendritic arborisations, which can be innervated by the parallel fibres. However, the climbing fibre input continuously exerts an activity-dependent repression on parallel fibre synapses, thus confining them to the distal Purkinje cell dendritic domain. Such repression persists after Eph receptor activation, but is overridden by Eph receptor inhibition with EphA4/Fc in neonatal cultured cerebellar slices as well as mature acute cerebellar slices, following in vivo infusion of the EphA4/Fc inhibitor and in EphB receptor-deficient mice. When electrical activity is blocked in vivo by tetrodotoxin leading to a high spine density in Purkinje cell proximal dendrites, stimulation of Eph receptor activation recapitulates the spine repressive effects of climbing fibres. These results suggest that Eph receptor signalling mediates the repression of spine proliferation induced by climbing fibre activity in Purkinje cell proximal dendrites. Such repression is necessary to maintain the correct architecture of the cerebellar cortex.

  19. Antihyperalgesic effect of CB1 receptor activation involves the modulation of P2X3 receptor in the primary afferent neuron.

    Oliveira-Fusaro, Maria Cláudia Gonçalves; Zanoni, Cristiane Isabel Silva; Dos Santos, Gilson Gonçalves; Manzo, Luis Paulo; Araldi, Dionéia; Bonet, Ivan José Magayewski; Tambeli, Cláudia Herrera; Dias, Elayne Vieira; Parada, Carlos Amilcar

    2017-03-05

    Cannabinoid system is a potential target for pain control. Cannabinoid receptor 1 (CB1) activation play a role in the analgesic effect of cannabinoids once it is expressed in primary afferent neurons. This study investigates whether the anti-hyperalgesic effect of CB1 receptor activation involves P2X3 receptor in primary afferent neurons. Mechanical hyperalgesia was evaluated by electronic von Frey test. Cannabinoid effect was evaluated using anandamide or ACEA, a non-selective or a selective CB1 receptor agonists, respectively; AM251, a CB1 receptor antagonist, and antisense ODN for CB1 receptor. Calcium imaging assay was performed to evaluated α,β-meATP-responsive cultured DRG neurons pretreated with ACEA. Anandamide or ACEA administered in peripheral tissue reduced the carrageenan-induced mechanical hyperalgesia. The reduction in the carrageenan-induced hyperalgesia induced by ACEA was completely reversed by administration of AM251 as well as by the intrathecal treatment with antisense ODN for CB1 receptor. Also, ACEA reduced the mechanical hyperalgesia induced by bradykinin and by α,β-meATP, a P2X3 receptor non-selective agonist, but not by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and chemokine-induced chemoattractant-1 (CINC-1). Finally, CB1 receptors are co-localized with P2X3 receptors in DRG small-diameter neurons and the treatment with ACEA reduced the number of α,β-meATP-responsive cultured DRG neurons. Our data suggest that the analgesic effect of CB1 receptor activation is mediated by a negative modulation of the P2X3 receptor in the primary afferent neurons.

  20. Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.

    Staus, Dean P; Strachan, Ryan T; Manglik, Aashish; Pani, Biswaranjan; Kahsai, Alem W; Kim, Tae Hun; Wingler, Laura M; Ahn, Seungkirl; Chatterjee, Arnab; Masoudi, Ali; Kruse, Andrew C; Pardon, Els; Steyaert, Jan; Weis, William I; Prosser, R Scott; Kobilka, Brian K; Costa, Tommaso; Lefkowitz, Robert J

    2016-07-21

    G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and β-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the β2-adrenergic receptor (β2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct β2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for β2AR in the presence of Nb80 compared to the affinity of isoprenaline for β2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the β2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of

  1. Activation of human tonsil and skin mast cells by agonists of proteinase activated receptor-2

    Shao-heng HE; Hua XIE; Yi-ling FU

    2005-01-01

    Aim: To investigate the effects of the agonists of proteinase activated receptor (PAR)-2,and histamine on degranulation of human mast cells. Methods: Human mast cells were enzymatically dispersed from tonsil and skin tissues. The dis persed cells were then cultured with various stimuli, and tryptase and histamine levels in cell supernatants collected from challenge tubes were measured. Results:PAR-2 agonist peptide SLIGKV provoked a dose-dependent release of histamine from skin mast cells. It also induced tryptase release from tonsil mast cells, tcLIGRLO appeared less potent than SLIGKV in induction of release of histamine and tryptase. Trypsin was able to induce a "bell" shape increase in tryptase release from tonsil mast cells. It was also able to induce a dose-dependent release of histamine from both tonsil and skin mast cells. The actions of trypsin on mast cells were inhibited by soy bean trypsin inhibitor (SBTI) or α1-antitrypsin (α1-AT).Time course study revealed that both stimulated tryptase or histamine release initiated within 10 s and reached their peak release between 4 and 6 min. Pretreatment of cells with metabolic inhibitors or pertussis toxin reduced the ability of mast cells to release tryptase or histamine. Conclusion: It was demonstrated that the in vitro tryptase release properties of human tonsil and skin mast cells suggested a novel type of mast cell heterogeneity. The activation of mast cells by PAR-2 agonists indicated a self-amplification mechanism of mast cell degranulation.

  2. Modulating activity of M1 receptor to the reaction of ileal smooth muscle

    Izabela Glaza

    2011-08-01

    Full Text Available Background:The subject of the study was determination of the effect of drugs on ileal smooth muscle contraction induced by activation of M1 type muscarinic receptors. Drugs that have an effect on muscarinic receptors are divided to agonists, with close ties to the receptor and high internal activity and antagonists, with no internal activity. Conducted experiments tested interactions between a broad-spectrum agonist of muscarinic receptors, carbachol and a selective muscarinic receptor antagonist of M1 type, pirenzepine.Material/Methods:Testing was conducted on tissues isolated from rat’s intestine. Male Wistar rats with weight between 220 g and 360 g were anesthetized by intraperitoneal injection of urethane (120 mg/kg. Concentration-effect curves were determined with the use of cumulated concentration method, in accordance with the van Rossum method (1963 in Kenakin modification (2006.Results:The purpose of the study was determination of concentration-effect curves for carbachol. This curve was compared with the curve of receptor occupation depending on concentration of this drug. Based on concentration-effect curves, the average value of EC50 was calculated for carbachol, amounting to 2.44×10–6 [M/l].Conclusions:The results confirmed that atropine is effective in stopping contractions caused by carbachol, meeting the conditions of competitive antagonists. Atropine caused the shift of curves for carbachol to the right. Pirenzepine, selectively blocking muscarinic receptors of M1 type gave similar results. It was proved that in the preparation of gastric fundus smooth muscle, M1 type receptors occur not only presynaptically, but also postsynaptically.

  3. Analysis of the heat shock response in mouse liver reveals dependence on the Nuclear Receptor Peroxisome Proliferator-Activated Receptor alpha (PPARalpha)

    Vallanat, B.; Anderson, S.P.; Brown-Borg, H.M.; Ren, H.; Kersten, A.H.; Jonnalagadda, S.; Srinivasan, S.; Corton, J.C.

    2010-01-01

    Background - The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARa) regulates responses to chemical or physical stress in part by altering expression of genes involved in proteome maintenance. Many of these genes are also transcriptionally regulated by heat shock (HS) through

  4. Peroxisome Proliferator-activated Receptor gamma Regulates Expression of the Anti-lipolytic G-protein-coupled Receptor 81 (GPR81/Gpr81)

    Jeninga, E.H.; Bugge, A.; Nielsen, R.; Kersten, A.H.; Hamers, N.; Dani, C.; Wabitsch, M.; Berger, R.; Stunnenberg, H.G.; Mandrup, S.; Kalkhoven, E.

    2009-01-01

    The ligand-inducible nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) plays a key role in the differentiation, maintenance, and function of adipocytes and is the molecular target for the insulin-sensitizing thiazoledinediones (TZDs). Although a number of PPAR gamma targ

  5. A novel signaling pathway of tissue kallikrein in promoting keratinocyte migration: Activation of proteinase-activated receptor 1 and epidermal growth factor receptor

    Gao, Lin; Chao, Lee [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States); Chao, Julie, E-mail: chaoj@musc.edu [Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC 29425-2211 (United States)

    2010-02-01

    Biological functions of tissue kallikrein (TK, KLK1) are mainly mediated by kinin generation and subsequent kinin B2 receptor activation. In this study, we investigated the potential role of TK and its signaling pathways in cultured human keratinocyte migration and in a rat skin wound healing model. Herein, we show that TK promoted cell migration and proliferation in a concentration- and time-dependent manner. Inactive TK or kinin had no significant effect on cell migration. Interestingly, cell migration induced by active TK was not blocked by icatibant or L-NAME, indicating an event independent of kinin B2 receptor and nitric oxide formation. TK's stimulatory effect on cell migration was inhibited by small interfering RNA for proteinase-activated receptor 1 (PAR{sub 1}), and by PAR{sub 1} inhibitor. TK-induced migration was associated with increased phosphorylation of epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK), which was blocked by inhibition of protein kinase C (PKC), Src, EGFR and ERK. TK-induced cell migration and EGFR phosphorylation were blocked by metalloproteinase (MMP) inhibitor, heparin, and antibodies against EGFR external domain, heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR). Local application of TK promoted skin wound healing in rats, whereas icatibant and EGFR inhibitor blocked TK's effect. Skin wound healing was further delayed by aprotinin and neutralizing TK antibody. This study demonstrates a novel role of TK in skin wound healing and uncovers new signaling pathways mediated by TK in promoting keratinocyte migration through activation of the PAR{sub 1}-PKC-Src-MMP pathway and HB-EGF/AR shedding-dependent EGFR transactivation.

