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Sample records for asialoglycoprotein receptor mediates

  1. Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications.

    PubMed

    D'Souza, Anisha A; Devarajan, Padma V

    2015-04-10

    Hepatocyte resident afflictions continue to affect the human population unabated. The asialoglycoprotein receptor (ASGPR) is primarily expressed on hepatocytes and minimally on extra-hepatic cells. This makes it specifically attractive for receptor-mediated drug delivery with minimum concerns of toxicity. ASGPR facilitates internalization by clathrin-mediated endocytosis and exhibits high affinity for carbohydrates specifically galactose, N-acetylgalactosamine and glucose. Isomeric forms of sugar, galactose density and branching, spatial geometry and galactose linkages are key factors influencing ligand-receptor binding. Popular ligands for ASGPR mediated targeting are carbohydrate polymers, arabinogalactan and pullulan. Other ligands include galactose-bearing glycoproteins, glycopeptides and galactose modified polymers and lipids. Drug-ligand conjugates provide a viable strategy; nevertheless ligand-anchored nanocarriers provide an attractive option for ASGPR targeted delivery and are widely explored. The present review details various ligands and nanocarriers exploited for ASGPR mediated delivery of drugs to hepatocytes. Nanocarrier properties affecting ASGPR mediated uptake are discussed at length. The review also highlights the clinical relevance of ASGPR mediated targeting and applications in diagnostics. ASGPR mediated hepatocyte targeting provides great promise for improved therapy of hepatic afflictions.

  2. A Drug Delivery Strategy: Binding Enkephalin to Asialoglycoprotein Receptor by Enzymatic Galactosylation

    PubMed Central

    Christie, Michelle P.; Simerská, Pavla; Jen, Freda E.-C.; Hussein, Waleed M.; Rawi, Mohamad F. M.; Hartley-Tassell, Lauren E.; Day, Christopher J.; Jennings, Michael P.; Toth, Istvan

    2014-01-01

    Glycosylation of biopharmaceuticals can mediate cell specific delivery by targeting carbohydrate receptors. Additionally, glycosylation can improve the physico-chemical (drug-like) properties of peptide based drug candidates. The main purpose of this study was to examine if glycosylation of the peptide enkephalin could facilitate its binding to the carbohydrate receptor, asialoglycoprotein. Firstly, we described the one-pot enzymatic galactosylation of lactose modified enkephalin in the presence of uridine-5′-diphosphogalactose 4-epimerase and lipopolysaccharyl α-1,4-galactosyltransferase. Stability experiments using human plasma and Caco-2 cell homogenates showed that glycosylation considerably improved the stability of enkephalin (at least 60% remained stable after a 2 hr incubation at 37°C). In vitro permeability experiments using Caco-2 cells revealed that the permeability of mono- and trisaccharide conjugated enkephalins was 14 and 28 times higher, respectively, than that of enkephalin alone (Papp 3.1×10−8 cm/s). By the methods of surface plasmon resonance and molecular modeling, we demonstrated that the enzymatic glycosylation of enkephalin enabled binding the asialoglycoprotein receptor. The addition of a trisaccharide moiety to enkephalin improved the binding of enkephalin to the asialoglycoprotein receptor two fold (KD = 91 µM). The docking scores from molecular modeling showed that the binding modes and affinities of the glycosylated enkephalin derivatives to the asialoglycoprotein receptor complemented the results from the surface plasmon resonance experiments. PMID:24736570

  3. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  4. Closely related mammals have distinct asialoglycoprotein receptor carbohydrate specificities.

    PubMed

    Park, Eric I; Baenziger, Jacques U

    2004-09-24

    We recently reported that the rat asialoglycoprotein receptor binds oligosaccharides terminating with sialic acid (Sia) alpha2,6GalNAc. Despite a high percentage of identical amino acids in their sequences, orthologues of the asialoglycoprotein receptor (ASGP-R) in different mammals differ in their specificity for terminal Siaalpha2,6GalNAc. The recombinant subunit 1 of the ASGP-R from the rat (RHL-1 or rat hepatic lectin) and the mouse (MHL-1 or mouse hepatic lectin), which differ at only 12 positions in the amino acid sequence of their carbohydrate recognition domains, binds Siaalpha2,6GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin and GalNAcbeta1,4GlcNAcbeta1,2Man-bovine serum albumin in ratios of 16:1.0 and 1.0:1.0, respectively. Mutagenesis was used to show that amino acids both in the immediate vicinity of the proposed binding site for terminal GalNAc and on the alpha2 helix that is distant from the binding site contribute to the specificity for terminal Siaalpha2,6GalNAc. Thus, multiple amino acid sequence alterations in two key locations contribute to the difference in specificity observed for the rat and mouse ASGP-Rs. We hypothesize that the altered specificity of ASPG-R orthologues in such evolutionarily closely related species reflects rapidly changing requirements for recognition of endogenous or exogenous oligosaccharides in vivo.

  5. Impact of asialoglycoprotein receptor deficiency on the development of liver injury

    PubMed Central

    Lee, Serene ML; Casey, Carol A; McVicker, Benita L

    2009-01-01

    The asialoglycoprotein (ASGP) receptor is a well-characterized hepatic receptor that is recycled via the common cellular process of receptor-mediated endocytosis (RME). The RME process plays an integral part in the proper trafficking and routing of receptors and ligands in the healthy cell. Thus, the mis-sorting or altered transport of proteins during RME is thought to play a role in several diseases associated with hepatocyte and liver dysfunction. Previously, we examined in detail alterations that occur in hepatocellular RME and associated receptor functions as a result of one particular liver injury, alcoholic liver disease (ALD). The studies revealed profound ethanol-mediated impairments to the ASGP receptor and the RME process, indicating the importance of this receptor and the maintenance of proper endocytic events in normal tissue. To further clarify these observations, studies were performed utilizing knockout mice (lacking a functional ASGP receptor) to which were administered several liver toxicants. In addition to alcohol, we examined the effects following administration of anti-Fas (CD95) antibody, carbon tetrachloride (CCl4) and lipopolysaccharide (LPS)/galactosamine. The results of these studies demonstrated that the knockout mice sustained enhanced liver injury in response to all of the treatments, as shown by increased indices of liver damage, such as enhancement of serum enzyme levels, histopathological scores, as well as hepatocellular death. Overall, the work completed to date suggests a possible link between hepatic receptors and liver injury. In particular, adequate function and content of the ASGP receptor may provide protection against various toxin-mediated liver diseases. PMID:19291819

  6. (18)F-FBHGal for asialoglycoprotein receptor imaging in a hepatic fibrosis mouse model.

    PubMed

    Kao, Hao-Wen; Chen, Chuan-Lin; Chang, Wen-Yi; Chen, Jenn-Tzong; Lin, Wuu-Jyh; Liu, Ren-Shyan; Wang, Hsin-Ell

    2013-02-15

    Quantification of the expression of asialoglycoprotein receptor (ASGPR), which is located on the hepatocyte membrane with high-affinity for galactose residues, can help assess ASGPR-related liver diseases. A hepatic fibrosis mouse model with lower asialoglycoprotein receptor expression was established by dimethylnitrosamine (DMN) administration. This study developed and demonstrated that 4-(18)F-fluoro-N-(6-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)hexyl)benzamide ((18)F-FBHGal), a new (18)F-labeled monovalent galactose derivative, is an asialoglycoprotein receptor (ASGPR)-specific PET probe in a normal and a hepatic fibrosis mouse models. Immunoassay exhibited a linear correlation between the accumulation of GalH-FITC, a fluorescent surrogate of FBHGal, and the amount of ASGPR. A significant reduction in HepG2 cellular uptake (P <0.0001) was observed using confocal microscopy when co-incubated with 0.5μM of asialofetuin, a well known ASGPR blocking agent. Animal studies showed the accumulation of (18)F-FBHGal in fibrosis liver (14.84±1.10 %ID/g) was appreciably decreased compared with that in normal liver (20.50±1.51 %ID/g, P <0.01) at 30min post-injection. The receptor indexes (liver/liver-plus-heart ratio at 30min post-injection) of hepatic fibrosis mice derived from both microPET imaging and biodistribution study were significantly lower (P <0.01) than those of normal mice. The pharmacokinetic parameters (T(1/2)α, T(1/2)β, AUC and Cl) derived from microPET images revealed prolonged systemic circulation of (18)F-FBHGal in hepatic fibrosis mice compared to that in normal mice. The findings in biological characterizations suggest that (18)F-FBHGal is a feasible agent for PET imaging of hepatic fibrosis in mice and may provide new insights into ASGPR-related liver dysfunction.

  7. Three-Dimensional Models of the Oligomeric Human Asialoglycoprotein Receptor (ASGP-R)

    PubMed Central

    Massarelli, Ilaria; Chiellini, Federica; Chiellini, Emo; Bianucci, Anna Maria

    2010-01-01

    The work presented here is aimed at suggesting plausible hypotheses for functional oligomeric forms of the human asialoglycoprotein receptor (ASGP-R), by applying a combination of different computational techniques. The functional ASGP-R is a hetero-oligomer, that comprises of several subunits of two different kinds (H1 and H2), which are highly homologous. Its stoichiometry is still unknown. An articulated step-wise modeling protocol was used in order to build the receptor model in a minimal oligomeric form, necessary for it to bind multi-antennary carbohydrate ligands. The ultimate target of the study is to contribute to increasing the knowledge of interactions between the human ASGP-R and carbohydrate ligands, at the molecular level, pertinent to applications in the field of hepatic tissue engineering. PMID:21152305

  8. Modulation of mannose and asialoglycoprotein receptor expression determines glycoprotein hormone half-life at critical points in the reproductive cycle.

    PubMed

    Mi, Yiling; Lin, Angela; Fiete, Dorothy; Steirer, Lindsay; Baenziger, Jacques U

    2014-04-25

    The rate at which glycoproteins are cleared from the circulation has a critical impact on their biologic activity in vivo. We have shown that clearance rates for glycoproteins such as luteinizing hormone (LH) that undergo regulated release into the circulation determine their potency. Two highly abundant, carbohydrate-specific, endocytic receptors, the asialoglycoprotein receptor (ASGR) and the mannose receptor (ManR) are expressed in the liver by parenchymal and sinusoidal endothelial cells, respectively. We demonstrate that the ManR mediates the clearance of glycoproteins such as LH that bear N-linked glycans terminating with β1,4-linked GalNAc-4-SO4, as well as glycoproteins bearing glycans that terminate with Man. Steady state levels of mRNA encoding the ASGR and the ManR are regulated by progesterone in pregnant mice, reaching maximal levels on day 12.5 of pregnancy. Protein expression and glycan-specific binding activity also increase in the livers of pregnant mice. In contrast, ManR mRNA, but not ASGR mRNA, decreases in male mice at the time of sexual maturation. We show that levels of ManR and ASGR expression control the clearance rate for glycoproteins bearing recognized glycans. Thus, reduced expression of the ManR at the time of sexual maturation will increase the potency of LH in vivo, whereas increased expression during pregnancy will reduce LH potency until progesterone and receptor levels fall prior to parturition.

  9. Liver asialoglycoprotein receptor levels correlate with severity of alcoholic liver damage in rats.

    PubMed

    Casey, Carol A; McVicker, Benita L; Donohue, Terrence M; McFarland, Melinda A; Wiegert, Robert L; Nanji, Amin A

    2004-01-01

    It has been demonstrated that the oral administration of ethanol (Lieber-DeCarli liquid diet) to rats results in a decreased expression and content of the asialoglycoprotein receptor (ASGP-R) in the resultant fatty liver. In the present study, we wanted to determine whether the extent of impaired receptor content was correlated with the severity of liver pathology by using the intragastric feeding model. When ASGP-R protein and mRNA levels were measured in animals infused with ethanol or dextrose in the presence of fish oil (FO) or medium-chain triglyceride as the source of fat, more significant impairments to the ASGP-R were observed in the FO-ethanol group compared with the medium-chain triglyceride-ethanol group. Furthermore, only the FO-ethanol group showed pathological liver changes. These results demonstrate that a correlation exists between the progression of alcohol-associated liver injury, as defined by the severity of liver pathology, and an ethanol-induced decline in ASGP-R content.

  10. Cloning, expression and polyclonal antibody preparation of the asialoglycoprotein receptor of Marmota himalayan.

    PubMed

    Yang, Yan; Huang, Huang; Zhang, Zhenghua; Wang, Baoju; Tian, Yongjun; Lu, Mengji; Yang, Dongliang

    2007-08-01

    The objective of this study is to express the carbohydrate recognition domain (CRD) of the asialoglycoprotein receptor (ASGPR) H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21(DE3)plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay (ELISA), Western blotting and immunohistochemical staining (IHC). The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marmota Himalayan-for the research of infectious diseases of hepatitis viruses and liver cancer treatment.

  11. Masking of an endoplasmic reticulum retention signal by its presence in the two subunits of the asialoglycoprotein receptor.

    PubMed

    Shenkman, M; Ehrlich, M; Lederkremer, G Z

    2000-01-28

    Human asialoglycoprotein receptor H1 and H2b subunits assemble into a hetero-oligomer that travels to the cell surface. The H2a variant on the other hand is a precursor of a cleaved soluble form that is secreted. Uncleaved H2a precursor molecules cannot exit the endoplasmic reticulum (ER), a lumenal juxtamembrane pentapeptide being responsible for their retention. Insertion of this pentapeptide into H1 (H1i5) causes its complete ER retention but not fast degradation as happens to H2a. Cotransfection of H2a elicited, by heterodimerization, the Golgi processing of H1i5 and its surface expression. This occurred to a much lesser extent by cotransfection of H2b. Likewise, coexpression of H1i5 and not H1 stabilized H2a and caused its export to the cell surface. Homodimerization of molecules containing the pentapeptide did not cancel the retention. Thus, only when the pentapeptide is present in both subunits is the ER retention efficiently abrogated. The results show the unexpected finding that identical ER retention signals present in two associated chains can mask and cancel each other's effect. This could have important implications as similar abrogation of ER retention of other proteins could eventually be obtained by engineering and coexpressing an associated protein containing the same retention signal.

  12. Asialoglycoprotein receptor facilitates infection of PLC/PRF/5 cells by HEV through interaction with ORF2.

    PubMed

    Zhang, Li; Tian, Yabin; Wen, Zhiheng; Zhang, Feng; Qi, Ying; Huang, Weijin; Zhang, Heqiu; Wang, Youchun

    2016-12-01

    Although the biological and epidemiological features of hepatitis E virus (HEV) have been studied extensively in recent years, the mechanism by which HEV infects cells is still poorly understood. In this study, coimmunoprecipitation, pull-down, and ELISA were used to show that the HEV ORF2 protein interacts directly with the ectodomain of both ASGR1 and ASGR2. Susceptibility to HEV correlated positively with the expression level of surface asialoglycoprotein receptor (ASGPR) in cell lines. ASGPR-directed small interfering RNA (siRNA) in HEV-infected PLC/PRF/5 cells had no significant effect on HEV release, suggesting that ASGPR mainly regulates the viral binding and entry steps. Both the purified ASGPR ectodomain and anti-ASGPR antibodies disturbed the binding of HEV to PLC/PRF/5 cells. The classic ASGPR ligands asialofetuin, asialoganglioside, and fibronectin competitively inhibited the binding of HEV to hepatocytes in the presence of calcium. HeLa cell lines stably expressing ASGPR displayed increased HEV-binding capacity, whereas ASGPR-knockout PLC/PRF/5 cell lines had lower HEV-binding capacity. Thus, our study demonstrates that ASGPR is involved in and facilitates HEV infection by binding to ORF2. J. Med. Virol. 88:2186-2195, 2016. © 2016 Wiley Periodicals, Inc.

  13. Development of T cells carrying two complementary chimeric antigen receptors against glypican-3 and asialoglycoprotein receptor 1 for the treatment of hepatocellular carcinoma.

    PubMed

    Chen, Cheng; Li, Kesang; Jiang, Hua; Song, Fei; Gao, Huiping; Pan, Xiaorong; Shi, Bizhi; Bi, Yanyu; Wang, Huamao; Wang, Hongyang; Li, Zonghai

    2017-04-01

    Adoptive immunotherapy leveraging chimeric antigen receptor-modified T (CAR-T) cells holds great promise for the treatment of cancer. However, tumor-associated antigens often have low expression levels in normal tissues, which can cause on-target, off-tumor toxicity. Recently, we reported that GPC3-targeted CAR-T cells could eradicate hepatocellular carcinoma (HCC) xenografts in mice. However, it remains unknown whether on-target, off-tumor toxicity can occur. Therefore, we proposed that dual-targeted CAR-T cells co-expressing glypican-3 (GPC3) and asialoglycoprotein receptor 1 (ASGR1) (a liver tissue-specific protein)-targeted CARs featuring CD3ζ and 28BB (containing both CD28 and 4-1BB signaling domains), respectively, may have reduced on-target, off-tumor toxicity. Our results demonstrated that dual-targeted CAR-T cells caused no cytotoxicity to ASGR1(+)GPC3(-) tumor cells, but they exhibited a similar cytotoxicity against GPC3(+)ASGR1(-) and GPC3(+)ASGR1(+) HCC cells in vitro. We found that dual-targeted CAR-T cells showed significantly higher cytokine secretion, proliferation and antiapoptosis ability against tumor cells bearing both antigens than single-targeted CAR-T cells in vitro. Furthermore, the dual-targeted CAR-T cells displayed potent growth suppression activity on GPC3(+)ASGR1(+) HCC tumor xenografts, while no obvious growth suppression was seen with single or double antigen-negative tumor xenografts. Additionally, the dual-targeted T cells exerted superior anticancer activity and persistence against single-targeted T cells in two GPC3(+)ASGR1(+) HCC xenograft models. Together, T cells carrying two complementary CARs against GPC3 and ASGR1 may reduce the risk of on-target, off-tumor toxicity while maintaining relatively potent antitumor activities on GPC3(+)ASGR1(+) HCC.

  14. Epitope Structure of the Carbohydrate Recognition Domain of Asialoglycoprotein Receptor to a Monoclonal Antibody Revealed by High-Resolution Proteolytic Excision Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Stefanescu, Raluca; Born, Rita; Moise, Adrian; Ernst, Beat; Przybylski, Michael

    2011-01-01

    Recent studies suggest that the H1 subunit of the carbohydrate recognition domain (H1CRD) of the asialoglycoprotein receptor is used as an entry site into hepatocytes by hepatitis A and B viruses and Marburg virus. Thus, molecules binding specifically to the CRD might exert inhibition towards these diseases by blocking the virus entry site. We report here the identification of the epitope structure of H1CRD to a monoclonal antibody by proteolytic epitope excision of the immune complex and high-resolution MALDI-FTICR mass spectrometry. As a prerequisite of the epitope determination, the primary structure of the H1CRD antigen was characterised by ESI-FTICR-MS of the intact protein and by LC-MS/MS of tryptic digest mixtures. Molecular mass determination and proteolytic fragments provided the identification of two intramolecular disulfide bridges (seven Cys residues), and a Cys-mercaptoethanol adduct formed by treatment with β-mercaptoethanol during protein extraction. The H1CRD antigen binds to the monoclonal antibody in both native and Cys-alkylated form. For identification of the epitope, the antibody was immobilized on N-hydroxysuccinimide (NHS)-activated Sepharose. Epitope excision and epitope extraction with trypsin and FTICR-MS of affinity-bound peptides provided the identification of two specific epitope peptides (5-16) and (17-23) that showed high affinity to the antibody. Affinity studies of the synthetic epitope peptides revealed independent binding of each peptide to the antibody.

  15. Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system.

    PubMed

    Li, Zhen; Deng, Si-Si; Zang, Yi; Gu, Zhen; He, Xiao-Peng; Chen, Guo-Rong; Chen, Kaixian; James, Tony D; Li, Jia; Long, Yi-Tao

    2013-01-01

    Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

  16. Capturing intercellular sugar-mediated ligand-receptor recognitions via a simple yet highly biospecific interfacial system

    NASA Astrophysics Data System (ADS)

    Li, Zhen; Deng, Si-Si; Zang, Yi; Gu, Zhen; He, Xiao-Peng; Chen, Guo-Rong; Chen, Kaixian; James, Tony D.; Li, Jia; Long, Yi-Tao

    2013-07-01

    Intercellular ligand-receptor recognitions are crucial natural interactions that initiate a number of biological and pathological events. We present here the simple construction of a unique class of biomimetic interfaces based on a graphene-mediated self-assembly of glycosyl anthraquinones to a screen-printed electrode for the detection of transmembrane glycoprotein receptors expressed on a hepatoma cell line. We show that an electroactive interface confined with densely clustered galactosyl ligands is able to ingeniously recognize the asialoglycoprotein receptors on live Hep-G2 cells employing simple electrochemical techniques. The only facility used is a personal laptop in connection with a cheap and portable electrochemical workstation.

  17. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  18. Simplified quantification method for in vivo SPECT/CT imaging of asialoglycoprotein receptor with 99mTc-p(VLA-co-VNI) to assess and stage hepatic fibrosis in mice

    PubMed Central

    Zhang, Deliang; Guo, Zhide; Zhang, Pu; Li, Yesen; Su, Xinhui; You, Linyi; Gao, Mengna; Liu, Chang; Wu, Hua; Zhang, Xianzhong

    2016-01-01

    The goal of this study is to develop a noninvasive method of SPECT imaging to quantify and stage liver fibrosis with an Asialoglycoprotein receptor (ASGP-R) targeting tracer—99mTc-p(VLA-co-VNI). ASGP-Rs are well known to specifically express in the mammalian liver. Here, we demonstrated ASGP-R expression decreased in carbon tetrachloride (CCl4)-induced mouse model. ASGP-R expression correlated with liver fibrosis progression. ASGP-R could be a useful marker in the stage of liver fibrosis. Liver uptake value (LUV) derived by SPECT imaging was used to assess liver fibrosis in the CCl4-induced mouse model. LUV = [radioactivity (liver uptake)/radioactivity (injected)] × 100/liver volume. The LUV decreased along with the disease progression. The relationships between LUV and liver hydroxyproline (i.e. collagen), as well as Sirius Red were established and verified. A strong negative linear correlation was found between LUV and hydroxyproline levels (r = −0.83) as well as LUV and Sirius Red quantification (r = −0.83). In conclusion, SPECT imaging with 99mTc-p(VLA-co-VNI) is useful in evaluating and staging liver fibrosis in vivo. PMID:27150943

  19. Receptor-Mediated Tobacco Toxicity

    PubMed Central

    Arredondo, Juan; Chernyavsky, Alexander I.; Marubio, Lisa M.; Beaudet, Arthur L.; Jolkovsky, David L.; Pinkerton, Kent E.; Grando, Sergei A.

    2005-01-01

    Tobacco is a known cause of oral disease but the mechanism remains elusive. Nicotine (Nic) is a likely culprit of pathobiological effects because it displaces the local cytotransmitter acetylcholine from the nicotinic receptors (nAChRs) expressed by oral keratinocytes (KCs). To gain a mechanistic insight into tobacco-induced morbidity in the oral cavity, we studied effects of exposures to environmental tobacco smoke (ETS) versus equivalent concentration of pure Nic on human and murine KCs. Both ETS and Nic up-regulated expression of cell cycle and apoptosis regulators, differentiation marker filaggrin, and signal transduction factors at both the mRNA and protein levels. These changes could be abolished in cultured human oral KCs transfected with anti-α3 small interfering RNA or treated with the α3β2-preferring antagonist α-conotoxin MII. Functional inactivation of α3-mediated signaling in α3−/− mutant KCs prevented most of the ETS/Nic-dependent changes in gene expression. To determine relevance of the in vitro findings to the in vivo situation, we studied gene expression in oral mucosa of neonatal α3+/+ and α3−/− littermates delivered by heterozygous mice soon after their exposures to ETS or equivalent concentration of pure Nic in drinking water. In addition to reverse transcriptase-polymerase chain reaction and Western blot, the ETS/Nic-dependent alterations in gene expression were also detected by semiquantitative immunofluorescence assay directly in KCs comprising murine oral mucosa. Only wild-type mice consistently developed significant (P < 0.05) changes in the gene expression. These results identified α3β2 nAChR as a major receptor mediating effects of tobacco products on KC gene expression. Real-time polymerase chain reaction demonstrated that in all three model systems the common genes targeted by α3β2-mediated ETS/Nic toxicity were p21, Bcl-2, NF-κB, and STAT-1. The expression of the nAChR subunits α5 and β2 and the muscarinic

  20. Toll-like receptor 7 mediates pruritus.

    PubMed

    Liu, Tong; Xu, Zhen-Zhong; Park, Chul-Kyu; Berta, Temugin; Ji, Ru-Rong

    2010-12-01

    Toll-like receptors are typically expressed in immune cells to regulate innate immunity. We found that functional Toll-like receptor 7 (TLR7) was expressed in C-fiber primary sensory neurons and was important for inducing itch (pruritus), but was not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Our results indicate that TLR7 mediates itching and is a potential therapeutic target for anti-itch treatment in skin disease conditions.

  1. Receptor-mediated DNA-targeted photoimmunotherapy.

    PubMed

    Karagiannis, Tom C; Lobachevsky, Pavel N; Leung, Brenda K Y; White, Jonathan M; Martin, Roger F

    2006-11-01

    We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.

  2. Receptor-mediated signaling in Aspergillus fumigatus

    PubMed Central

    Grice, C. M.; Bertuzzi, M.; Bignell, E. M.