  6. Glucose activation of islets of Langerhans up-regulates Toll-like receptor 5: possible mechanism of protection

    Weile, Christian Roar Andersen; Josefsen, Knud Elnegaard; Buschard, Karsten Stig

    2011-01-01

    Toll-like receptors are pattern-recognition receptors of the innate immune system that are activated during viral, bacterial or other infections, as well as during disease progression of type 1 and type 2 diabetes. Toll-like receptor 5 (TLR-5) specifically recognizes bacterial infection through b...

  7. Transmembrane α-Helix 2 and 7 Are Important for Small Molecule-Mediated Activation of the GLP-1 Receptor

    Underwood, Christina Rye; Møller Knudsen, Sanne; Schjellerup Wulff, Birgitte;

    2011-01-01

    , the structurally related small molecule, compound 3, stimulated cAMP production from GLP-1R, but not from the homologous glucagon receptor (GluR). The receptor selectivity encouraged a chimeric receptor approach to identify domains important for compound 3-mediated activation of GLP-1R. A subsegment of the GLP-1R...

  8. Investigation of in vitro Opioid Receptor Binding Activities of Some Turkish Salvia species

    Özge Gündüz Çınar

    2011-01-01

    Full Text Available Kappa Opioid Peptide Receptor (KOPr activation produces analgesic, psychotomimetic, diuretic and antipruritic effects. KOPr ligands are investigated for their potential roles in the treatment of addiction, depression, feeding behavior, psychosis and schizophrenia. In this study the methanolic extracts of a number of Salvia species which are native to Turkey (S. tomentosa, S. tchihatcheffii , S. rosifolia, S. dichroantha and S. sclarea were tested for their potential binding to opioid receptors in rat brain membranes and Chinese Hamster Ovary Cells expressing human KOPr (CHO-KOPh. [ 3H]Diprenorphine, an unselective opioid antagonist, was utilized in the radioligand receptor binding assays. All extracts (0.11 mg/ml inhibited the [ 3H]Diprenorphine binding with ranging KOPr binding affinities. More than 50% inhibition of diprenorphine binding was shown only with Salvia dichroantha and Salvia sclarea both in rat brain membranes and CHO-KOPh membranes.Among them Salvia sclarea deserves further investigation for its active component(s and its pharmacological characterization. This study clearly demonstrates the potential opioid receptor binding activities of several Turkish Salvia species. This work constitutes the first study on in vitro opioid receptor binding activities of Salvia species from the Turkish flora.

  9. Pericellular proteolytic cascade by plasmin/plasminogen activator system

    2001-01-01

    Plasmin/plasminogen activators (PA) are the serine enzyme which digests fibrin and/or fibrinogen. Plasmin is produced by the cleavage of its precursor, plasminogen by PAs (urokinase-type PA and tissue-type PA). These events are expected in the thrmbolytic therapy for thromboembolic deseases. Apart from the blood fibrinolysis mentioned above, new role of plasmin/plasminogen activators has been extensively investigated in the field of cellular biology. On the cell surface, the receptor for urokinase-type PA (u-PAR) was found (that for t-PA has not cloned yet). Then, plasmin as well as u-PA itself activates pro-form of matrix metalloproteinases (MMPs) around the pericellular space. These proteolytic activities by u-PA, plasmin and MMPs induce the degradation of extracellular matrix (ECM), affording the cells certain enviroment for their biological function. Further, the coupling of u-PA/u-PAR system and integrins can generate intracellular signal transductions which take part in the regulation of cell proliferation, attachment or migration followed by various physiological and pathophysiological functions. These serial mechanisms are the principle of pericellular proteolytic cascade.

  10. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

    Tunaru, Sorin; Althoff, Till F; Nüsing, Rolf M; Diener, Martin; Offermanns, Stefan

    2012-06-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

  11. Prejunctional inhibition of sympathetically evoked pupillary dilation in cats by activation of histamine H3 receptors.

    Koss, M C; Hey, J A

    1993-08-01

    Frequency-dependent pupillary dilations were evoked by electrical stimulation of the pre- or post-ganglionic cervical sympathetic nerve (sympatho-excitation) or the hypothalamus (parasympatho-inhibition) in sympathectomized anesthetized cats. Systemic administration of the selective histamine H3 receptor agonist (R)-alpha-methylhistamine (R alpha MeHA) produced a dose-dependent depression of mydriasis due to direct neural sympathetic activation but had no effect on responses elicited by parasympathetic withdrawal. The histamine H2 receptor agonist, dimaprit, was inactive. R alpha MeHA was much more effective in depressing sympathetic responses obtained at lower frequencies when compared to higher frequencies of stimulation. Responses evoked both pre- and postganglionically were inhibited by R alpha MeHA. This peripheral sympatho-inhibitory action of R alpha MeHA was antagonized by the histamine H3 receptor blocker thioperamide but not by intravenous pretreatment with the histamine H1 receptor antagonist chlorpheniramine. Histamine H2 receptor blockers cimetidine and ranitidine were also without effect. R alpha MeHA did not depress pupillary responses elicited by i.v. (-)-adrenaline. The results demonstrate that histamine H3 receptors modulate sympathetic activation of the iris at a site proximal to the iris dilator muscle. The predominant mechanism of action appears to the prejunctional inhibition of noradrenaline release from postganglionic sympathetic nerve endings. However, a concomitant ganglionic inhibitory action cannot be excluded.

  12. Phasic and tonic mGlu7 receptor activity modulates the thalamocortical network

    Valériane eTassin

    2016-04-01

    Full Text Available Mutation of the metabotropic glutamate receptor type 7 (mGlu7 induces absence-like epileptic seizures, but its precise role in the somatosensory thalamocortical network remains unknown. By combining electrophysiological recordings, optogenetics and pharmacology we dissected the contribution of the mGlu7 receptor at mouse thalamic synapses. We found that mGlu7 is functionally expressed at both glutamatergic and GABAergic synapses, where it can inhibit neurotransmission and regulate short-term plasticity. These effects depend on the PDZ-ligand of the receptor, as they are lost in mutant mice. Interestingly, the very low affinity of mGlu7 receptors for glutamate raises the question of how it can be activated, namely at GABAergic synapses and in basal conditions. Inactivation of the receptor activity with the mGlu7 negative allosteric modulator (NAM, ADX71743, enhances thalamic synaptic transmission. In vivo administration of the NAM induces a lethargic state with spindle and/or spike-and-wave discharges accompanied by a behavioral arrest typical of absence epileptic seizures. This provides evidence for mGlu7 receptor-mediated tonic modulation of a physiological function in vivo preventing synchronous and potentially pathological oscillations.

  13. Prostaglandin E2 regulates angiogenesis via activation of fibroblast growth factor receptor-1.

    Finetti, Federica; Solito, Raffaella; Morbidelli, Lucia; Giachetti, Antonio; Ziche, Marina; Donnini, Sandra

    2008-01-25

    Prostaglandin E(2) (PGE(2)) behaves as a mitogen in epithelial tumor cells as well as in many other cell types. We investigated the actions of PGE(2) on microvascular endothelial cells (capillary venular endothelial cells) with the purpose of delineating the signaling pathway leading to the acquisition of the angiogenic phenotype and to new vessel formation. PGE(2) (100 nM) produced activation of the fibroblast growth factor receptor 1 (FGFR-1), as measured by its phosphorylation, but not of vascular endothelial growth factor receptor 2. PGE(2) stimulated the EP3 subtype receptor, as deduced by abrogation of EP3 Galpha(i) subunit activity through pertussis toxin. Consistent with this result, in human umbilical venular endothelial cells missing the EP3 receptor, PGE(2) did not phosphorylate FGFR-1. Upon binding to its receptor, PGE(2) initiated an autocrine/paracrine signaling cascade involving the intracellular activation of c-Src, activation of matrix metalloproteinase (predominantly MMP2), which in turn caused the mobilization of membrane-anchored fibroblast growth factor-2 (FGF-2). In fact, in cells unable to release FGF-2 the transfection with both FGFR-1 and EP3 did not result in FGFR-1 phosphorylation in response to PGE(2). Relevance for the FGF2-FGFR-1 system was highlighted by confocal analysis, showing receptor internalization after cell exposure to the prostanoid. ERK1/2 appeared to be the distal signal involved, its phosphorylation being sensitive to either cSrc inhibitor or FGFR-1 blocker. Finally, PGE(2) stimulated cell migration and capillary formation in aortic rings, which were severely reduced by inhibitors of signaling molecules or by receptor antagonist. In conclusion, this study provides evidence for the involvement of FGFR-1 through FGF2 in eliciting PGE(2) angiogenic responses. This signaling pattern is similar to the autocrine-paracrine mechanism which operates in endothelial cells to support neovascular growth.