    2013-01-01

    Aspergillus fumigatus is the most pathogenic species among the Aspergilli, and the major fungal agent of human pulmonary infection. To prosper in diverse ecological niches, Aspergilli have evolved numerous mechanisms for adaptive gene regulation, some of which are also crucial for mammalian infection. Among the molecules which govern such responses, integral membrane receptors are thought to be the most amenable to therapeutic modulation. This is due to the localization of these molecular sensors at the periphery of the fungal cell, and to the prevalence of small molecules and licensed drugs which target receptor-mediated signaling in higher eukaryotic cells. In this review we highlight the progress made in characterizing receptor-mediated environmental adaptation in A. fumigatus and its relevance for pathogenicity in mammals. By presenting a first genomic survey of integral membrane proteins in this organism, we highlight an abundance of putative seven transmembrane domain (7TMD) receptors, the majority of which remain uncharacterized. Given the dependency of A. fumigatus upon stress adaptation for colonization and infection of mammalian hosts, and the merits of targeting receptor-mediated signaling as an antifungal strategy, a closer scrutiny of sensory perception and signal transduction in this organism is warranted. PMID:23430083

  3. Synaptic NMDA Receptors Mediate Hypoxic Excitotoxic Death

    PubMed Central

    Wroge, Christine M.; Hogins, Joshua; Eisenman, Larry; Mennerick, Steven

    2012-01-01

    Excessive NMDA receptor activation and excitotoxicity underlies pathology in many neuropsychiatric and neurological disorders, including hypoxia/ischemia. Thus, the development of effective therapeutics for these disorders demands a complete understanding of NMDA receptor (NMDAR) activation during excitotoxic insults. The extrasynaptic NMDAR hypothesis posits that synaptic NMDARs are neurotrophic/neuroprotective and extrasynaptic NMDARs are neurotoxic. In part, the extrasynaptic hypothesis is built on observed selectivity for extrasynaptic receptors of a neuroprotective use-dependent NMDAR channel blocker, memantine. In rat hippocampal neurons we found that a neuroprotective concentration of memantine shows little selectivity for extrasynaptic NMDARs when all receptors are tonically activated by exogenous glutamate. This led us to test the extrasynaptic NMDAR hypothesis using metabolic challenge, where the source of excitotoxic glutamate buildup may be largely synaptic. Three independent approaches suggest strongly that synaptic receptors participate prominently in hypoxic excitotoxicity. First, block of glutamate transporters with a non-substrate antagonist exacerbated rather than prevented damage, consistent with a primarily synaptic source of glutamate. Second, selective, preblock of synaptic NMDARs with a slowly reversible, use-dependent antagonist protected nearly fully against prolonged hypoxic insult. Third, glutamate pyruvate transaminase (GPT), which degrades ambient but not synaptic glutamate, did not protect against hypoxia but protected against exogenous glutamate damage. Together, these results suggest that synaptic NMDARs can mediate excitotoxicity, particularly when the glutamate source is synaptic and when synaptic receptor contributions are rigorously defined. Moreover, the results suggest that in some situations therapeutically targeting extrasynaptic receptors may be inappropriate. PMID:22573696

  4. Toll-like Receptor-7 Mediates Pruritus

    PubMed Central

    Liu, Tong; Xu, Zhen-Zhong; Park, Chul-Kyu; Berta, Temugin; Ji, Ru-Rong

    2010-01-01

    Toll-like receptors (TLRs) are typically expressed in immune cells to regulate innate immunity. Here we report that functional TLR7 is expressed in C-fiber primary sensory neurons and important for inducing itch (pruritis) but not necessary for eliciting mechanical, thermal, inflammatory and neuropathic pain in mice. Thus, we have uncovered TLR7 as a novel itch mediator and a potential therapeutic target for anti-itch treatment in skin disease conditions. PMID:21037581

  5. Receptors mediating toxicity and their involvement in endocrine disruption.

    PubMed

    Rüegg, Joëlle; Penttinen-Damdimopoulou, Pauliina; Mäkelä, Sari; Pongratz, Ingemar; Gustafsson, Jan-Ake

    2009-01-01

    Many toxic compounds exert their harmful effects by activating of certain receptors, which in turn leads to dysregulation of transcription. Some of these receptors are so called xenosensors. They are activated by external chemicals and evoke a cascade of events that lead to the elimination of the chemical from the system. Other receptors that are modulated by toxic substances are hormone receptors, particularly the ones of the nuclear receptor family. Some environmental chemicals resemble endogenous hormones and can falsely activate these receptors, leading to undesired activity in the cell. Furthermore, excessive activation of the xenosensors can lead to disturbances of the integrity of the system as well. In this chapter, the concepts of receptor-mediated toxicity and hormone disruption are introduced. We start by describing environmental chemicals that can bind to xenosensors and nuclear hormone receptors. We then describe the receptors most commonly targeted by environmental chemicals. Finally, the mechanisms by which receptor-mediated events can disrupt the system are depicted.

  6. Receptor-Mediated Drug Delivery Systems Targeting to Glioma

    PubMed Central

    Wang, Shanshan; Meng, Ying; Li, Chengyi; Qian, Min; Huang, Rongqin

    2015-01-01

    Glioma has been considered to be the most frequent primary tumor within the central nervous system (CNS). The complexity of glioma, especially the existence of the blood-brain barrier (BBB), makes the survival and prognosis of glioma remain poor even after a standard treatment based on surgery, radiotherapy, and chemotherapy. This provides a rationale for the development of some novel therapeutic strategies. Among them, receptor-mediated drug delivery is a specific pattern taking advantage of differential expression of receptors between tumors and normal tissues. The strategy can actively transport drugs, such as small molecular drugs, gene medicines, and therapeutic proteins to glioma while minimizing adverse reactions. This review will summarize recent progress on receptor-mediated drug delivery systems targeting to glioma, and conclude the challenges and prospects of receptor-mediated glioma-targeted therapy for future applications.

  7. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    PubMed

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  8. NMDA Receptors Mediate Synaptic Competition in Culture

    PubMed Central

    She, Kevin; Craig, Ann Marie

    2011-01-01

    Background Activity through NMDA type glutamate receptors sculpts connectivity in the developing nervous system. This topic is typically studied in the visual system in vivo, where activity of inputs can be differentially regulated, but in which individual synapses are difficult to visualize and mechanisms governing synaptic competition can be difficult to ascertain. Here, we develop a model of NMDA-receptor dependent synaptic competition in dissociated cultured hippocampal neurons. Methodology/Principal Findings GluN1 -/- (KO) mouse hippocampal neurons lacking the essential NMDA receptor subunit were cultured alone or cultured in defined ratios with wild type (WT) neurons. The absence of functional NMDA receptors did not alter neuron survival. Synapse development was assessed by immunofluorescence for postsynaptic PSD-95 family scaffold and apposed presynaptic vesicular glutamate transporter VGlut1. Synapse density was specifically enhanced onto minority wild type neurons co-cultured with a majority of GluN1 -/- neighbour neurons, both relative to the GluN1 -/- neighbours and relative to sister pure wild type cultures. This form of synaptic competition was dependent on NMDA receptor activity and not conferred by the mere physical presence of GluN1. In contrast to these results in 10% WT and 90% KO co-cultures, synapse density did not differ by genotype in 50% WT and 50% KO co-cultures or in 90% WT and 10% KO co-cultures. Conclusions/Significance The enhanced synaptic density onto NMDA receptor-competent neurons in minority coculture with GluN1 -/- neurons represents a cell culture paradigm for studying synaptic competition. Mechanisms involved may include a retrograde ‘reward’ signal generated by WT neurons, although in this paradigm there was no ‘punishment’ signal against GluN1 -/- neurons. Cell culture assays involving such defined circuits may help uncover the rules and mechanisms of activity-dependent synaptic competition in the developing nervous

  9. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  10. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  11. Glutamate receptor-mediated toxicity in optic nerve oligodendrocytes

    PubMed Central

    Matute, Carlos; Sánchez-Gómez, M. Victoria; Martínez-Millán, Luis; Miledi, Ricardo

    1997-01-01

    In cultured oligodendrocytes isolated from perinatal rat optic nerves, we have analyzed the expression of ionotropic glutamate receptor subunits as well as the effect of the activation of these receptors on oligodendrocyte viability. Reverse transcription–PCR, in combination with immunocytochemistry, demonstrated that most oligodendrocytes differentiated in vitro express the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluR3 and GluR4 and the kainate receptor subunits GluR6, GluR7, KA1 and KA2. Acute and chronic exposure to kainate caused extensive oligodendrocyte death in culture. This effect was partially prevented by the AMPA receptor antagonist GYKI 52466 and was completely abolished by the non-N-methyl-d-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), suggesting that both AMPA and kainate receptors mediate the observed kainate toxicity. Furthermore, chronic application of kainate to optic nerves in vivo resulted in massive oligodendrocyte death which, as in vitro, could be prevented by coinfusion of the toxin with CNQX. These findings suggest that excessive activation of the ionotropic glutamate receptors expressed by oligodendrocytes may act as a negative regulator of the size of this cell population. PMID:9238063

  12. Retinoic Acid-mediated Nuclear Receptor Activation and Hepatocyte Proliferation

    PubMed Central

    Bushue, Nathan; Wan, Yu-Jui Yvonne

    2016-01-01

    Due to their well-known differentiation and apoptosis-inducing abilities, retinoic acid (RA) and its analogs have strong anti-cancer efficacy in human cancers. However, in vivo RA is a liver mitogen. While speculation has persisted that RA-mediated signaling is likely involved in hepatocyte proliferation during liver regeneration, direct evidence is still required. Findings in support of this proposition include observations that a release of retinyl palmitate (the precursor of RA) occurs in liver stellate cells following liver injury. Nevertheless, the biological action of this released vitamin A is virtually unknown. More likely is that the released vitamin A is converted to RA, the biological form, and then bound to a specific receptor (retinoid x receptor; RXRα), which is most abundantly expressed in the liver. Considering the mitogenic effects of RA, the RA-activated RXRα would likely then influence hepatocyte proliferation and liver tissue repair. At present, the mechanism by which RA stimulates hepatocyte proliferation is largely unknown. This review summarizes the activation of nuclear receptors (peroxisome proliferator activated receptor-α, pregnane x receptor, constitutive androstane receptor, and farnesoid x receptor) in an RXRα dependent manner to induce hepatocyte proliferation, providing a link between RA and its proliferative role. PMID:27635169

  13. The receptors that mediate the direct lethality of anthrax toxin.

    PubMed

    Liu, Shihui; Zhang, Yi; Hoover, Benjamin; Leppla, Stephen H

    2012-12-27

    Tumor endothelium marker-8 (TEM8) and capillary morphogenesis protein-2 (CMG2) are the two well-characterized anthrax toxin receptors, each containing a von Willebrand factor A (vWA) domain responsible for anthrax protective antigen (PA) binding. Recently, a cell-based analysis was used to implicate another vWA domain-containing protein, integrin β1 as a third anthrax toxin receptor. To explore whether proteins other than TEM8 and CMG2 function as anthrax toxin receptors in vivo, we challenged mice lacking TEM8 and/or CMG2. Specifically, we used as an effector protein the fusion protein FP59, a fusion between the PA-binding domain of anthrax lethal factor (LF) and the catalytic domain of Pseudomonas aeruginosa exotoxin A. FP59 is at least 50-fold more potent than LF in the presence of PA, with 2 μg PA + 2 μg FP59 being sufficient to kill a mouse. While TEM8(-/-) and wild type control mice succumbed to a 5 μg PA + 5 μg FP59 challenge, CMG2(-/-) mice were completely resistant to this dose, confirming that CMG2 is the major anthrax toxin receptor in vivo. To detect whether any toxic effects are mediated by TEM8 or other putative receptors such as integrin β1, CMG2(-/-)/TEM8(-/-) mice were challenged with as many as five doses of 50 μg PA + 50 μg FP59. Strikingly, the CMG2(-/-)/TEM8(-/-) mice were completely resistant to the 5-dose challenge. These results strongly suggest that TEM8 is the only minor anthrax toxin receptor mediating direct lethality in vivo and that other proteins implicated as receptors do not play this role.

  14. Toll Like Receptor-9 Mediated Invasion in Breast Cancer

    DTIC Science & Technology

    2012-07-01

    breast cancers. A likely mechanism of this clinical finding involves dif- ferential responses to hypoxia. Our pre-clinical studies indicate that while...outcome of TLR activation is an innate immune reaction characterized by increased production of inflammatory mediators [2]. Recent studies indicate that...A 14. ABSTRACT TLR9 is a cellular DNA-receptor that is widely expressed in breast cancers. The aim of this work was to study whether DNA

  15. Nicotinic and opioid receptor regulation of striatal dopamine D2-receptor mediated transmission

    PubMed Central

    Mamaligas, Aphroditi A.; Cai, Yuan; Ford, Christopher P.

    2016-01-01

    In addition to dopamine neuron firing, cholinergic interneurons (ChIs) regulate dopamine release in the striatum via presynaptic nicotinic receptors (nAChRs) on dopamine axon terminals. Synchronous activity of ChIs is necessary to evoke dopamine release through this pathway. The frequency-dependence of disynaptic nicotinic modulation has led to the hypothesis that nAChRs act as a high-pass filter in the dopaminergic microcircuit. Here, we used optogenetics to selectively stimulate either ChIs or dopamine terminals directly in the striatum. To measure the functional consequence of dopamine release, D2-receptor synaptic activity was assessed via virally overexpressed potassium channels (GIRK2) in medium spiny neurons (MSNs). We found that nicotinic-mediated dopamine release was blunted at higher frequencies because nAChRs exhibit prolonged desensitization after a single pulse of synchronous ChI activity. However, when dopamine neurons alone were stimulated, nAChRs had no effect at any frequency. We further assessed how opioid receptors modulate these two mechanisms of release. Bath application of the κ opioid receptor agonist U69593 decreased D2-receptor activation through both pathways, whereas the μ opioid receptor agonist DAMGO decreased D2-receptor activity only as a result of cholinergic-mediated dopamine release. Thus the release of dopamine can be independently modulated when driven by either dopamine neurons or cholinergic interneurons. PMID:27886263

  16. A classification of opiate receptors that mediate antinociception in animals.

    PubMed Central

    Tyers, M. B.