  14. SIRTUIN 1 (SIRT1) AND STEROID HORMONE RECEPTOR ACTIVITY IN CANCER

    Moore, R.L.; Dai, Y.; Faller, D.V.

    2013-01-01

    Sirtuins, which are class III NAD-dependent histone deacetylases (HDACs) that regulate a number of physiological processes, play important roles in the regulation of metabolism, aging, oncogenesis and cancer progression. More recently, a role for the sirtuins in the regulation of steroid hormone receptor signaling is emerging. In this mini-review, we will summarize current research into the regulation of estrogen, androgen, progesterone, mineralocorticoid and glucocorticoid signaling by sirtuins in cancer. Sirtuins can regulate steroid hormone signaling through a variety of molecular mechanisms, including acting as co-regulatory transcription factors, deacetylating histones in the promoters of genes with nuclear receptor binding sites, directly deacetylating steroid hormone nuclear receptors, and regulating pathways which modify steroid hormone receptors through phosphorylation. Furthermore, disruption of sirtuin activity may be an important step in the development of steroid hormone-refractory cancers. PMID:22159506

  15. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inh...

  16. Cannabinoid receptor 1 signalling dampens activity and mitochondrial transport in networks of enteric neurones.

    Boesmans, W; Ameloot, K; van den Abbeel, V; Tack, J; Vanden Berghe, P

    2009-09-01

    Cannabinoid (CB) receptors are expressed in the enteric nervous system (ENS) and CB(1) receptor activity slows down motility and delays gastric emptying. This receptor system has become an important target for GI-related drug development such as in obesity treatment. The aim of the study was to investigate how CB(1) ligands and antagonists affect ongoing activity in enteric neurone networks, modulate synaptic vesicle cycling and influence mitochondrial transport in nerve processes. Primary cultures of guinea-pig myenteric neurones were loaded with different fluorescent markers: Fluo-4 to measure network activity, FM1-43 to image synaptic vesicles and Mitotracker green to label mitochondria. Synaptic vesicle cluster density was assessed by immunohistochemistry and expression of CB(1) receptors was confirmed by RT-PCR. Spontaneous network activity, displayed by both excitatory and inhibitory neurones, was significantly increased by CB(1) receptor antagonists (AM-251 and SR141716), abolished by CB(1) activation (methanandamide, mAEA) and reduced by two different inhibitors (arachidonylamide serotonin, AA-5HT and URB597) of fatty acid amide hydrolase. Antagonists reduced the number of synaptic vesicles that were recycled during an electrical stimulus. CB(1) agonists (mAEA and WIN55,212) reduced and antagonists enhanced the fraction of transported mitochondria in enteric nerve fibres. We found immunohistochemical evidence for an enhancement of synaptophysin-positive release sites with SR141716, while WIN55,212 caused a reduction. The opposite effects of agonists and antagonists suggest that enteric nerve signalling is under the permanent control of CB(1) receptor activity. Using inhibitors of the endocannabinoid degrading enzyme, we were able to show there is endogenous production of a CB ligand in the ENS.

  17. Analysis of the activation mechanism of the guinea-pig Histamine H1-receptor

    Straßer, Andrea; Wittmann, Hans-Joachim

    2007-09-01

    The Histamine H1-receptor (H1R), belonging to the amine receptor-class of family A of the G-protein coupled receptors (GPCRs) gets activated by agonists. The consequence is a conformational change of the receptor, which may involve the binding-pocket. So, for a good prediction of the binding-mode of an agonist, it is necessary to have knowledge about these conformational changes. Meanwhile some experimental data about the structural changes of GPCRs during activation exist. Based on homology modeling of the guinea-pig H1R (gpH1R), using the crystal structure of bovine rhodopsin as template, we performed several MD simulations with distance restraints in order to get an inactive and an active structure of the gpH1R. The calculations led to a Phe6.44/Trp6.48/Phe6.52-switch and linearization of the proline kinked transmembrane helix VI during receptor activation. Our calculations showed that the Trp6.48/Phe6.52-switch induces a conformational change in Phe6.44, which slides between transmembrane helices III and VI. Additionally we observed a hydrogen bond interaction of Ser3.39 with Asn7.45 in the inactive gpH1R, but because of a counterclockwise rotation of transmembrane helix III Ser3.39 establishes a water-mediated hydrogen bond to Asp2.50 in the active gpH1R. Additionally we simulated a possible mechanism for receptor activation with a modified LigPath-algorithm.

  18. Functional Selectivity and Antidepressant Activity of Serotonin 1A Receptor Ligands

    Zdzisław Chilmonczyk

    2015-08-01

    Full Text Available Serotonin (5-HT is a monoamine neurotransmitter that plays an important role in physiological functions. 5-HT has been implicated in sleep, feeding, sexual behavior, temperature regulation, pain, and cognition as well as in pathological states including disorders connected to mood, anxiety, psychosis and pain. 5-HT1A receptors have for a long time been considered as an interesting target for the action of antidepressant drugs. It was postulated that postsynaptic 5-HT1A agonists could form a new class of antidepressant drugs, and mixed 5-HT1A receptor ligands/serotonin transporter (SERT inhibitors seem to possess an interesting pharmacological profile. It should, however, be noted that 5-HT1A receptors can activate several different biochemical pathways and signal through both G protein-dependent and G protein-independent pathways. The variables that affect the multiplicity of 5-HT1A receptor signaling pathways would thus result from the summation of effects specific to the host cell milieu. Moreover, receptor trafficking appears different at pre- and postsynaptic sites. It should also be noted that the 5-HT1A receptor cooperates with other signal transduction systems (like the 5-HT1B or 5-HT2A/2B/2C receptors, the GABAergic and the glutaminergic systems, which also contribute to its antidepressant and/or anxiolytic activity. Thus identifying brain specific molecular targets for 5-HT1A receptor ligands may result in a better targeting, raising a hope for more effective medicines for various pathologies.

  19. Similar activation of signal transduction pathways by the herpesvirus-encoded chemokine receptors US28 and ORF74

    McLean, Katherine A; Holst, Peter J; Martini, Lene;

    2004-01-01

    The virally encoded chemokine receptors US28 from human cytomegalovirus and ORF74 from human herpesvirus 8 are both constitutively active. We show that both receptors constitutively activate the transcription factors nuclear factor of activated T cells (NFAT) and cAMP response element binding pro...

  20. DMPD: Toll receptors, CD14, and macrophage activation and deactivation by LPS. [Dynamic Macrophage Pathway CSML Database

    Full Text Available 12106783 Toll receptors, CD14, and macrophage activation and deactivation by LPS. D...ceptors, CD14, and macrophage activation and deactivation by LPS. PubmedID 12106783 Title Toll receptors, CD14, and macrophage activa...tion and deactivation by LPS. Authors Dobrovolskaia MA,

  1. Activating killer cell Ig-like receptors in health and disease

    Martin A Ivarsson

    2014-04-01

    Full Text Available Expression of non-rearranged HLA class I-binding receptors characterizes human and mouse NK cells. The postulation of the missing-self hypothesis some 30 years ago triggered the subsequent search and discovery of inhibitory MHC-receptors, both in humans and mice. These receptors have two functions; i to control the threshold for NK cell activation, a process termed licensing or education, and ii to inhibit NK cell activation during interactions with healthy HLA class I-expressing cells. The discovery of activating forms of KIRs (aKIR challenged the concept of NK cell tolerance in steady state, as well as during immune challenge: what is the biological role of the activating KIR, in particular when NK cells express aKIRs in the absence of inhibitory receptors? Recently it was shown that aKIRs also participate in the education of NK cells. However, instead of lowering the threshold of activation like iKIRs, the expression of aKIRs has the opposite effect, i.e. rendering NK cells hyporesponsive. These findings may have consequences during NK cell response to viral infection, in cancer development, and in the initial stages of pregnancy. Here we review the current knowledge of activating KIRs, including the biological concept of aKIR-dependent NK cell education, and their impact in health and disease.