    1980-01-01

    1 To investigate the opiate receptors that mediate antinociception, the activity profiles of opioid analgesic drugs have been determined against different nociceptive stimuli in the mouse and rat. 2 In tests that employ heat as the nociceptive stimulus, mu-opiate receptor agonists, such as morphine, pethidine and dextropropoxyphene, had steep and parallel dose-response curves and were capable of achieving maximum effects. In addition, the antinociceptive potency ratios of these drugs in heat tests were similar to those for analgesia in man. 3 The kappa-agonists, such as ethylketazocine, nalorphine, Mr2034 and pentazocine, were essentially inactive against heat nociception except at doses that caused sedation and motor incapacitation. 4 In the writhing and paw pressure tests both mu- and kappa-agonists produced steep and parallel dose-response curves. 5 It is concluded that both mu- and kappa-opiate receptors mediate antinociception in animals and that the interactions of analgesic drugs with these receptors may be classified in terms of their antinociceptive activities against qualitatively different nociceptive stimuli. PMID:6249436

  17. Receptor-mediated choreography of life and death.

    PubMed

    Bhardwaj, Anjana; Aggarwal, Bharat B

    2003-09-01

    The cytokine tumor necrosis factor was originally identified as a protein that kills tumor cells. So far, 18 distinct members of this family have been identified. All of them regulate cell survival, proliferation, differentiation, and cell death, also called apoptosis. The apoptosis induced by TNF, and other members of the family, for example, FasL, VEGI, and TRAIL is mediated through death receptors. The apoptotic signals by these cytokines are transduced by eight different death domain- (DD) containing receptors (TNFR1, also called DR1; Fas, also called DR2; DR3, DR4, DR5, DR6, NGFR, and EDAR). The intracellular portion of all these receptors contains a region approximately 80 amino acids long referred to as the "death domain." Upon activation by its ligand, the DD recruits various proteins that mediate both death and proliferation of the cells. These proteins in turn recruit other proteins via their DDs or death effector domains. The actual destruction of the cell, however, is accomplished by serial activation of a family of proteases referred to as caspases. Cell death is negatively regulated by a family of proteins that includes decoy receptors, silencer of DD, sentrin, cellular FLICE inhibitory protein, cellular inhibitors of apoptosis, and survivin. This review is an attempt to describe how these negative and positive players of cell death perform a harmonious dance with each other.

  18. Of pheromones and kairomones: what receptors mediate innate emotional responses?

    PubMed

    Fortes-Marco, Lluis; Lanuza, Enrique; Martinez-Garcia, Fernando

    2013-09-01

    Some chemicals elicit innate emotionally laden behavioral responses. Pheromones mediate sexual attraction, parental care or agonistic confrontation, whereas predators' kairomones elicit defensive behaviors in their preys. This essay explores the hypothesis that the detection of these semiochemicals relies on highly specific olfactory and/or vomeronasal receptors. The V1R, V2R, and formyl-peptide vomeronasal receptors bind their ligands in highly specific and sensitive way, thus being good candidates for pheromone- or kairomone-detectors (e.g., secreted and excreted proteins, peptides and lipophilic volatiles). The olfactory epithelium also expresses specific receptors, for example trace amine-associated receptors (TAAR) and guanylyl cyclase receptors (GC-D and other types), some of which bind kairomones and putative pheromones. However, most of the olfactory neurons express canonical olfactory receptors (ORs) that bind many ligands with different affinity, being not suitable for mediating responses to pheromones and kairomones. In this respect, trimethylthiazoline (TMT) is considered a fox-derived kairomone for mice and rats, but it seems to be detected by canonical ORs. Therefore, we have reassessed the kairomonal nature of TMT by analyzing the behavioral responses of outbred (CD1) and inbred mice (C57BL/J6) to TMT. Our results confirm that both mouse strains avoid TMT, which increases immobility in C57BL/J6, but not CD1 mice. However, mice of both strains sniff at TMT throughout the test and show no trace of TMT-induced contextual conditioning (immobility or avoidance). This suggests that TMT is not a kairomone but, similar to a loud noise, in high concentrations it induces aversion and stress as unspecific responses to a strong olfactory stimulation.

  19. Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

    PubMed Central

    Cattaneo, Fabio; Guerra, Germano; Parisi, Melania; De Marinis, Marta; Tafuri, Domenico; Cinelli, Mariapia; Ammendola, Rosario

    2014-01-01

    G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca2+ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we

  20. Induction of aryl hydrocarbon receptor-mediated and estrogen receptor-mediated activities, and modulation of cell proliferation by dinaphthofurans.

    PubMed

    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.

  1. TRPA1 receptors mediate environmental irritant-induced meningeal vasodilatation

    PubMed Central

    Kunkler, Phillip Edward; Ballard, Carrie Jo; Oxford, Gerry Stephen; Hurley, Joyce Harts

    2010-01-01

    The TRPA1 receptor is a member of the transient receptor potential (TRP) family of ion channels expressed in nociceptive neurons. TRPA1 receptors are targeted by pungent compounds from mustard and garlic and environmental irritants such as formaldehyde and acrolein. Ingestion or inhalation of these chemical agents causes irritation and burning in the nasal and oral mucosa and respiratory lining. Headaches have been widely reported to be induced by inhalation of environmental irritants, but it is unclear how these agents produce headache. Stimulation of trigeminal neurons releases CGRP and substance P and induces neurogenic inflammation associated with the pain of migraine. Here we test the hypothesis that activation of TRPA1 receptors are the mechanistic link between environmental irritants and peptide mediated neurogenic inflammation. Known TRPA1 agonists and environmental irritants stimulate CGRP release from dissociated rat trigeminal ganglia neurons and this release is blocked by a selective TRPA1 antagonist, HC-030031. Further, TRPA1 agonists and environmental irritants increase meningeal blood flow following intranasal administration. Prior dural application of the CGRP antagonist, CGRP8–37, or intranasal or dural administration of HC-030031, blocks the increases in blood flow elicited by environmental irritants. Together these results demonstrate that TRPA1 receptor activation by environmental irritants stimulates CGRP release and increases cerebral blood flow. We suggest that these events contribute to headache associated with environmental irritants. PMID:21075522

  2. Purine receptor mediated actin cytoskeleton remodeling of human fibroblasts

    PubMed Central

    Goldman, Nanna; Chandler-Militello, Devin; Langevin, Helene; Nedergaard, Maiken; Takano, Takahiro

    2013-01-01

    Earlier studies have shown that activation of adenosine A1 receptors on peripheral pain fibers contributes to acupuncture-induced suppression of painful input. In addition to adenosine, acupuncture triggers the release of other purines, including ATP and ADP that may bind to purine receptors on nearby fibroblasts. We here show that purine agonists trigger increase in cytosolic Ca 2+ signaling in a cultured human fibroblasts cell line. The profile of agonist-induced Ca2+ increases indicates that the cells express functional P2yR2 and P2yR4 receptors, as well as P2yR1 and P2xR7 receptors. Unexpectedly, purine-induced Ca2+ signaling was associated with a remodeling of the actin cytoskeleton. ATP induced a transient loss in F-actin stress fiber. The changes of actin cytoskeleton occurred slowly and peaked at 10 min after agonist exposure. Inhibition of ATP-induced increases in Ca2+ by cyclopiazonic acid blocked receptor-mediated cytoskeleton remodeling. The Ca2+ ionophore failed to induce cytoskeletal remodeling despite triggering robust increases in cytosolic Ca2+. These observations indicate that purine signaling induces transient changes in fibroblast cytoarchitecture that could be related to the beneficial effects of acupuncture. PMID:23462235

  3. Receptor- and reactive intermediate-mediated mechanisms of teratogenesis.

    PubMed

    Wells, Peter G; Lee, Crystal J J; McCallum, Gordon P; Perstin, Julia; Harper, Patricia A

    2010-01-01

    Drugs and environmental chemicals can adversely alter the development of the fetus at critical periods during pregnancy, resulting in death, or in structural and functional birth defects in the surviving offspring. This process of teratogenesis may not be evident until a decade or more after birth. Postnatal functional abnormalities include deficits in brain function, a variety of metabolic diseases, and cancer. Due to the high degree of fetal cellular division and differentiation, and to differences from the adult in many biochemical pathways, the fetus is highly susceptible to teratogens, typically at low exposure levels that do not harm the mother. Insights into the mechanisms of teratogenesis come primarily from animal models and in vitro systems, and involve either receptor-mediated or reactive intermediate-mediated processes. Receptor-mediated mechanisms involving the reversible binding of xenobiotic substrates to a specific receptor are exemplified herein by the interaction of the environmental chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or "dioxin") with the cytosolic aryl hydrocarbon receptor (AHR), which translocates to the nucleus and, in association with other proteins, binds to AH-responsive elements (AHREs) in numerous genes, initiating changes in gene transcription that can perturb development. Alternatively, many xenobiotics are bioactivated by fetal enzymes like the cytochromes P450 (CYPs) and prostaglandin H synthases (PHSs) to highly unstable electrophilic or free radical reactive intermediates. Electrophilic reactive intermediates can covalently (irreversibly) bind to and alter the function of essential cellular macromolecules (proteins, DNA), causing developmental anomalies. Free radical reactive intermediates can enhance the formation of reactive oxygen species (ROS), resulting in oxidative damage to cellular macromolecules and/or altered signal transduction. The teratogenicity of reactive intermediates is determined to a large extent

  4. Opioid receptor internalization contributes to dermorphin-mediated antinociception

    PubMed Central

    Macey, Tara A.; Ingram, Susan L.; Bobeck, Erin N.; Hegarty, Deborah M.; Aicher, Sue A.; Arttamangkul, Seksiri; Morgan, Michael M.

    2010-01-01

    Microinjection of opioids into the ventrolateral periaqueductal gray (vlPAG) produces antinociception in part by binding to mu-opioid receptors (MOPrs). Although both high and low efficacy agonists produce antinociception, low efficacy agonists such as morphine produce limited MOPr internalization suggesting that MOPr internalization and signaling leading to antinociception are independent. This hypothesis was tested in awake, behaving rats using DERM-A594, a fluorescently labeled dermorphin analog, and internalization blockers. Microinjection of DERM-A594 into the vlPAG produced both antinociception and internalization of DERM-A594. Administration of the irreversible opioid receptor antagonist beta-CNA prior to DERM-A594 microinjection reduced both the antinociceptive effect and the number of DERM-A594 labeled cells demonstrating that both effects are opioid receptor-mediated. Pretreatment with the internalization blockers dynamin dominant-negative inhibitory peptide (dynamin-DN) and concanavalinA (ConA) attenuated both DERM-A594 internalization and antinociception. Microinjection of dynamin-DN and ConA also decreased the antinociceptive potency of the unlabeled opioid agonist dermorphin when microinjected into the vlPAG as demonstrated by rightward shifts in the dose-response curves. In contrast, administration of dynamin-DN had no effect on the antinociceptive effect of microinjecting the GABAA antagonist bicuculline into the vlPAG. The finding that dermorphin-induced antinociception is attenuated by blocking receptor internalization indicates that key parts of opioid receptor-mediated signaling depend on internalization. PMID:20394808

  5. Menthol inhibits 5-HT3 receptor-mediated currents.

    PubMed

    Ashoor, Abrar; Nordman, Jacob C; Veltri, Daniel; Yang, Keun-Hang Susan; Shuba, Yaroslav; Al Kury, Lina; Sadek, Bassem; Howarth, Frank C; Shehu, Amarda; Kabbani, Nadine; Oz, Murat

    2013-11-01

    The effects of alcohol monoterpene menthol, a major active ingredient of the peppermint plant, were tested on the function of human 5-hydroxytryptamine type 3 (5-HT3) receptors expressed in Xenopus laevis oocytes. 5-HT (1 μM)-evoked currents recorded by two-electrode voltage-clamp technique were reversibly inhibited by menthol in a concentration-dependent (IC50 = 163 μM) manner. The effects of menthol developed gradually, reaching a steady-state level within 10-15 minutes and did not involve G-proteins, since GTPγS activity remained unaltered and the effect of menthol was not sensitive to pertussis toxin pretreatment. The actions of menthol were not stereoselective as (-), (+), and racemic menthol inhibited 5-HT3 receptor-mediated currents to the same extent. Menthol inhibition was not altered by intracellular 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid injections and transmembrane potential changes. The maximum inhibition observed for menthol was not reversed by increasing concentrations of 5-HT. Furthermore, specific binding of the 5-HT3 antagonist [(3)H]GR65630 was not altered in the presence of menthol (up to 1 mM), indicating that menthol acts as a noncompetitive antagonist of the 5-HT3 receptor. Finally, 5-HT3 receptor-mediated currents in acutely dissociated nodose ganglion neurons were also inhibited by menthol (100 μM). These data demonstrate that menthol, at pharmacologically relevant concentrations, is an allosteric inhibitor of 5-HT3 receptors.

  6. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  7. Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.

    PubMed

    Lopez, Joëlle; Gamache, Karine; Schneider, Rilla; Nader, Karim

    2015-02-11

    Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

  8. The brain hypocretins and their receptors: mediators of allostatic arousal.

    PubMed

    Carter, Matthew E; Schaich Borg, Jana; de Lecea, Luis

    2009-02-01

    The hypocretins (abbreviated 'Hcrts' - also called 'orexins') are two neuropeptides secreted exclusively by a small population of neurons in the lateral hypothalamus. These peptides bind to two receptors located throughout the brain in nuclei associated with diverse cognitive and physiological functions. Initially, the brain Hcrt system was found to have a major role in the regulation of sleep/wake transitions. More recent studies indicate Hcrts may play a role in other physiological functions, including food intake, addiction, and stress. Taken together, these studies suggest a general role for Hcrts in mediating arousal, especially when an organism must respond to unexpected stressors and challenges in the environment.

  9. Origin of Toll-like receptor-mediated innate immunity.

    PubMed

    Kanzok, Stefan M; Hoa, Ngo T; Bonizzoni, Mariangela; Luna, Coralia; Huang, Yaming; Malacrida, Anna R; Zheng, Liangbiao

    2004-04-01

    Toll-related receptors (TLR) have been found in four animal phyla: Nematoda, Arthropoda, Echinodermata, and Chordata. No TLR has been identified thus far in acoelomates. TLR genes play a pivotal role in the innate immunity in both fruit fly and mammals. The prevailing view is that TLR-mediated immunity is ancient. The two pseudocoelomate TLRs, one each from Caenorhabditis elegans and Strongyloides stercoralis, were distinct from the coelomate ones. Further, the only TLR gene (Tol-1) in Ca. elegans did not appear to play a role in innate immunity. We argue that TLR-mediated innate immunity developed only in the coelomates, after they split from pseudocoelomates and acoelomates. We hypothesize that the function of TLR-mediated immunity is to prevent microbial infection in the body cavity present only in the coelomates. Phylogenetic analysis showed that almost all arthropod TLRs form a separate cluster from the mammalian counterparts. We further hypothesize that TLR-mediated immunity developed independently in the protostomia and deuterostomia coelomates.

  10. Drosophila Ryanodine Receptors Mediate General Anesthesia by Halothane

    PubMed Central

    Gao, Shuying; Sandstrom, David J.; Smith, Harold E.; High, Brigit; Marsh, Jon W.; Nash, Howard A.

    2012-01-01

    Background Although in vitro studies have identified numerous possible targets, the molecules that mediate the in vivo effects of volatile anesthetics remain largely unknown. The mammalian ryanodine receptor (Ryr) is a known halothane target, and we hypothesized that it has a central role in anesthesia. Methods Gene function of the Drosophila Ryr (dRyr) was manipulated in the whole body or in specific tissues using a collection of mutants and transgenes, and responses to halothane were measured with a reactive climbing assay. Cellular responses to halothane were studied using Ca2+ imaging and patch clamp electrophysiology. Results Halothane potency strongly correlates with dRyr gene copy number, and missense mutations in regions known to be functionally important in mammalian Ryrs gene cause dominant hypersensitivity. Tissue-specific manipulation of dRyr shows that expression in neurons and glia, but not muscle, mediates halothane sensitivity. In cultured cells, halothane-induced Ca2+ efflux is strictly dRyr-dependent, suggesting a close interaction between halothane and dRyr. Ca2+ imaging and electrophysiology of Drosophila central neurons reveal halothane-induced Ca2+ flux that is altered in dRyr mutants and correlates with strong hyperpolarization. Conclusions In Drosophila, neurally-expressed dRyr mediates a substantial proportion of halothane's anesthetic effects in vivo, is potently activated by halothane in vitro, and activates an inhibitory conductance. Our results provide support for Ryr as an important mediator of immobilization by volatile anesthetics. PMID:23254148

  11. Extracellular loop 2 in the FSH receptor is crucial for ligand mediated receptor activation.

    PubMed

    Dupakuntla, Madhavi; Pathak, Bhakti; Roy, Binita Sur; Mahale, Smita D

    2012-10-15

    The present study aims to determine the role of the specific residues of the extracellular loops (ELs) of the FSH receptor (FSHR) in hormone binding and receptor activation. By substituting the sequences of each of the ELs of human FSHR with those of the luteinizing hormone/choriogonadotropin receptor (LH/CGR), we generated three mutant constructs where the three ELs were individually replaced. A fourth construct had all the three substituted ELs. The receptor expression and hormone binding ability of the mutants were comparable to that of the wild type. Hormone-induced signaling and internalization were lower in the EL2 substitution mutant (EL2M). In this mutant, the EL2 of FSHR was substituted with the corresponding loop of LH/CGR. Interestingly, homology modeling revealed a change in the orientation of EL2 in the mutant receptor. Thus, disruption of EL2 affected overall receptor function, suggesting the role of FSHR specific residues of the loop in ligand mediated signaling.

  12. CXCR4 chemokine receptor signaling mediates pain in diabetic neuropathy

    PubMed Central

    2014-01-01

    Background Painful Diabetic Neuropathy (PDN) is a debilitating syndrome present in a quarter of diabetic patients that has a substantial impact on their quality of life. Despite this significant prevalence and impact, current therapies for PDN are only partially effective. Moreover, the cellular mechanisms underlying PDN are not well understood. Neuropathic pain is caused by a variety of phenomena including sustained excitability in sensory neurons that reduces the pain threshold so that pain is produced in the absence of appropriate stimuli. Chemokine signaling has been implicated in the pathogenesis of neuropathic pain in a variety of animal models. We therefore tested the hypothesis that chemokine signaling mediates DRG neuronal hyperexcitability in association with PDN. Results We demonstrated that intraperitoneal administration of the specific CXCR4 antagonist AMD3100 reversed PDN in two animal models of type II diabetes. Furthermore DRG sensory neurons acutely isolated from diabetic mice displayed enhanced SDF-1 induced calcium responses. Moreover, we demonstrated that CXCR4 receptors are expressed by a subset of DRG sensory neurons. Finally, we observed numerous CXCR4 expressing inflammatory cells infiltrating into the DRG of diabetic mice. Conclusions These data suggest that CXCR4/SDF-1 signaling mediates enhanced calcium influx and excitability in DRG neurons responsible for PDN. Simultaneously, CXCR4/SDF-1 signaling may coordinate inflammation in diabetic DRG that could contribute to the development of pain in diabetes. Therefore, targeting CXCR4 chemokine receptors may represent a novel intervention for treating PDN. PMID:24961298

  13. Receptor-mediated toxicity of pahutoxin, a marine trunkfish surfactant.

    PubMed

    Kalmanzon, Eliahu; Rahamim, Yocheved; Barenholz, Yechezkel; Carmeli, Shmuel; Zlotkin, Eliahu

    2003-07-01

    Pahutoxin (PHN, choline chloride ester of 3-acetoxypalmitic acid) is a natural fish-killing (ichthyotoxic) agent derived from the defensive secretions of trunkfish. In spite of its obvious structural resemblance to synthetic cationic long-chain quaternary ammonium detergents, we show that PHN's action does not rely on its surfactant properties and is in fact, receptor-mediated. The above conclusion is supported by the following data: 1. Ichthyotoxicity is not related to its detergency or surfactivity, as indicated by the fact that the lethal concentration is about 1.5 orders of magnitude below its critical micelle concentration value (69 microM) and its liposomal/seawater partition coefficient is low (62-85); 2. The trunkfish is tolerant to its own pahutoxin; 3. Ichthyotoxicity occurs only upon application to the surrounding water, suggesting the existence of externally located receptors; 4. The receptor hypothesis was supported by the aid of equilibrium saturation binding assays revealing the presence of specific binding sites to PHN on the fish gill membranes; 5. The PHN tolerant trunkfish was shown to be devoid of PHN-binding sites. Some chemo-ecological, and environmental implications are discussed.

  14. Lamprey variable lymphocyte receptors mediate complement-dependent cytotoxicity.

    PubMed

    Wu, Fenfang; Chen, Liyong; Liu, Xin; Wang, Huaying; Su, Peng; Han, Yinglun; Feng, Bo; Qiao, Xu; Zhao, Jing; Ma, Ning; Liu, Huijie; Zheng, Zhen; Li, Qingwei

    2013-02-01

    An alternative adaptive-immune system is present in the most basal vertebrates--lampreys and hagfish--the only surviving jawless vertebrates. These eel-like fish use leucine-rich repeat-based receptors for Ag recognition instead of the Ig-based receptors used in jawed vertebrates. We report that in Japanese lamprey (Lampetra japonica), variable lymphocyte receptor (VLR)B interacts with C1q and C3 proteins to mediate complement-dependent cytotoxicity for bacteria and tumor cells. The immune-based lysis involves deposition of VLRB and C1q-like protein complex on the surface of target cells, activation of C3, and ultimate disruption of cell wall integrity. The demonstration of functional interaction between VLRB and complement components in lamprey provides evidence for the emergence of cooperative innate and adaptive-immune responses at a pivotal point in vertebrate evolution, before or in parallel with the evolution of Ig-based Abs and the classical complement-activation pathway.

  15. Visualization of Receptor-mediated Endocytosis in Yeast

    PubMed Central

    Mulholland, Jon; Konopka, James; Singer-Kruger, Birgit; Zerial, Marino; Botstein, David

    1999-01-01

    We studied the ligand-induced endocytosis of the yeast α-factor receptor Ste2p by immuno-electron microscopy. We observed and quantitated time-dependent loss of Ste2p from the plasma membrane of cells exposed to α-factor. This ligand-induced internalization of Ste2p was blocked in the well-characterized endocytosis-deficient mutant sac6Δ. We provide evidence that implicates furrow-like invaginations of the plasma membrane as the site of receptor internalization. These invaginations are distinct from the finger-like plasma membrane invaginations within actin cortical patches. Consistent with this, we show that Ste2p is not located within the cortical actin patch before and during receptor-mediated endocytosis. In wild-type cells exposed to α-factor we also observed and quantitated a time-dependent accumulation of Ste2p in intracellular, membrane-bound compartments. These compartments have a characteristic electron density but variable shape and size and are often located adjacent to the vacuole. In immuno-electron microscopy experiments these compartments labeled with antibodies directed against the rab5 homologue Ypt51p (Vps21p), the resident vacuolar protease carboxypeptidase Y, and the vacuolar H+-ATPase Vph1p. Using a new double-labeling technique we have colocalized antibodies against Ste2p and carboxypeptidase Y to this compartment, thereby identifying these compartments as prevacuolar late endosomes. PMID:10069819

  16. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines.

    PubMed

    Hussain, Ashiq; Zhang, Mo; Üçpunar, Habibe K; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C

    2016-05-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly's high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans.

  17. Ionotropic Chemosensory Receptors Mediate the Taste and Smell of Polyamines

    PubMed Central

    Üçpunar, Habibe K.; Svensson, Thomas; Quillery, Elsa; Gompel, Nicolas; Ignell, Rickard; Grunwald Kadow, Ilona C.

    2016-01-01

    The ability to find and consume nutrient-rich diets for successful reproduction and survival is fundamental to animal life. Among the nutrients important for all animals are polyamines, a class of pungent smelling compounds required in numerous cellular and organismic processes. Polyamine deficiency or excess has detrimental effects on health, cognitive function, reproduction, and lifespan. Here, we show that a diet high in polyamine is beneficial and increases reproductive success of flies, and we unravel the sensory mechanisms that attract Drosophila to polyamine-rich food and egg-laying substrates. Using a combination of behavioral genetics and in vivo calcium imaging, we demonstrate that Drosophila uses multisensory detection to find and evaluate polyamines present in overripe and fermenting fruit, their favored feeding and egg-laying substrate. In the olfactory system, two coexpressed ionotropic receptors (IRs), IR76b and IR41a, mediate the long-range attraction to the odor. In the gustatory system, multimodal taste sensation by IR76b receptor and GR66a bitter receptor neurons is used to evaluate quality and valence of the polyamine providing a mechanism for the fly’s high attraction to polyamine-rich and sweet decaying fruit. Given their universal and highly conserved biological roles, we propose that the ability to evaluate food for polyamine content may impact health and reproductive success also of other animals including humans. PMID:27145030

  18. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by adenosine receptors in the rat hippocampus.

    PubMed Central

    Morton, R A; Davies, C H

    1997-01-01

    1. Intracellular current clamp recordings were made from CA1 pyramidal neurones in rat hippocampal slices. Experiments were performed in the presence of ionotropic glutamate receptor antagonists and gamma-aminobutyric acid (GABA) receptor antagonists to block all fast excitatory and inhibitory synaptic transmission. A single stimulus, delivered extracellularly in the stratum oriens, caused a reduction in spike frequency adaptation in response to a depolarizing current step delivered 2 s after the stimulus. A 2- to 10-fold increase in stimulus intensity evoked a slow excitatory postsynaptic potential (EPSP) which was associated with a small increase in input resistance. The peak amplitude of the EPSP occurred approximately 2.5 s after the stimulus and its magnitude (up to 30 mV) and duration (10-50 s) increased with increasing stimulus intensity. 2. The slow EPSP was unaffected by the metabotropic glutamate receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG; 1000 microM) but was greatly enhanced by the acetylcholinesterase inhibitor physostigmine (1-5 microM). Both the slow EPSP and the stimulus-evoked reduction in spike frequency adaptation were inhibited by the muscarinic acetylcholine receptor (mAChR) antagonist atropine (1-5 microM). These results are consistent with these effects being mediated by mAChRs. 3. Both the mAChR-mediated EPSP (EPSPm) and the associated reduction in spike frequency adaptation were reversibly depressed (up to 97%) by either adenosine (100 microM) or its non-hydrolysable analogue 2-chloroadenosine (CADO; 0.1-5.0 microM). These effects were often accompanied by postsynaptic hyperpolarization (up to 8 mV) and a reduction in input resistance (up to 11%). The selective adenosine A1 receptor agonists 2-chloro-N6-cyclopentyladenosine (CCPA; 0.1-0.4 microM) and R(-)N6-(2-phenylisopropyl)-adenosine (R-PIA; 1 microM) both depressed the EPSPm. In contrast, the adenosine A2A receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5

  19. Dynamic kisspeptin receptor trafficking modulates kisspeptin-mediated calcium signaling.

    PubMed

    Min, Le; Soltis, Kathleen; Reis, Ana Claudia S; Xu, Shuyun; Kuohung, Wendy; Jain, Manisha; Carroll, Rona S; Kaiser, Ursula B

    2014-01-01

    Kisspeptin receptor (KISS1R) signaling plays a critical role in the regulation of reproduction. We investigated the role of kisspeptin-stimulated KISS1R internalization, recycling, and degradation in the modulation of KISS1R signaling. Kisspeptin stimulation of Chinese hamster ovary or GT1-7 cells expressing KISS1R resulted in a biphasic increase in intracellular Ca(2+) ([Ca(2+)]i), with a rapid acute increase followed by a more sustained second phase. In contrast, stimulation of the TRH receptor, another Gq/11-coupled receptor, resulted in a much smaller second-phase [Ca(2+)]i response. The KISS1R-mediated second-phase [Ca(2+)]i response was abolished by removal of kisspeptin from cell culture medium. Notably, the second-phase [Ca(2+)]i response was also inhibited by dynasore, brefeldin A, and phenylarsine oxide, which inhibit receptor internalization and recycling, suggesting that KISS1R trafficking contributes to the sustained [Ca(2+)]i response. We further demonstrated that KISS1R undergoes dynamic ligand-dependent and -independent recycling. We next investigated the fate of the internalized kisspeptin-KISS1R complex. Most internalized kisspeptin was released extracellularly in degraded form within 1 hour, suggesting rapid processing of the internalized kisspeptin-KISS1R complex. Using a biotinylation assay, we demonstrated that degradation of cell surface KISS1R was much slower than that of the internalized ligand, suggesting dissociated processing of the internalized kisspeptin-KISS1R complex. Taken together, our results suggest that the sustained calcium response to kisspeptin is dependent on the continued presence of extracellular ligand and is the result of dynamic KISS1R trafficking.