  2. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    Glas, Evi; Mückter, Harald; Gudermann, Thomas; Breit, Andreas

    2016-01-01

    Gs protein-coupled receptors regulate many vital body functions by activation of cAMP response elements (CRE) via cAMP-dependent kinase A (PKA)-mediated phosphorylation of the CRE binding protein (CREB). Melanocortin 4 receptors (MC4R) are prototypical Gs-coupled receptors that orchestrate the hypothalamic control of food-intake and metabolism. Remarkably, the significance of PKA for MC4R-induced CRE-dependent transcription in hypothalamic cells has not been rigorously interrogated yet. In two hypothalamic cell lines, we observed that blocking PKA activity had only weak or no effects on reporter gene expression. In contrast, inhibitors of exchange factors directly activated by cAMP-1/2 (EPAC-1/2) mitigated MC4R-induced CRE reporter activation and mRNA induction of the CREB-dependent genes c-fos and thyrotropin-releasing hormone. Furthermore, we provide first evidence that extracellular-regulated kinases-1/2 (ERK-1/2) activated by EPACs and not PKA are the elusive CREB kinases responsible for MC4R-induced CREB/CRE activation in hypothalamic cells. Overall, these data emphasize the pivotal role of EPACs rather than PKA in hypothalamic gene expression elicited by a prototypical Gs-coupled receptor. PMID:27612207

  3. Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process

    Carlos de Torre-Minguela; Maria Barberà-Cremades; Gómez, Ana I.; Fátima Martín-Sánchez; Pablo Pelegrín

    2016-01-01

    The activation of P2X7 receptor (P2X7R) on M1 polarized macrophages induces the assembly of the NLRP3 inflammasome leading to the release of pro-inflammatory cytokines and the establishment of the inflammatory response. However, P2X7R signaling to the NLRP3 inflammasome is uncoupled on M2 macrophages without changes on receptor activation. In this study, we analyzed P2X7R secretome in wild-type and P2X7R-deficient macrophages polarized either to M1 or M2 and proved that proteins released afte...

  4. The role of transmembrane segment II in 7TM receptor activation

    Benned-Jensen, Tau; Rosenkilde, M M

    2009-01-01

    During the two past decades tremendous effort has been put into uncovering the activation mechanism of 7TM receptors. The majority of such studies have focused on the major binding pocket, comprised of transmembrane segments (TM) -III through -VII, as most non-peptide and peptide ligands, in addi......During the two past decades tremendous effort has been put into uncovering the activation mechanism of 7TM receptors. The majority of such studies have focused on the major binding pocket, comprised of transmembrane segments (TM) -III through -VII, as most non-peptide and peptide ligands...

  5. CLE Peptides in Plants: Proteolytic Processing,Structure-Activity Relationship, and Ligand-Receptor Interaction

    Xiaoming Gao; Yongfeng Guo

    2012-01-01

    Ligand-receptor signaling initiated by the CLAVATA3/ENDOSPERM SURROUNDING REGION (CLE) family peptides is critical in regulating cell division and differentiation in meristematic tissues in plants.Biologically active CLE peptides are released from precursor proteins via proteolytic processing.The mature form of CLE ligands consists of 12-13 amino acids with several post-translational modifications.This review summarizes recent progress toward understanding the proteolytic activities that cleave precursor proteins to release CLE peptides,the molecular structure and function of mature CLE ligands,and interactions between CLE ligands and corresponding leucine-rich repeat (LRR) receptor-like kinases (RLKs).

  6. Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators.

    Schlienger, Nathalie; Lund, Birgitte W; Pawlas, Jan; Badalassi, Fabrizio; Bertozzi, Fabio; Lewinsky, Rasmus; Fejzic, Alma; Thygesen, Mikkel B; Tabatabaei, Ali; Bradley, Stefania Risso; Gardell, Luis R; Piu, Fabrice; Olsson, Roger

    2009-11-26

    Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.

  7. The orphan nuclear receptor Rev-Erbalpha is a peroxisome proliferator-activated receptor (PPAR) gamma target gene and promotes PPARgamma-induced adipocyte differentiation

    Fontaine, Coralie; Dubois, Guillaume; Duguay, Yannick;

    2003-01-01

    Rev-Erbalpha (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor alpha gene. Rev-Erbalpha mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor gamma...... (PPARgamma) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbalpha expression is induced by PPARgamma activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro...... for this nuclear receptor as a promoter of adipocyte differentiation....

  8. Synthetic studies of neoclerodane diterpenes from Salvia divinorum: preparation and opioid receptor activity of salvinicin analogues.

    Simpson, Denise S; Katavic, Peter L; Lozama, Anthony; Harding, Wayne W; Parrish, Damon; Deschamps, Jeffrey R; Dersch, Christina M; Partilla, John S; Rothman, Richard B; Navarro, Hernan; Prisinzano, Thomas E

    2007-07-26

    Further modification of salvinorin A (1a), the major active component of Salvia divinorum, has resulted in the synthesis of novel neoclerodane diterpenes with opioid receptor affinity and activity. We report in this study that oxadiazole 11a and salvidivin A (12a), a photooxygenation product of 1a, have been identified as the first neoclerodane diterpenes with kappa antagonist activity. This indicates that additional structural modifications of 1a may lead to analogues with higher potency and utility as drug abuse medications.

  9. receptores

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  10. Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

    Dienger Krista

    2011-09-01

    Full Text Available Abstract Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2; however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient were sensitized using German cockroach (GC feces (frass, the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production, serum IgE levels and airway hyperresponsiveness (AHR were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice

  11. Second extracellular loop of human glucagon-like peptide-1 receptor (GLP-1R) has a critical role in GLP-1 peptide binding and receptor activation.

    Koole, Cassandra; Wootten, Denise; Simms, John; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M

    2012-02-03

    The glucagon-like peptide-1 receptor (GLP-1R) is a therapeutically important family B G protein-coupled receptor (GPCR) that is pleiotropically coupled to multiple signaling effectors and, with actions including regulation of insulin biosynthesis and secretion, is one of the key targets in the management of type II diabetes mellitus. However, there is limited understanding of the role of the receptor core in orthosteric ligand binding and biological activity. To assess involvement of the extracellular loop (ECL) 2 in ligand-receptor interactions and receptor activation, we performed alanine scanning mutagenesis of loop residues and assessed the impact on receptor expression and GLP-1(1-36)-NH(2) or GLP-1(7-36)-NH(2) binding and activation of three physiologically relevant signaling pathways as follows: cAMP formation, intracellular Ca(2+) (Ca(2+)(i)) mobilization, and phosphorylation of extracellular signal-regulated kinases 1 and 2 (pERK1/2). Although antagonist peptide binding was unaltered, almost all mutations affected GLP-1 peptide agonist binding and/or coupling efficacy, indicating an important role in receptor activation. However, mutation of several residues displayed distinct pathway responses with respect to wild type receptor, including Arg-299 and Tyr-305, where mutation significantly enhanced both GLP-1(1-36)-NH(2)- and GLP-1(7-36)-NH(2)-mediated signaling bias for pERK1/2. In addition, mutation of Cys-296, Trp-297, Asn-300, Asn-302, and Leu-307 significantly increased GLP-1(7-36)-NH(2)-mediated signaling bias toward pERK1/2. Of all mutants studied, only mutation of Trp-306 to alanine abolished all biological activity. These data suggest a critical role of ECL2 of the GLP-1R in the activation transition(s) of the receptor and the importance of this region in the determination of both GLP-1 peptide- and pathway-specific effects.

  12. The structural basis of androgen receptor activation: Intramolecular and intermolecular amino–carboxy interactions

    Schaufele, Fred; Carbonell, Xavier; Guerbadot, Martin; Borngraeber, Sabine; Chapman, Mark S.; Ma, Aye Aye K.; Miner, Jeffrey N.; Diamond, Marc I.