  20. Ligand Receptor-Mediated Regulation of Growth in Plants.

    PubMed

    Haruta, Miyoshi; Sussman, Michael R

    2017-01-01

    Growth and development of multicellular organisms are coordinately regulated by various signaling pathways involving the communication of inter- and intracellular components. To form the appropriate body patterns, cellular growth and development are modulated by either stimulating or inhibiting these pathways. Hormones and second messengers help to mediate the initiation and/or interaction of the various signaling pathways in all complex multicellular eukaryotes. In plants, hormones include small organic molecules, as well as larger peptides and small proteins, which, as in animals, act as ligands and interact with receptor proteins to trigger rapid biochemical changes and induce the intracellular transcriptional and long-term physiological responses. During the past two decades, the availability of genetic and genomic resources in the model plant species, Arabidopsis thaliana, has greatly helped in the discovery of plant hormone receptors and the components of signal transduction pathways and mechanisms used by these immobile but highly complex organisms. Recently, it has been shown that two of the most important plant hormones, auxin and abscisic acid (ABA), act through signaling pathways that have not yet been recognized in animals. For example, auxins stimulate cell elongation by bringing negatively acting transcriptional repressor proteins to the proteasome to be degraded, thus unleashing the gene expression program required for increasing cell size. The "dormancy" inducing hormone, ABA, binds to soluble receptor proteins and inhibits a specific class of protein phosphatases (PP2C), which activates phosphorylation signaling leading to transcriptional changes needed for the desiccation of the seeds prior to entering dormancy. While these two hormone receptors have no known animal counterparts, there are also many similarities between animal and plant signaling pathways. For example, in plants, the largest single gene family in the genome is the protein kinase

  1. NFAT regulates calcium-sensing receptor-mediated TNF production.

    PubMed

    Abdullah, Huda Ismail; Pedraza, Paulina L; Hao, Shoujin; Rodland, Karin D; McGiff, John C; Ferreri, Nicholas R

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca(2+) (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca(2+) were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  2. NFAT regulates calcium-sensing receptor-mediated TNF production

    SciTech Connect

    abdullah, huda ismail; Pedraza, Paulina L.; Hao, Shoujin; Rodland, Karin D.; McGiff, John C.; Ferreri, Nicholas R.

    2006-05-01

    Because nuclear factor of activated T cells (NFAT) has been implicated in TNF production as well as osmoregulation and salt and water homeostasis, we addressed whether calcium-sensing receptor (CaR)-mediated TNF production in medullary thick ascending limb (mTAL) cells was NFAT dependent. TNF production in response to addition of extracellular Ca2+ (1.2 mM) was abolished in mTAL cells transiently transfected with a dominant-negative CaR construct (R796W) or pretreated with the phosphatidylinositol phospholipase C (PI-PLC) inhibitor U-73122. Cyclosporine A (CsA), an inhibitor of the serine/threonine phosphatase calcineurin, and a peptide ligand, VIVIT, that selectively inhibits calcineurin-NFAT signaling, also prevented CaR-mediated TNF production. Increases in calcineurin activity in cells challenged with Ca2+ were inhibited after pretreatment with U-73122 and CsA, suggesting that CaR activation increases calcineurin activity in a PI-PLC-dependent manner. Moreover, U-73122, CsA, and VIVIT inhibited CaR-dependent activity of an NFAT construct that drives expression of firefly luciferase in transiently transfected mTAL cells. Collectively, these data verify the role of calcineurin and NFAT in CaR-mediated TNF production by mTAL cells. Activation of the CaR also increased the binding of NFAT to a consensus oligonucleotide, an effect that was blocked by U-73122 and CsA, suggesting that a calcineurin- and NFAT-dependent pathway increases TNF production in mTAL cells. This mechanism likely regulates TNF gene transcription as U-73122, CsA, and VIVIT blocked CaR-dependent activity of a TNF promoter construct. Elucidating CaR-mediated signaling pathways that regulate TNF production in the mTAL will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.

  3. Herpes virus entry mediator synergizes with Toll-like receptor mediated neutrophil inflammatory responses

    PubMed Central

    Haselmayer, Philipp; Tenzer, Stefan; Kwon, Byoung S; Jung, Gundram; Schild, Hansjörg; Radsak, Markus P

    2006-01-01

    In microbial infections polymorphnuclear neutrophils (PMN) constitute a major part of the innate host defence, based upon their ability to rapidly accumulate in inflamed tissues and clear the site of infection from microbial pathogens by their potent effector mechanisms. The recently described transmembrane receptor herpes virus entry mediator (HVEM) is a member of the tumour necrosis factor receptor super family and is expressed on many haematopoietic cells, including T cells, B cells, natural killer cells, monocytes and PMN. Interaction of HVEM with the natural ligand LIGHT on T cells has a costimulatory effect, and increases the bactericidal activity of PMN. To further characterize the function of HVEM on PMN, we evaluated the effect of receptor ligation on human PMN effector functions using an agonistic monoclonal antibody. Here we demonstrate that activation of HVEM causes activation of neutrophil effector functions, including respiratory burst, degranulation and release of interleukin-8 in synergy with ligands for Toll-like receptors or GM-CSF. In addition, stimulation via HVEM enhanced neutrophil phagocytic activity of complement opsonized, but not of non-opsonized, particles. In conclusion, these results indicate a new, as yet unknown, participation of HVEM in the innate immune response and points to a new link between innate and adaptive immunity. PMID:17067315

  4. Brain prostanoid TP receptor-mediated adrenal noradrenaline secretion and EP3 receptor-mediated sympathetic noradrenaline release in rats.

    PubMed

    Yokotani, Keiko; Okada, Shoshiro; Nakamura, Kumiko; Yamaguchi-Shima, Naoko; Shimizu, Takahiro; Arai, Junichi; Wakiguchi, Hiroshi; Yokotani, Kunihiko

    2005-04-04

    Sympathetic nerves release noradrenaline, whereas adrenal medullary chromaffin cells secrete noradrenaline and adrenaline. Therefore, plasma noradrenaline reflects the secretion from adrenal medulla in addition to the release from sympathetic nerves, however the exact mechanisms of adrenal noradrenaline secretion remain to be elucidated. The present study was designated to characterize the source of plasma noradrenaline induced by intracerebroventricularly (i.c.v.) administered bombesin and prostaglandin E2 in urethane-anesthetized rats. Bombesin (1.0 nmol/animal, i.c.v.) elevated plasma noradrenaline and adrenaline, while prostaglandin E2 (0.3 nmol/animal, i.c.v.) elevated only plasma noradrenaline. The bombesin-induced elevations of both catecholamines were attenuated by pretreatments with furegrelate (an inhibitor of thromboxane A2 synthase) [250 and 500 microg (0.9 and 1.8 micromol)/animal, i.c.v.)] and [(+)-S-145] [(+)-(1R,2R,3S,4S)-(5Z)-7-(3-[4-3H]-phenylsulphonyl-aminobicyclo[2.2.1]hept-2-yl)hept-5-enoic acid sodium salt] (an antagonist of prostanoid TP receptors) [100 and 250 microg (250 and 625 nmol)/animal)], and abolished by acute bilateral adrenalectomy. On the other hand, the prostaglandin E2-induced elevation of plasma noradrenaline was not influenced by acute bilateral adrenalectomy. These results suggest that adrenal noradrenaline secretion and sympathetic noradrenaline release are mediated by differential central mechanisms; brain prostanoid TP receptors activated by bombesin are involved in the adrenal noradrenaline secretion, while brain prostanoid EP (probably EP3) receptors activated by prostaglandin E2 are involved in the sympathetic noradrenaline release in rats. Brain prostanoid TP receptors activated by bombesin are also involved in the adrenal adrenaline secretion.

  5. Determinants of oligomeric structure in the chicken liver glycoprotein receptor.

    PubMed Central

    Verrey, F; Drickamer, K

    1993-01-01

    The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation. Images Figure 1 Figure 3 PMID:8503842

  6. Mefloquine neurotoxicity is mediated by non-receptor tyrosine kinase.

    PubMed

    Milatovic, Dejan; Jenkins, Jerry W; Hood, Jonathan E; Yu, Yingchun; Rongzhu, Lu; Aschner, Michael

    2011-10-01

    Among several available antimalarial drugs, mefloquine has proven to be effective against drug-resistant Plasmodium falciparum and remains the drug of choice for both therapy and chemoprophylaxis. However, mefloquine is known to cause adverse neurological and/or psychiatric symptoms, which offset its therapeutic advantage. The exact mechanisms leading to the adverse neurological effects of mefloquine are poorly defined. Alterations in neurotransmitter release and calcium homeostasis, the inhibition of cholinesterases and the interaction with adenosine A(2A) receptors have been hypothesized to play prominent roles in mediating the deleterious effects of this drug. Our recent data have established that mefloquine can also trigger oxidative damage and subsequent neurodegeneration in rat cortical primary neurons. Furthermore, we have utilized a system biology-centered approach and have constructed a pathway model of cellular responses to mefloquine, identifying non-receptor tyrosine kinase 2 (Pyk2) as a critical target in mediating mefloquine neurotoxicity. In this study, we sought to establish an experimental validation of Pyk2 using gene-silencing techniques (siRNA). We have examined whether the downregulation of Pyk2 in primary rat cortical neurons alters mefloquine neurotoxicity by evaluating cell viability, apoptosis and oxidative stress. Results from our study have confirmed that mefloquine neurotoxicity is associated with apoptotic response and oxidative injury, and we have demonstrated that mefloquine affects primary rat cortical neurons, at least in part, via Pyk2. The implication of these findings may prove beneficial in suppressing the neurological side effects of mefloquine and developing effective therapeutic modalities to offset its adverse effects.

  7. Retromer-Mediated Trafficking of Transmembrane Receptors and Transporters

    PubMed Central

    Klinger, Stine C.; Siupka, Piotr; Nielsen, Morten S.

    2015-01-01

    Transport between the endoplasmatic reticulum, the Golgi-network, the endo-lysosomal system and the cell surface can be categorized as anterograde or retrograde, describing traffic that goes forward or backward, respectively. Traffic going from the plasma membrane to endosomes and lysosomes or the trans-Golgi network (TGN) constitutes the major retrograde transport routes. Several transmembrane proteins undergo retrograde transport as part of a recycling mechanism that contributes to reutilization and maintenance of a steady-state protein localization. In addition, some receptors are hijacked by exotoxins and used for entry and intracellular transport. The physiological relevance of retrograde transport cannot be overstated. Retrograde trafficking of the amyloid precursor protein determines the distribution between organelles, and hence the possibility of cleavage by γ-secretase. Right balancing of the pathways is critical for protection against Alzheimer’s disease. During embryonic development, retrograde transport of Wntless to the TGN is essential for the following release of Wnt from the plasma membrane. Furthermore, overexpression of Wntless has been linked to oncogenesis. Here, we review relevant aspects of the retrograde trafficking of mammalian transmembrane receptors and transporters, with focus on the retromer-mediated transport between endosomes and the TGN. PMID:26154780

  8. Scavenger receptor mediates systemic RNA interference in ticks.

    PubMed

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Xuenan, Xuan; Suzuki, Hiroshi; Galay, Remil Linggatong; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-01-01

    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks.

  9. Scavenger Receptor Mediates Systemic RNA Interference in Ticks

    PubMed Central

    Aung, Kyaw Min; Boldbaatar, Damdinsuren; Umemiya-Shirafuji, Rika; Liao, Min; Xuenan, Xuan; Suzuki, Hiroshi; Linggatong Galay, Remil; Tanaka, Tetsuya; Fujisaki, Kozo

    2011-01-01

    RNA interference is an efficient method to silence gene and protein expressions. Here, the class B scavenger receptor CD36 (SRB) mediated the uptake of exogenous dsRNAs in the induction of the RNAi responses in ticks. Unfed female Haemaphysalis longicornis ticks were injected with a single or a combination of H. longicornis SRB (HlSRB) dsRNA, vitellogenin-1 (HlVg-1) dsRNA, and vitellogenin receptor (HlVgR) dsRNA. We found that specific and systemic silencing of the HlSRB, HlVg-1, and HlVgR genes was achieved in ticks injected with a single dsRNA of HlSRB, HlVg-1, and HlVgR. In ticks injected first with HlVg-1 or HlVgR dsRNA followed 96 hours later with HlSRB dsRNA (HlVg-1/HlSRB or HlVgR/HlSRB), gene silencing of HlSRB was achieved in addition to first knockdown in HlVg-1 or HlVgR, and prominent phenotypic changes were observed in engorgement, mortality, and hatchability, indicating that a systemic and specific double knockdown of target genes had been simultaneously attained in these ticks. However, in ticks injected with HlSRB dsRNA followed 96 hours later with HlVg-1 or HlVgR dsRNAs, silencing of HlSRB was achieved, but no subsequent knockdown in HlVgR or HlVg-1 was observed. The Westernblot and immunohistochemical examinations revealed that the endogenous HlSRB protein was fully abolished in midguts of ticks injected with HlSRB/HlVg-1 dsRNAs but HlVg-1 was normally expressed in midguts, suggesting that HlVg-1 dsRNA-mediated RNAi was fully inhibited by the first knockdown of HlSRB. Similarly, the abolished localization of HlSRB protein was recognized in ovaries of ticks injected with HlSRB/HlVgR, while normal localization of HlVgR was observed in ovaries, suggesting that the failure to knock-down HlVgR could be attributed to the first knockdown of HlSRB. In summary, we demonstrated for the first time that SRB may not only mediate the effective knock-down of gene expression by RNAi but also play essential roles for systemic RNAi of ticks. PMID:22145043

  10. Rapid and estrogen receptor beta mediated actions in the hippocampus mediate some functional effects of estrogen

    PubMed Central

    Walf, Alicia A.; Frye, Cheryl A.

    2008-01-01

    The steroid hormone, estradiol (E2), has numerous targets in the central nervous system, including the hippocampus, which plays a key role in cognition and affective behavior. This review focuses on our evidence from studies in rodents that E2 has diverse mechanisms in the hippocampus for its functional effects E2 has rapid, membrane-mediated effects in the hippocampus to enhance cognitive performance. Administration of E2 to the hippocampus of rats for 10 minutes following training enhances performance in a hippocampus-mediated task. Increased cell firing in the hippocampus occurs within this short time frame. Furthermore, administration of free E2 or an E2 conjugate, E2:bovine serum albumin (BSA), to the hippocampus produces similar performance-enhancing effects in this task, suggesting that E2 has membrane actions in the hippocampus for these effects. Further evidence that E2 has rapid, membrane-mediated effects is that co-administration of E2 and inhibitors of mitogen activated protein kinase (MAPK), rather than intracellular E2 receptors (ERs) or protein synthesis, attenuate the enhancing effects of E2 in this task. Despite these data that demonstrate E2 can have rapid and/or membrane-mediated effects in the hippocampus, there is clear evidence to suggest that intracellular ERs, particularly the β (rather than α) isoform of ERs, may be important targets for E2’s functional effects for hippocampal processes. Administration of ligands that are specific for ERβ, but not ERα, have enhancing effects on hippocampal processes similar to that of E2 (which has similar affinity for ERα and ERβ). These effects are attenuated when ERβ expression is knocked down in transgenic models or with central administration of antisense oligonucleotides. Thus, there may be a convergence of E2’s actions through rapid, membrane-mediated effects and intracellular ERs and in the hippocampus for these functional effects. PMID:18342348

  11. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    EPA Science Inventory

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
    Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

  12. Neonatal Fc Receptor Promotes Immune Complex–Mediated Glomerular Disease

    PubMed Central

    Olaru, Florina; Luo, Wentian; Suleiman, Hani; St. John, Patricia L.; Ge, Linna; Mezo, Adam R.; Shaw, Andrey S.; Abrahamson, Dale R.; Miner, Jeffrey H.

    2014-01-01

    The neonatal Fc receptor (FcRn) is a major regulator of IgG and albumin homeostasis systemically and in the kidneys. We investigated the role of FcRn in the development of immune complex–mediated glomerular disease in mice. C57Bl/6 mice immunized with the noncollagenous domain of the α3 chain of type IV collagen (α3NC1) developed albuminuria associated with granular capillary loop deposition of exogenous antigen, mouse IgG, C3 and C5b-9, and podocyte injury. High-resolution imaging showed abundant IgG deposition in the expanded glomerular basement membrane, especially in regions corresponding to subepithelial electron dense deposits. FcRn-null and -humanized mice immunized with α3NC1 developed no albuminuria and had lower levels of serum IgG anti-α3NC1 antibodies and reduced glomerular deposition of IgG, antigen, and complement. Our results show that FcRn promotes the formation of subepithelial immune complexes and subsequent glomerular pathology leading to proteinuria, potentially by maintaining higher serum levels of pathogenic IgG antibodies. Therefore, reducing pathogenic IgG levels by pharmacologic inhibition of FcRn may provide a novel approach for the treatment of immune complex–mediated glomerular diseases. As proof of concept, we showed that a peptide inhibiting the interaction between human FcRn and human IgG accelerated the degradation of human IgG anti-α3NC1 autoantibodies injected into FCRN-humanized mice as effectively as genetic ablation of FcRn, thus preventing the glomerular deposition of immune complexes containing human IgG. PMID:24357670

  13. Thyrotropin modulates receptor-mediated processing of the atrial natriuretic peptide receptor in cultured thyroid cells

    SciTech Connect

    Tseng, Y.L.; Burman, K.D.; Lahiri, S.; Abdelrahim, M.M.; D'Avis, J.C.; Wartofsky, L. )

    1991-03-01

    In a prior study of atrial natriuretic peptide (ANP) binding to cultured thyroid cells, we reported that at 4 C, more than 95% of bound ANP is recovered on cell membranes, with negligible ANP internalization observed. Since ANP binding was inhibited by TSH, we have further studied TSH effects on postbinding ANP processing to determine whether this phenomenon reflects enhanced endocytosis of the ANP-receptor complex. An ANP chase study was initiated by binding (125I) ANP to thyroid cells at 4 C for 2 h, followed by incubation at 37 C. ANP processing was then traced by following 125I activity at various time intervals in three fractions: cell surface membranes, incubation medium, and inside the cells. Radioactivity released into medium represented processed ANP rather than ANP dissociated from surface membranes, since prebound (125I)ANP could not be competitively dissociated by a high concentration of ANP (1 mumol/L) at 37 C. Chase study results showed that prebound ANP quickly disappeared from cell membranes down to 34% by 30 min. Internalized ANP peaked at 10 min, with 21% of initial prebound ANP found inside the cells. At the same time, radioactivity recovered in incubation medium sharply increased between 10-30 min from 8% to 52%. Preincubation of cells with chloroquine (which blocks degradation of the ANP-receptor complex by inhibiting lysosomal hydrolase) caused a 146% increase in internalized (125I)ANP by 30 min (39% compared to 15% control), while medium radioactivity decreased from 52% to 16%, suggesting that processing of the receptor complex is mediated via lysosomal enzymes. In chase studies employing cells pretreated with chloroquine, TSH stimulated the internalization rate of ANP-receptor complex. By 30 min, TSH significantly reduced the membrane-bound ANP, and the decrease was inversely correlated to the increase in internalized radioactivity.

  14. Biologically bounded risk assessment for receptor-mediated nongenotoxic carcinogens.

    PubMed

    Gastel, J A; Sutter, T R

    1995-12-01

    We have developed a biologically bounded marginal effect model for use in risk assessment of human exposure to receptor-mediated nongenotoxic carcinogens. Schematically this model can be reduced to four components: CI, the absence of an observable biological response; CII, observable biochemical responses but no observable pathology; CIII, observable pathology; and CIV, both observable pathology and lethality. The inflection point in the marginal response curve between CI and CII is defined as the biologically evaluated scientifically tested no observable effect level (BESTNOEL). We demonstrate the utility of this approach by applying it to the well-studied nongenotoxic carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using a well-developed mechanistic understanding of the initial interactions of TCDD with the cell, we justify the selection of the minimal effective dose for CYP1A1 mRNA induction as the BESTNOEL. With allowance for variation in human sensitivity to TCDD, the BESTNOEL is assigned a human liver tissue burden of approximately 0.25-25 ppt and an allowable daily intake level in the range of 15-1500 fg/kg/day. In the future, the BESTNOEL can help establish a lower boundary for acceptable extrapolation when using either statistical or biologically based attributable risk models.

  15. Ryanodine receptor-2 upregulation and nicotine-mediated plasticity.

    PubMed

    Ziviani, Elena; Lippi, Giordano; Bano, Daniele; Munarriz, Eliana; Guiducci, Stefania; Zoli, Michele; Young, Kenneth W; Nicotera, Pierluigi

    2011-01-05

    Nicotine, the major psychoactive component of cigarette smoke, modulates neuronal activity to produce Ca2+-dependent changes in gene transcription. However, the downstream targets that underlie the long-term effects of nicotine on neuronal function, and hence behaviour, remain to be elucidated. Here, we demonstrate that nicotine administration to mice upregulates levels of the type 2 ryanodine receptor (RyR2), a Ca2+-release channel present on the endoplasmic reticulum, in a number of brain areas associated with cognition and addiction, notably the cortex and ventral midbrain. Nicotine-mediated RyR2 upregulation was driven by CREB, and caused a long-lasting reinforcement of Ca2+ signalling via the process of Ca2+-induced Ca2+ release. RyR2 upregulation was itself required for long-term phosphorylation of CREB in a positive-feedback signalling loop. We further demonstrate that inhibition of RyR-activation in vivo abolishes sensitization to nicotine-induced habituated locomotion, a well-characterised model for onset of drug dependence. Our findings, therefore, indicate that gene-dependent reprogramming of Ca2+ signalling is involved in nicotine-induced behavioural changes.

  16. Secreted PCSK9 downregulates low density lipoprotein receptor through receptor-mediated endocytosis.

    PubMed

    Qian, Yue-Wei; Schmidt, Robert J; Zhang, Youyan; Chu, Shaoyou; Lin, Aimin; Wang, He; Wang, Xiliang; Beyer, Thomas P; Bensch, William R; Li, Weiming; Ehsani, Mariam E; Lu, Deshun; Konrad, Robert J; Eacho, Patrick I; Moller, David E; Karathanasis, Sotirios K; Cao, Guoqing

    2007-07-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a protease that regulates low density lipoprotein receptor (LDLR) protein levels. The mechanisms of this action, however, remain to be defined. We show here that recombinant human PCSK9 expressed in HEK293 cells was readily secreted into the medium, with the prosegment associated with the C-terminal domain. Secreted PCSK9 mediated cell surface LDLR degradation in a concentration- and time-dependent manner when added to HEK293 cells. Accordingly, cellular LDL uptake was significantly reduced as well. When infused directly into C57B6 mice, purified human PCSK9 substantially reduced hepatic LDLR protein levels and resulted in increased plasma LDL cholesterol. When added to culture medium, fluorescently labeled PCSK9 was endocytosed and displayed endosomal-lysosomal intracellular localization in HepG2 cells, as was demonstrated by colocalization with DiI-LDL. PCSK9 endocytosis was mediated by LDLR as LDLR deficiency (hepatocytes from LDLR null mice), or RNA interference-mediated knockdown of LDLR markedly reduced PCSK9 endocytosis. In addition, RNA interference knockdown of the autosomal recessive hypercholesterolemia (ARH) gene product also significantly reduced PCSK9 endocytosis. Biochemical analysis revealed that the LDLR extracellular domain interacted directly with secreted PCSK9; thus, overexpression of the LDLR extracellular domain was able to attenuate the reduction of cell surface LDLR levels by secreted PCSK9. Together, these results reveal that secreted PCSK9 retains biological activity, is able to bind directly to the LDLR extracellular domain, and undergoes LDLR-ARH-mediated endocytosis, leading to accelerated intracellular degradation of the LDLR.

  17. Anti-nociception mediated by a κ opioid receptor agonist is blocked by a δ receptor agonist

    PubMed Central

    Taylor, A M W; Roberts, K W; Pradhan, A A; Akbari, H A; Walwyn, W; Lutfy, K; Carroll, F I; Cahill, C M; Evans, C J

    2015-01-01

    BACKGROUND AND PURPOSE The opioid receptor family comprises four structurally homologous but functionally distinct sub-groups, the μ (MOP), δ (DOP), κ (KOP) and nociceptin (NOP) receptors. As most opioid agonists are selective but not specific, a broad spectrum of behaviours due to activation of different opioid receptors is expected. In this study, we examine whether other opioid receptor systems influenced KOP-mediated antinociception. EXPERIMENTAL APPROACH We used a tail withdrawal assay in C57Bl/6 mice to assay the antinociceptive effect of systemically administered opioid agonists with varying selectivity at KOP receptors. Pharmacological and genetic approaches were used to analyse the interactions of the other opioid receptors in modulating KOP-mediated antinociception. KEY RESULTS Etorphine, a potent agonist at all four opioid receptors, was not anti-nociceptive in MOP knockout (KO) mice, although etorphine is an efficacious KOP receptor agonist and specific KOP receptor agonists remain analgesic in MOP KO mice. As KOP receptor agonists are aversive, we considered KOP-mediated antinociception might be a form of stress-induced analgesia that is blocked by the anxiolytic effects of DOP receptor agonists. In support of this hypothesis, pretreatment with the DOP antagonist, naltrindole (10 mg·kg−1), unmasked etorphine (3 mg·kg−1) antinociception in MOP KO mice. Further, in wild-type mice, KOP-mediated antinociception by systemic U50,488H (10 mg·kg−1) was blocked by pretreatment with the DOP agonist SNC80 (5 mg·kg−1) and diazepam (1 mg·kg−1). CONCLUSIONS AND IMPLICATIONS Systemic DOP receptor agonists blocked systemic KOP antinociception, and these results identify DOP receptor agonists as potential agents for reversing stress-driven addictive and depressive behaviours mediated through KOP receptor activation. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles

  18. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  19. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis

    PubMed Central

    Gonçalves-Carneiro, Daniel; McKeating, Jane A.