    2005-01-01

    Nuclear receptors (NRs) are ligand-regulated transcription factors important in human physiology and disease. In certain NRs, including the androgen receptor (AR), ligand binding to the carboxy-terminal domain (LBD) regulates transcriptional activation functions in the LBD and amino-terminal domain (NTD). The basis for NTD–LBD communication is unknown but may involve NTD–LBD interactions either within a single receptor or between different members of an AR dimer. Here, measurement of FRET between fluorophores attached to the NTD and LBD of the AR established that agonist binding initiated an intramolecular NTD–LBD interaction in the nucleus and cytoplasm. This intramolecular folding was followed by AR self-association, which occurred preferentially in the nucleus. Rapid, ligand-induced intramolecular folding and delayed association also were observed for estrogen receptor-α but not for peroxisome proliferator activated receptor-γ2. An antagonist ligand, hydroxyflutamide, blocked the NTD–LBD association within AR. NTD–LBD association also closely correlated with the transcriptional activation by heterologous ligands of AR mutants isolated from hormone-refractory prostate tumors. Intramolecular folding, but not AR–AR affinity, was disrupted by mutation of an α-helical (23FQNLF27) motif in the AR NTD previously described to interact with the AR LBD in vitro. This work establishes an intramolecular NTD–LBD conformational change as an initial component of ligand-regulated NR function. PMID:15994236

  13. Investigation of Prolactin Receptor Activation and Blockade Using Time-Resolved Fluorescence Resonance Energy Transfer

    Estelle eTallet

    2011-09-01

    Full Text Available The prolactin receptor (PRLR is emerging as a therapeutic target in oncology. Knowledge-based drug design led to the development of a pure PRLR antagonist (Del1-9-G129R-hPRL that was recently shown to prevent PRL-induced mouse prostate tumorogenesis. In humans, the first gain-of-function mutation of the PRLR (PRLRI146L was recently identified in breast tumor patients. At the molecular level, the actual mechanism of action of these two novel players in the PRL system remains elusive. In this study, we addressed whether constitutive PRLR activation (PRLRI146L or PRLR blockade (antagonist involved alteration of receptor oligomerization and/or of inter-chain distances compared to unstimulated and PRL-stimulated PRLR. Using a combination of various biochemical and spectroscopic approaches (co-IP, blue-native electrophoresis, BRET1, we demonstrated that preformed PRLR homodimers are altered neither by PRL- or I146L-induced receptor triggering, nor by antagonist-mediated blockade. These findings were confirmed using a novel time-resolved fluorescence resonance energy transfer (TR-FRET technology that allows monitoring distance changes between cell-surface tagged receptors. This technology revealed that PRLR blockade or activation did not involve detectable distance changes between extracellular domains of receptor chains within the dimer. This study merges with our previous structural investigations suggesting that the mechanism of PRLR activation solely involves intermolecular contact adaptations leading to subtle intramolecular rearrangements.

  14. Allosteric activation of the 5-HT3AB receptor by mCPBG.

    Miles, Timothy F; Lester, Henry A; Dougherty, Dennis A

    2015-04-01

    The 5-HT3AB receptor contains three A and two B subunits in an A-A-B-A-B order. However, serotonin function at the 5-HT3AB receptor has been shown to depend solely on the A-A interface present in the homomeric receptor. Using mutations at sites on both the primary (E122) and complementary (Y146) faces of the B subunit, we demonstrate that meta-chlorophenyl biguanide (mCPBG), a 5-HT3 selective agonist, is capable of binding to and activating the 5-HT3AB receptor at all five subunit interfaces of the heteromer. Further, mCPBG is capable of allosterically modulating the activity of serotonin from these sites. While these five binding sites are similar enough that they conform to a monophasic dose - response relationship, we uncover subtle differences in the heteromeric binding sites. We also find that the A-A interface appears to contribute disproportionately to the efficacy of 5-HT3AB receptor activation.

  15. Motor activity affects adult skeletal muscle re-innervation acting via tyrosine kinase receptors.

    Sartini, Stefano; Bartolini, Fanny; Ambrogini, Patrizia; Betti, Michele; Ciuffoli, Stefano; Lattanzi, Davide; Di Palma, Michael; Cuppini, Riccardo

    2013-05-01

    Recently, muscle expression of brain-derived neurotrophic factor (BDNF) mRNA and protein under activity control has been reported. BDNF is a neurotrophin known to be involved in axon sprouting in the CNS. Hence, we set out to study the effect of chronic treadmill mid-intensity running on adult rat muscle re-innervation, and to explore the involvement of BDNF and tropomyosin-related kinase (Trk) receptors. After nerve crush, muscle re-innervation was evaluated using intracellular recordings, tension recordings, immunostaining and Western blot analyses. An enhanced muscle multiple innervation was found in running rats that was fully reversed to control values blocking Trk receptors or interrupting the running activity. An increase in muscle multiple innervation was also found in sedentary rats treated with a selective TrkB receptor agonist. The expression of TrkB receptors by intramuscular axons was demonstrated, and increased muscle expression of BDNF was found in running animals. The increase in muscle multiple innervation was consistent with the faster muscle re-innervation that we found in running animals. We conclude that, when regenerating axons contact muscle cells, muscle activity progressively increases modulating BDNF and possibly other growth factors, which in turn, acting via Trk receptors, induce axon sprouting to re-innervate skeletal muscle.

  16. No more pain upon Gq-protein-coupled receptor activation: role of endocannabinoids.

    Hu, Sherry Shu-Jung; Ho, Yu-Cheng; Chiou, Lih-Chu

    2014-02-01

    Marijuana has been used to relieve pain for centuries. The analgesic mechanism of its constituents, the cannabinoids, was only revealed after the discovery of cannabinoid receptors (CB1 and CB2) two decades ago. The subsequent identification of the endocannabinoids, anandamide and 2-arachidonoylglycerol (2-AG), and their biosynthetic and degradation enzymes discloses the therapeutic potential of compounds targeting the endocannabinoid system for pain control. Inhibitors of the anandamide and 2-AG degradation enzymes, fatty acid amide hydrolase and monoacylglycerol lipase, respectively, may be superior to direct cannabinoid receptor ligands as endocannabinoids are synthesized on demand and rapidly degraded, focusing action at generating sites. Recently, a promising strategy for pain relief was revealed in the periaqueductal gray (PAG). It is initiated by Gq-protein-coupled receptor (Gq PCR) activation of the phospholipase C-diacylglycerol lipase enzymatic cascade, generating 2-AG that produces inhibition of GABAergic transmission (disinhibition) in the PAG, thereby leading to analgesia. Here, we introduce the antinociceptive properties of exogenous cannabinoids and endocannabinoids, involving their biosynthesis and degradation processes, particularly in the PAG. We also review recent studies disclosing the Gq PCR-phospholipase C-diacylglycerol lipase-2-AG retrograde disinhibition mechanism in the PAG, induced by activating several Gq PCRs, including metabotropic glutamatergic (type 5 metabotropic glutamate receptor), muscarinic acetylcholine (M1/M3), and orexin 1 receptors. Disinhibition mediated by type 5 metabotropic glutamate receptor can be initiated by glutamate transporter inhibitors or indirectly by substance P, neurotensin, cholecystokinin and capsaicin. Finally, the putative role of 2-AG generated after activating the above neurotransmitter receptors in stress-induced analgesia is discussed.

  17. Activation of the G protein-coupled estrogen receptor, but not estrogen receptor α or β, rapidly enhances social learning.

    Ervin, Kelsy Sharice Jean; Mulvale, Erin; Gallagher, Nicola; Roussel, Véronique; Choleris, Elena

    2015-08-01

    Social learning is a highly adaptive process by which an animal acquires information from a conspecific. While estrogens are known to modulate learning and memory, much of this research focuses on individual learning. Estrogens have been shown to enhance social learning on a long-term time scale, likely via genomic mechanisms. Estrogens have also been shown to affect individual learning on a rapid time scale through cell-signaling cascades, rather than via genomic effects, suggesting they may also rapidly influence social learning. We therefore investigated the effects of 17β-estradiol and involvement of the estrogen receptors (ERs) using the ERα agonist propyl pyrazole triol, the ERβ agonist diarylpropionitrile, and the G protein-coupled ER 1 (GPER1) agonist G1 on the social transmission of food preferences (STFP) task, within a time scale that focused on the rapid effects of estrogens. General ER activation with 17β-estradiol resulted in a modest facilitation of social learning, with mice showing a preference up to 30min of testing. Specific activation of the GPER1 also rapidly enhanced social learning, with mice showing a socially learned preference up to 2h of testing. ERα activation instead shortened the expression of a socially learned food preference, while ERβ activation had little to no effects. Thus, rapid estrogenic modulation of social learning in the STFP may be the outcome of competing action at the three main receptors. Hence, estrogens' rapid effects on social learning likely depend on the specific ERs present in brain regions recruited during social learning.

  18. Norepinephrine activates dopamine D4 receptors in the rat lateral habenula.

    Root, David H; Hoffman, Alexander F; Good, Cameron H; Zhang, Shiliang; Gigante, Eduardo; Lupica, Carl R; Morales, Marisela

    2015-02-25

    The lateral habenula (LHb) is involved in reward and aversion and is reciprocally connected with dopamine (DA)-containing brain regions, including the ventral tegmental area (VTA). We used a multidisciplinary approach to examine the properties of DA afferents to the LHb in the rat. We find that >90% of VTA tyrosine hydroxylase (TH) neurons projecting to the LHb lack vesicular monoamine transporter 2 (VMAT2) mRNA, and there is little coexpression of TH and VMAT2 protein in this mesohabenular pathway. Consistent with this, electrical stimulation of LHb did not evoke DA-like signals, assessed with fast-scan cyclic voltammetry. However, electrophysiological currents that were inhibited by L741,742, a DA-D4-receptor antagonist, were observed in LHb neurons when DA uptake or degradation was blocked. To prevent DA activation of D4 receptors, we repeated this experiment in LHb slices from DA-depleted rats. However, this did not disrupt D4 receptor activation initiated by the dopamine transporter inhibitor, GBR12935. As the LHb is also targeted by noradrenergic afferents, we examined whether GBR12935 activation of DA-D4 receptors occurred in slices depleted of norepinephrine (NE). Unlike DA, NE depletion prevented the activation of DA-D4 receptors. Moreover, direct application of NE elicited currents in LHb neurons that were blocked by L741,742, and GBR12935 was found to be a more effective blocker of NE uptake than the NE-selective transport inhibitor nisoxetine. These findings demonstrate that NE is released in the rat LHb under basal conditions and that it activates DA-D4 receptors. Therefore, NE may be an important regulator of LHb function.

  19. Rifaximin is a gut-specific human pregnane X receptor activator.

    Ma, Xiaochao; Shah, Yatrik M; Guo, Grace L; Wang, Ting; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2007-07-01

    Rifaximin, a rifamycin analog approved for the treatment of travelers' diarrhea, is also beneficial in the treatment of multiple chronic gastrointestinal disorders. However, the mechanisms contributing to the effects of rifaximin on chronic gastrointestinal disorders are not fully understood. In the current study, rifaximin was investigated for its role in activation of the pregnane X receptor (PXR), a nuclear receptor that regulates genes involved in xenobiotic and limited endobiotic deposition and detoxication. PXR-humanized (hPXR), Pxr-null, and wild-type mice were treated orally with rifaximin, and rifampicin, a well characterized human PXR ligand. Rifaximin was highly concentrated in the intestinal tract compared with rifampicin. Rifaximin treatment resulted in significant induction of PXR target genes in the intestine of hPXR mice, but not in wild-type and Pxr-null mice. However, rifaximin treatment demonstrated no significant effect on hepatic PXR target genes in wild-type, Pxr-null, and hPXR mice. Consistent with the in vivo data, cell-based reporter gene assay revealed rifaximin-mediated activation of human PXR, but not the other xenobiotic nuclear receptors constitutive androstane receptor, peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, and farnesoid X receptor. Pretreatment with rifaximin did not affect the pharmacokinetics of the CYP3A substrate midazolam, but it increased the C(max) and decreased T(max) of 1'-hydroxymidazolam. Collectively, the current study identified rifaximin as a gut-specific human PXR ligand, and it provided further evidence for the utility of hPXR mice as a critical tool for the study of human PXR activators. Further human studies are suggested to assess the potential role of rifaximin-mediated gut PXR activation in therapeutics of chronic gastrointestinal disorders.

  20. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus.

  1. GABAA receptors in visual and auditory cortex and neural activity changes during basic visual stimulation

    Pengmin eQin

    2012-12-01

    Full Text Available Recent imaging studies have demonstrated that levels of resting GABA in the visual cortex predict the degree of stimulus-induced activity in the same region. These studies have used the presentation of discrete visual stimulus; the change from closed eyes to open also represents a simple visual stimulus, however, and has been shown to induce changes in local brain activity and in functional connectivity between regions. We thus aimed to investigate the role of the GABA system, specifically GABAA receptors, in the changes in brain activity between the eyes closed (EC and eyes open (EO state in order to provide detail at the receptor level to complement previous studies of GABA concentrations. We conducted an fMRI study involving two different modes of the change from EC to EO: An EO and EC block design, allowing the modelling of the haemodynamic response, followed by longer periods of EC and EO to allow the measuring of functional connectivity. The same subjects also underwent [18F]Flumazenil PET measure GABAA receptor binding potentials. It was demonstrated that the local-to-global ratio of GABAA receptor binding potential in the visual cortex predicted the degree of changes in neural activity from EC to EO. This same relationship was also shown in the auditory cortex. Furthermore, the local-to-global ratio of GABAA receptor binding potential in the visual cortex also predicts the change of functional connectivity between visual and auditory cortex from EC to EO. These findings contribute to our understanding of the role of GABAA receptors in stimulus-induced neural activity in local regions and in inter-regional functional connectivity.

  2. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  3. Suppression of pancreatic carcinoma growth by activating peroxisome proliferator-activated receptor γ involves angiogenesis inhibition

    Yu-Wei Dong; Xing-Peng Wang; Kai Wu

    2009-01-01

    AIM: To study the possible actions and mechanisms of peroxisome proliferator-activated receptor γ (PPARγ), a ligand-activated transcription factor, in pancreatic carcinogenesis,especially in angiogenesis.METHODS: Expressions of PPARγ and retinoid acid receptor (RXRα) were examined by reverse-transcription polymerase chain reaction (RT-PCR) with immunocytochemical staining. Pancreatic carcinoma cells, PANC-1,were treated either with 9-cis-RA, a ligand of RXRα,or with 15-deoxy-Δ12,14 prostaglandin J2(15d-PGJ2), a ligand of PPARγ, or both. Antiproliferative effect was evaluated by cell viability using methyltetrazolium (MTT) assay. A pancreatic carcinoma xenograft tumor model of nude mice was established by inoculating PANC-1 cells subcutaneously. Rosiglitazone, a specific ligand of PPARγ, was administered via water drinking in experimental group of nude mice. After 75 d, all mice were sacrificed. Expression of proliferating cell nuclear antigen (PCNA) in tumor tissue was examined with immunohistochemical staining. Expression of vascular endothelial growth factor (VEGF) mRNA in PANC-1 cells, which were treated with 15d-PGJ2 or 9-cis-RA at variousconcentrations or different duration, was detected by semi-quantitative RT-PCR. Effects of Rosiglitazone on changes of microvascular density (MVD) and VEGF expression were investigated in xenograft tumor tissue. Neovasculature was detected with immunohistochemistry staining labeled with anti-Ⅳ collagen antibody, and indicated by MVD.RESULTS: RT-PCR and immunocytochemical staining showed that PPARγ and RXRα were expressed in PANC-1 cells at both transcription level and translation level. MTT assay demonstrated that 15d-PGJ2, 9-cis-RA and their combination inhibited the growth of PANC-1 cells in a dose-dependent manner. 9-cis-RA had a combined inhibiting action with 15d-PGJ2 on the growth of pancreatic carcinoma. In vivo studies revealed that Rosiglitazone significantly suppressed the growth of pancreatic carcinoma

  4. Medium chain fatty acids are selective peroxisome proliferator activated receptor (PPAR) γ activators and pan-PPAR partial agonists

    Liberato, Marcelo Vizoná; Nascimento, Alessandro S; Ayers, Steven D; Lin, Jean Z; Cvoro, Aleksandra; Silveira, Rodrigo L; Martínez, Leandro; Souza, Paulo C T; Saidemberg, Daniel; Deng, Tuo; Amato, Angela Angelica; Togashi, Marie; Hsueh, Willa A; Phillips, Kevin; Palma, Mário Sérgio; Neves, Francisco A R; Skaf, Munir S; Webb, Paul; Polikarpov, Igor

    2012-01-01

    Thiazolidinediones (TZDs) act through peroxisome proliferator activated receptor (PPAR) γ to increase insulin sensitivity in type 2 diabetes (T2DM), but deleterious effects of these ligands mean that selective modulators with improved clinical profiles are needed. We obtained a crystal structure of

  5. Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

    Zuo, Wanhong; Zhang, Yong; Xie, Guiqin; Gregor, Danielle; Bekker, Alex; Ye, Jiang-Hong

    2016-02-01

    There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I((5-HTi))) and accelerated spontaneous firing in ∼80% of LHb neurons in rat brain slices. I((5-HTi)) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I((5-HTi)) was diminished by 5-HT(2/3) receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT(2/3) agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I((5-HTi)) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression.

  6. Interaction between Calpain 5, Peroxisome proliferator-activated receptor-gamma and Peroxisome proliferator-activated receptor-delta genes: a polygenic approach to obesity

    Ruiz Agustín

    2008-07-01

    Full Text Available Abstract Context Obesity is a multifactorial disorder, that is, a disease determined by the combined effect of genes and environment. In this context, polygenic approaches are needed. Objective To investigate the possibility of the existence of a crosstalk between the CALPAIN 10 homologue CALPAIN 5 and nuclear receptors of the peroxisome proliferator-activated receptors family. Design Cross-sectional, genetic association study and gene-gene interaction analysis. Subjects The study sample comprise 1953 individuals, 725 obese (defined as body mass index ≥ 30 and 1228 non obese subjects. Results In the monogenic analysis, only the peroxisome proliferator-activated receptor delta (PPARD gene was associated with obesity (OR = 1.43 [1.04–1.97], p = 0.027. In addition, we have found a significant interaction between CAPN5 and PPARD genes (p = 0.038 that reduces the risk for obesity in a 55%. Conclusion Our results suggest that CAPN5 and PPARD gene products may also interact in vivo.

  7. Learning and memory deficits in mice lacking protease activated receptor-1.

    Almonte, Antoine G; Hamill, Cecily E; Chhatwal, Jasmeer P; Wingo, Thomas S; Barber, Jeremy A; Lyuboslavsky, Polina N; David Sweatt, J; Ressler, Kerry J; White, David A; Traynelis, Stephen F

    2007-10-01

    The roles of serine proteases and protease activated receptors have been extensively studied in coagulation, wound healing, inflammation, and neurodegeneration. More recently, serine proteases have been suggested to influence synaptic plasticity. In this context, we examined the role of protease activated receptor 1 (PAR1), which is activated following proteolytic cleavage by thrombin and plasmin, in emotionally motivated learning. We were particularly interested in PAR1 because its activation enhances the function of NMDA receptors, which are required for some forms of synaptic plasticity. We examined several baseline behavioral measures, including locomotor activity, expression of anxiety-like behavior, motor task acquisition, nociceptive responses, and startle responses in C57Bl/6 mice in which the PAR1 receptor has been genetically deleted. In addition, we evaluated learning and memory in these mice using two memory tasks, passive avoidance and cued fear-conditioning. Whereas locomotion, pain response, startle, and measures of baseline anxiety were largely unaffected by PAR1 removal, PAR1-/- animals showed significant deficits in a passive avoidance task and in cued fear conditioning. These data suggest that PAR1 may play an important role in emotionally motivated learning.

  8. Histamine and spontaneous motor activity: biphasic changes, receptors involved and participation of the striatal dopamine system.

    Chiavegatto, S; Nasello, A G; Bernardi, M M

    1998-01-01

    The time- and dose-related effects of exogenous histamine on spontaneous motor activity and receptors involved were evaluated in male rats. Intracerebroventricular administration of histamine (5.4 and 54.3 nmol) produced a biphasic effect with initial transitory hypoactivity and later hyperactivity expressed by locomotion frequency in an open-field. The rearing frequencies were only reduced by all doses of histamine used. The histamine-induced hypoactivity was inhibited by the H3-antagonist thioperamide and was also induced by the H3-agonist N-alpha-methylhistamine. The histamine-induced hyperactivity phase was blocked by the H1-antagonist mepyramine. The H2-antagonist ranitidine increased locomotion and rearing frequencies. The participation of other neurotransmitters in the persistent hypokinetic effect induced by 135.8 nmol of histamine was determined by HPLC in the striatum and hypothalamus as counter-proof. A decreased DOPAC/DA ratio was observed only in the striatum. In the hypothalamus, low levels of 5HT were detected, probably not correlated with motor activity. In conclusion, the present results suggest that the exogenous histamine-induced hypoactivity response is probably due to activation of H3-receptors as heteroreceptors reducing the activity of the striatal dopaminergic system. This effect can partially overlap with the expression of the hyperactivity induced by H1-receptor activation. The participation of H2-receptors requires further investigation.

  9. Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1 by Diacylglycerol (DAG

    Oh Seog

    2008-10-01

    Full Text Available Abstract The capsaicin receptor, known as transient receptor potential channel vanilloid subtype 1 (TRPV1, is activated by a wide range of noxious stimulants and putative ligands such as capsaicin, heat, pH, anandamide, and phosphorylation by protein kinase C (PKC. However, the identity of endogenous activators for TRPV1 under physiological condition is still debated. Here, we report that diacylglycerol (DAG directly activates TRPV1 channel in a membrane-delimited manner in rat dorsal root ganglion (DRG neurons. 1-oleoyl-2-acetyl-sn-glycerol (OAG, a membrane-permeable DAG analog, elicited intracellular Ca2+ transients, cationic currents and cobalt uptake that were blocked by TRPV1-selective antagonists, but not by inhibitors of PKC and DAG lipase in rat DRG neurons or HEK 293 cells heterologously expressing TRPV1. OAG induced responses were about one fifth of capsaicin induced signals, suggesting that OAG displays partial agonism. We also found that endogenously produced DAG can activate rat TRPV1 channels. Mutagenesis of rat TRPV1 revealed that DAG-binding site is at Y511, the same site for capsaicin binding, and PtdIns(4,5P2binding site may not be critical for the activation of rat TRPV1 by DAG in heterologous system. We propose that DAG serves as an endogenous ligand for rat TRPV1, acting as an integrator of Gq/11-coupled receptors and receptor tyrosine kinases that are linked to phospholipase C.

  10. Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

    Matsui, Takanori [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp [Department of Pathophysiology and Therapeutics of Diabetic Vascular Complications, Kurume University School of Medicine, Kurume (Japan); Takeuchi, Masayoshi [Department of Pathophysiological Science, Faculty of Pharmaceutical Science, Hokuriku University, Kanazawa (Japan); Ueda, Seiji; Fukami, Kei; Okuda, Seiya [Department of Medicine, Kurume University School of Medicine, Kurume (Japan)

    2010-07-23

    Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenic reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE expression

  11. The active analog approach applied to the pharmacophore identification of benzodiazepine receptor ligands

    Tebib, Souhail; Bourguignon, Jean-Jacques; Wermuth, Camille-Georges

    1987-07-01

    Applied to seven potent benzodiazepine-receptor ligands belonging to chemically different classes, the active analog approach allowed the stepwise identification of the pharmacophoric pattern associated with the recognition by the benzodiazepine receptor. A unique pharmacophore model was derived which involves six critical zones: (a) a π-electron rich aromatic (PAR) zone; (b) two electron-rich zones δ1 and δ2 placed at 5.0 and 4.5 Å respectively from the reference centroid in the PAR zone; (c) a freely rotating aromatic ring (FRA) region; (d) an out-of-plane region (OPR), strongly associated with agonist properties; and (e) an additional hydrophobic region (AHR). The model accommodates all presently known ligands of the benzodiazepine receptor, identifies sensitivity to steric hindrance close to the δ1 zone, accounts for R and S differential affinities and distinguishes requirements for agonist versus non-agonist activity profiles.

  12. Synthesis and biological activity of novel small peptides with aminophosphonates moiety as NOP receptor ligands.

    Naydenova, Emilia D; Todorov, Petar T; Mateeva, Polina I; Zamfirova, Rositza N; Pavlov, Nikola D; Todorov, Simeon B

    2010-11-01

    The aim of the present study was the synthesis and the biological screening of new analogs of Ac-RYYRWK-NH2, modified at the N-terminal with 1-[(methoxyphosphono)methylamino]cycloalkanecarboxylic acids. The four newly synthesized ligands for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP) have been prepared by solid-phase peptide synthesis--Fmoc-strategy. These compounds were tested for agonistic activity in vitro on electrically stimulated smooth-muscle preparations isolated from vas deferens of Wistar rats. Our data showed that substitution of Arg at position 1 with aminophosphonates moiety decreased significantly the affinity of ligands to the NOP receptor. Furthermore, the enlargement of the cycle (with 5-8 carbon atoms) additionally diminished both the activity and the selectivity for NOP-receptor.

  13. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation

    Lotti, L V; Lanfrancone, L; Migliaccio, E;

    1996-01-01

    area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane......The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear....... The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein....

  14. Activation of the sphingomyelin cycle through the low-affinity neurotrophin receptor.

    Dobrowsky, R T; Werner, M H; Castellino, A M; Chao, M V; Hannun, Y A

    1994-09-09

    The role of the low-affinity neurotrophin receptor (p75NTR) in signal transduction is undefined. Nerve growth factor can activate the sphingomyelin cycle, generating the putative-lipid second messenger ceramide. In T9 glioma cells, addition of a cell-permeable ceramide analog mimicked the effects of nerve growth factor on cell growth inhibition and process formation. This signaling pathway appears to be mediated by p75NTR in T9 cells and NIH 3T3 cells overexpressing p75NTR. Expression of an epidermal growth factor receptor-p75NTR chimera in T9 cells imparted to epidermal growth factor the ability to activate the sphingomyelin cycle. These data demonstrate that p75NTR is capable of signaling independently of the trk neurotrophin receptor (p140trk) and that ceramide may be a mediator in neurotrophin biology.

  15. Haemolysis induced by α-toxin from Staphylococcus aureus requires P2X receptor activation

    Skals, Marianne Gerberg; Leipziger, Jens Georg; Prætorius, Helle

    2011-01-01

    -forming bacterial toxins. In this context, it is essential to know whether this is specific to HlyA-induced cell damage or if other bacterial pore-forming toxins involve purinergic signals to orchestrate haemolysis. Here, we investigate if the haemolysis produced by α-toxin from Staphylococcus aureus involves P2...... receptor activation. We observed that α-toxin-induced haemolysis is completely blocked by the unselective P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid. Moreover, several selective blockers of P2X1 and P2X7 ionotropic receptors abolished haemolysis in murine and equine...... erythrocytes. Inhibitors of pannexin channels partially reduced the α-toxin induced lysis. Thus, we conclude that α-toxin, similar to HlyA from E. coli produces cell damage by specific activation of a purinergic signalling cascade. These data indicate that pore-forming toxins in general require purinergic...

  16. Activation of mu opioid receptors sensitizes transient receptor potential vanilloid type 1 (TRPV1 via β-arrestin-2-mediated cross-talk.

    Matthew P Rowan

    Full Text Available The transient receptor potential family V1 channel (TRPV1 is activated by multiple stimuli, including capsaicin, acid, endovanilloids, and heat (>42C. Post-translational modifications to TRPV1 result in dynamic changes to the sensitivity of receptor activation. We have previously demonstrated that β-arrestin2 actively participates in a scaffolding mechanism to inhibit TRPV1 phosphorylation, thereby reducing TRPV1 sensitivity. In this study, we evaluated the effect of β-arrestin2 sequestration by G-protein coupled receptors (GPCRs on thermal and chemical activation of TRPV1. Here we report that activation of mu opioid receptor by either morphine or DAMGO results in β-arrestin2 recruitment to mu opioid receptor in sensory neurons, while activation by herkinorin does not. Furthermore, treatment of sensory neurons with morphine or DAMGO stimulates β-arrestin2 dissociation from TRPV1 and increased sensitivity of the receptor. Conversely, herkinorin treatment has no effect on TRPV1 sensitivity. Additional behavioral studies indicate that GPCR-driven β-arrestin2 sequestration plays an important peripheral role in the development of thermal sensitivity. Taken together, the reported data identify a novel cross-talk mechanism between GPCRs and TRPV1 that may contribute to multiple clinical conditions.

  17. GLP-1 receptor activation modulates appetite- and reward-related brain areas in humans.

    van Bloemendaal, Liselotte; IJzerman, Richard G; Ten Kulve, Jennifer S; Barkhof, Frederik; Konrad, Robert J; Drent, Madeleine L; Veltman, Dick J; Diamant, Michaela

    2014-12-01

    Gut-derived hormones, such as GLP-1, have been proposed to relay information to the brain to regulate appetite. GLP-1 receptor agonists, currently used for the treatment of type 2 diabetes (T2DM), improve glycemic control and stimulate satiety, leading to decreases in food intake and body weight. We hypothesized that food intake reduction after GLP-1 receptor activation is mediated through appetite- and reward-related brain areas. Obese T2DM patients and normoglycemic obese and lean individuals (n = 48) were studied in a randomized, crossover, placebo-controlled trial. Using functional MRI, we determined the acute effects of intravenous administration of the GLP-1 receptor agonist exenatide, with or without prior GLP-1 receptor blockade using exendin 9-39, on brain responses to food pictures during a somatostatin pancreatic-pituitary clamp. Obese T2DM patients and normoglycemic obese versus lean subjects showed increased brain responses to food pictures in appetite- and reward-related brain regions (insula and amygdala). Exenatide versus placebo decreased food intake and food-related brain responses in T2DM patients and obese subjects (in insula, amygdala, putamen, and orbitofrontal cortex). These effects were largely blocked by prior GLP-1 receptor blockade using exendin 9-39. Our findings provide novel insights into the mechanisms by which GLP-1 regulates food intake and how GLP-1 receptor agonists cause weight loss.

  18. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  19. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  20. 5-HT7 receptor activation attenuates thermal hyperalgesia in streptozocin-induced diabetic mice.

    Ulugol, Ahmet; Oltulu, Cagatay; Gunduz, Ozgur; Citak, Cihad; Carrara, Roberto; Shaqaqi, Mohammad Reza; Sanchez, Alicia Mansilla; Dogrul, Ahmet

    2012-08-01

    The role of 5-HT7 receptors in the nociceptive processing received most attention during the last few years. The involvement of 5-HT₇ receptors in nerve injury-induced neuropathic pain states have been reported only recently; however, there are no reports on its contribution in diabetic neuropathic pain. We therefore planned to investigate the effect of 5-HT₇ receptor activation on the changes of nociceptive threshold in diabetic mice. Diabetes was induced by a single intraperitoneal injection of streptozocin (150 mg/kg, i.p.). The nociceptive responses in normal and diabetic animals were tested in the hot-plate and tail-flick assays. Both hot-plate and tail-flick latencies significantly shortened at 1-3/4 weeks (thermal hyperalgesia) and prolonged at 6-7 weeks (thermal hypoalgesia) after streptozocin administration. At the dose of 10 mg/kg, systemic injections of AS-19, a selective 5-HT₇ receptor agonist, reduced thermal hyperalgesia at early stage of diabetes, but did not influence thermal hypoalgesia at late stage. Co-administration of SB-258719, a selective 5-HT₇ receptor antagonist, at a dose that had no effect on its own (10 mg/kg), reversed the anti-hyperalgesic effect of AS-19. Our results indicate that systemic administration of 5-HT₇ receptor agonists may have clinical utility in treating diabetic neuropathic pain.

  1. Internalization mechanisms of the epidermal growth factor receptor after activation with different ligands.

    Lasse Henriksen

    Full Text Available The epidermal growth factor receptor (EGFR regulates normal growth and differentiation, but dysregulation of the receptor or one of the EGFR ligands is involved in the pathogenesis of many cancers. There are eight ligands for EGFR, however most of the research into trafficking of the receptor after ligand activation focuses on the effect of epidermal growth factor (EGF and transforming growth factor-α (TGF-α. For a long time it was believed that clathrin-mediated endocytosis was the major pathway for internalization of the receptor, but recent work suggests that different pathways exist. Here we show that clathrin ablation completely inhibits internalization of EGF- and TGF-α-stimulated receptor, however the inhibition of receptor internalization in cells treated with heparin-binding EGF-like growth factor (HB-EGF or betacellulin (BTC was only partial. In contrast, clathrin knockdown fully inhibits EGFR degradation after all ligands tested. Furthermore, inhibition of dynamin function blocked EGFR internalization after stimulation with all ligands. Knocking out a number of clathrin-independent dynamin-dependent pathways of internalization had no effect on the ligand-induced endocytosis of the EGFR. We suggest that EGF and TGF-α lead to EGFR endocytosis mainly via the clathrin-mediated pathway. Furthermore, we suggest that HB-EGF and BTC also lead to EGFR endocytosis via a clathrin-mediated pathway, but can additionally use an unidentified internalization pathway or better recruit the small amount of clathrin remaining after clathrin knockdown.

  2. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation.

    Chen, Han-Ting; Ruan, Nan-Yu; Chen, Jin-Chung; Lin, Tzu-Yung

    2012-09-24

    The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  3. Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

    Jin‑Chung Chen

    2012-09-01

    Full Text Available The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3. To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2 activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser473 phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.

  4. Cannabinoid receptor-2 selective antagonist negatively regulates receptor activator of nuclear factor kappa B ligand mediated osteoclastogenesis

    GENG De-chun; XU Yao-zeng; YANG Hui-lin; ZHU Guang-ming; WANG Xian-bin; ZHU Xue-song

    2011-01-01

    Background The cannabinoid receptor-2 (CB2) is important for bone remodeling. In this study, we investigated the effects of CB2 selective antagonist (AM630) on receptor activator of nuclear factor kappa B (RANK) ligand (RANKL)induced osteoclast differentiation and the underlying signaling pathway using a monocyte-macrophage cell line-RAW264.7.Methods RAW264.7 was cultured with RANKL for 6 days and then treated with AM630 for 24 hours. Mature osteoclasts were measured by tartrate-resistant acid phosphatase (TRAP) staining using a commercial kit. Total ribonucleic acid (RNA)was isolated and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was done to examine the expression of RANK, cathepsin K (CPK) and nuclear factor kappa B (NF-κB). The extracellular signal-regulated kinase (ERK),phosphorylation of ERK (P-ERK) and NF-κB production were tested by Western blotting. The effect of AM630 on RAW264.7 viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay.Results AM630 did not affect the viability of RAW264.7. However, this CB2 selective antagonist markedly inhibited osteoclast formation and the inhibition rate was dose-dependent. The dose of >100 nmol/L could reduce TRAP positive cells to the levels that were significantly l