    2017-01-01

    ABSTRACT The signaling lymphocyte activation molecule F1 (SLAMF1) is both a microbial sensor and entry receptor for measles virus (MeV). Herein, we describe a new role for SLAMF1 to mediate MeV endocytosis that is in contrast with the alternative, and generally accepted, model that MeV genome enters cells only after fusion at the cell surface. We demonstrated that MeV engagement of SLAMF1 induces dramatic but transient morphological changes, most prominently in the formation of membrane blebs, which were shown to colocalize with incoming viral particles, and rearrangement of the actin cytoskeleton in infected cells. MeV infection was dependent on these dynamic cytoskeletal changes as well as fluid uptake through a macropinocytosis-like pathway as chemical inhibition of these processes inhibited entry. Moreover, we identified a role for the RhoA-ROCK-myosin II signaling axis in this MeV internalization process, highlighting a novel role for this recently characterized pathway in virus entry. Our study shows that MeV can hijack a microbial sensor normally involved in bacterial phagocytosis to drive endocytosis using a complex pathway that shares features with canonical viral macropinocytosis, phagocytosis, and mechanotransduction. This uptake pathway is specific to SLAMF1-positive cells and occurs within 60 min of viral attachment. Measles virus remains a significant cause of mortality in human populations, and this research sheds new light on the very first steps of infection of this important pathogen. IMPORTANCE Measles is a significant disease in humans and is estimated to have killed over 200 million people since records began. According to current World Health Organization statistics, it still kills over 100,000 people a year, mostly children in the developing world. The causative agent, measles virus, is a small enveloped RNA virus that infects a broad range of cells during infection. In particular, immune cells are infected via interactions between

  20. Histamine H3 receptor-mediated inhibition of noradrenaline release in the human brain.

    PubMed

    Schlicker, E; Werthwein, S; Zentner, J

    1999-01-01

    Stimulation-evoked 3H-noradrenaline release in human cerebrocortical slices was inhibited by histamine (in a manner sensitive to clobenpropit) and by imetit, suggesting H3 receptor-mediated inhibition of noradrenaline release in human brain.

  1. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy

    PubMed Central

    Pedrozo, Zully; Torrealba, Natalia; Fernández, Carolina; Gatica, Damian; Toro, Barbra; Quiroga, Clara; Rodriguez, Andrea E.; Sanchez, Gina; Gillette, Thomas G.; Hill, Joseph A.; Donoso, Paulina; Lavandero, Sergio

    2013-01-01

    Time for primary review: 15 days Aims Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of soluble cytosolic proteins bearing the sequence KFERQ. These proteins are targeted by chaperones and delivered to lysosomes where they are translocated into the lysosomal lumen and degraded via the lysosome-associated membrane protein type 2A (LAMP-2A). Mutations in LAMP2 that inhibit autophagy result in Danon disease characterized by hypertrophic cardiomyopathy. The ryanodine receptor type 2 (RyR2) plays a key role in cardiomyocyte excitation–contraction and its dysfunction can lead to cardiac failure. Whether RyR2 is degraded by CMA is unknown. Methods and results To induce CMA, cultured neonatal rat cardiomyocytes were treated with geldanamycin (GA) to promote protein degradation through this pathway. GA increased LAMP-2A levels together with its redistribution and colocalization with Hsc70 in the perinuclear region, changes indicative of CMA activation. The inhibition of lysosomes but not proteasomes prevented the loss of RyR2. The recovery of RyR2 content after incubation with GA by siRNA targeting LAMP-2A suggests that RyR2 is degraded via CMA. In silico analysis also revealed that the RyR2 sequence harbours six KFERQ motifs which are required for the recognition Hsc70 and its degradation via CMA. Our data suggest that presenilins are involved in RyR2 degradation by CMA. Conclusion These findings are consistent with a model in which oxidative damage of the RyR2 targets it for turnover by presenilins and CMA, which could lead to removal of damaged or leaky RyR2 channels. PMID:23404999

  2. NMDA receptor-mediated excitotoxicity depends on the coactivation of synaptic and extrasynaptic receptors.

    PubMed

    Zhou, X; Hollern, D; Liao, J; Andrechek, E; Wang, H

    2013-03-28

    N-methyl-D-aspartate receptors (NMDAR) overactivation is linked to neurodegeneration. The current prevailing theory suggests that synaptic and extrasynaptic NMDAR (syn- and ex-NMDAR) impose counteracting effects on cell fate, and neuronal cell death is mainly mediated by the activation of ex-NMDAR. However, several lines of evidence implicate the limitation of this theory. Here, we demonstrate that activation of NMDAR bi-directionally regulated cell fate through stimulating pro-survival or pro-death signaling. While low-dose NMDA preferentially activated syn-NMDAR and stimulated the extracellular signal-regulated kinase ½-cAMP responsive element-binding protein-brain-derived neurotrophic factor pro-survival signaling, higher doses progressively activated increasing amount of ex-NMDAR along with syn-NMDAR and triggered cell death program. Interestingly, the activation of syn- or ex-NMDAR alone did not cause measurable cell death. Consistently, activation of syn- or ex-NMDAR alone stimulated pro-survival but not pro-death signaling. Next, we found that memantine, which was previously identified as an ex-NMDAR blocker, inhibited intracellular signaling mediated by syn- or ex-NMDAR. Simultaneous blockade of syn- and ex-NMDAR by memantine dose-dependently attenuated NMDAR-mediated death. Moreover, long- but not short-term treatment with high-dose NMDA or oxygen-glucose deprivation triggered cell death and suppressed pro-survival signaling. These data implicate that activation of syn- or ex-NMDAR alone is not neurotoxic. The degree of excitotoxicity depends on the magnitude and duration of syn- and ex-NMDAR coactivation. Finally, genome-wide examination demonstrated that the activation of syn- and ex-NMDAR lead to significant overlapping rather than counteracting transcriptional responses.

  3. Slowly developing depression of N-methyl-D-aspartate receptor mediated responses in young rat hippocampi

    PubMed Central

    Dozmorov, Mikhail; Li, Rui; Xu, Hui-Ping; Jilderos, Barbro; Wigström, Holger

    2004-01-01

    Background Activation of N-methyl-D-aspartate (NMDA) type glutamate receptors is essential in triggering various forms of synaptic plasticity. A critical issue is to what extent such plasticity involves persistent changes of glutamate receptor subtypes and many prior studies have suggested a main role for alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in mediating the effect. Our previous work in hippocampal slices revealed that, under pharmacological unblocking of NMDA receptors, both AMPA and NMDA receptor mediated responses undergo a slowly developing depression. In the present study we have further adressed this phenomenon, focusing on the contribution via NMDA receptors. Pharmacologically isolated NMDA receptor mediated excitatory postsynaptic potentials (EPSPs) were recorded for two independent synaptic pathways in CA1 area using perfusion with low Mg2+ (0.1 mM) to unblock NMDA receptors. Results Following unblocking of NMDA receptors, there was a gradual decline of NMDA receptor mediated EPSPs for 2–3 hours towards a stable level of ca. 60–70 % of the maximal size. If such an experimental session was repeated twice in the same pathway with a period of NMDA receptor blockade in between, the depression attained in the first session was still evident in the second one and no further decay occurred. The persistency of the depression was also validated by comparison between pathways. It was found that the responses of a control pathway, unstimulated in the first session of receptor unblocking, behaved as novel responses when tested in association with the depressed pathway under the second session. In similar experiments, but with AP5 present during the first session, there was no subsequent difference between NMDA EPSPs. Conclusions Our findings show that merely evoking NMDA receptor mediated responses results in a depression which is input specific, induced via NMDA receptor activation, and is maintained for several hours through

  4. GABAA Receptor-Mediated Activity in a Model of Cortical Dysplasia

    DTIC Science & Technology

    2012-06-29

    a model of cortical dysplasia" Name of Candidate: Joseph Abbah Doctor of Philosophy Degree June 29, 2012 DISSERTATION AND ABSTRACT APPROVED...receptor-mediated activity in a model of cortical dysplasia’ is appropriately acknowledged and, beyond brief excerpts, is with the permission of the...certifies that the use of any copyrighted material in this dissertation manuscript entitled: “GABAA receptor-mediated activity in a model of

  5. SMRT isoforms mediate repression and anti-repression of nuclear receptor heterodimers.

    PubMed Central

    Chen, J D; Umesono, K; Evans, R M

    1996-01-01

    Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. We demonstrate that SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 PMID:8755515

  6. PKCɛ mediates substance P inhibition of GABAA receptors-mediated current in rat dorsal root ganglion.

    PubMed

    Li, Li; Zhao, Lei; Wang, Yang; Ma, Ke-tao; Shi, Wen-yan; Wang, Ying-zi; Si, Jun-qiang

    2015-02-01

    The mechanism underlying the modulatory effect of substance P (SP) on GABA-activated response in rat dorsal root ganglion (DRG) neurons was investigated. In freshly dissociated rat DRG neurons, whole-cell patch-clamp technique was used to record GABA-activated current and sharp electrode intracellular recording technique was used to record GABA-induced membrane depolarization. Application of GABA (1-1000 μmol/L) induced an inward current in a concentration-dependent manner in 114 out of 127 DRG neurons (89.8 %) examined with whole-cell patch-clamp recordings. Bath application of GABA (1-1000 μmol/L) evoked a depolarizing response in 236 out of 257 (91.8%) DRG neurons examined with intracellular recordings. Application of SP (0.001-1 μmol/L) suppressed the GABA-activated inward current and membrane depolarization. The inhibitory effects were concentration-dependent and could be blocked by the selective neurokinin 1 (NK1) receptors antagonist spantide but not by L659187 and SR142801 (1 μmol/L, n=7), selective antagonists of NK2 and NK3. The inhibitory effect of SP was significantly reduced by the calcium chelator BAPTA-AM, phospholipase C (PLC) inhibitor U73122, and PKC inhibitor chelerythrine, respectively. The PKA inhibitor H-89 did not affect the SP effect. Remarkably, the inhibitory effect of SP on GABA-activated current was nearly completely removed by a selective PKCε inhibitor epilon-V1-2 but not by safingol and LY333531, selective inhibitors of PKCα and PKCβ. Our results suggest that NK1 receptor mediates SP-induced inhibition of GABA-activated current and membrane depolarization by activating intracellular PLC-Ca²⁺-PKCε cascade. SP might regulate the excitability of peripheral nociceptors through inhibition of the "pre-synaptic inhibition" evoked by GABA, which may explain its role in pain and neurogenic inflammation.

  7. Opioid Receptors Mediate Direct Predictive Fear Learning: Evidence from One-Trial Blocking

    ERIC Educational Resources Information Center

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including [mu]-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear…

  8. Inhibition of pattern recognition receptor-mediated inflammation by bioactive phytochemicals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Emerging evidence reveals that pattern-recognition receptors (PRRs), Toll-like receptors (TLRs) and Nucleotide-binding oligomerization domain proteins (NODs) mediate both infection-induced and sterile inflammation by recognizing pathogen-associated molecular patterns (PAMPs) and endogenous molecules...

  9. Ionotropic glutamate receptors mediate OFF responses in light-adapted ON bipolar cells

    PubMed Central

    Pang, Ji-Jie; Gao, Fan; Wu, Samuel M.

    2013-01-01

    Previous studies have suggested that photoreceptor synaptic inputs to depolarizing bipolar cells (DBCs or ON bipolar cells) are mediated by mGluR6 receptors and those to hyperpolarizing bipolar cells (HBCs or OFF bipolar cells) are mediated by AMPA/kainate receptors. Here we show that in addition to mGluR6 receptors which mediate the sign-inverting, depolarizing light responses, subpopulations of cone-dominated and rod/cone mixed DBCs use GluR4 AMPA receptors to generate a transient sign-preserving OFF response under light adapted conditions. These AMPA receptors are located at the basal junctions postsynaptic to rods and they are silent under dark-adapted conditions, as tonic glutamate release in darkness desensitizes these receptors. Light adaptation enhances rod-cone coupling and thus allows cone photocurrents with an abrupt OFF depolarization to enter the rods. The abrupt rod depolarization triggers glutamate activation of unoccupied AMPA receptors, resulting in a transient OFF response in DBCs. It has been widely accepted that the DNQX-sensitive, OFF transient responses in retinal amacrine cells and ganglion cells are mediated exclusively by HBCs. Our results suggests that this view needs revision as AMPA receptors in subpopulations of DBCs are likely to significantly contribute to the DNQX-sensitive OFF transient responses in light-adapted third- and higher-order visual neurons. PMID:22842089

  10. Crosstalk Between Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2011-04-01

    the connection between obesity and breast cancer (5). Leptin , a product of the obese (ob) gene, is an adipocytokine that regulates appetite , bone... Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance Dr. Rita Nahta Emory University Atlanta, GA 30322 Obesity...hormones IGF-I and leptin and their receptors, IGF-IR and leptin receptor (Ob-R), are elevated in breast cancer. Co-immunoprecipitation and

  11. Glutamate mediates the function of melanocortin receptor 4 on sim1 neurons in body weight regulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing ...

  12. Steroid receptor coactivator-1 mediates estrogenic actions to prevent body weight gain in female mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Estrogen receptor-alpha (ERalpha) expressed by hypothalamic proopiomelanocortin and steroidogenic factor-1 neurons largely mediates the antiobesity effects of estrogens in females. However, the critical molecular events that are coupled to ERalpha and mediate estrogenic effects on energy balance rem...

  13. DEPENDENCE OF PPAR LIGAND-INDUCED MAPK SIGNALING ON EPIDERMAL GROWTH FACTOR RECEPTOR TRANSACTIVATION HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

    EPA Science Inventory

    Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that function as ligand-activated transcription factors regulating lipid metabolism and homeostasis. In addition to their ability to regulate PPAR-mediated gene transcription, PPARalpha and gamma li...

  14. Mannose receptor-mediated gene delivery into antigen presenting dendritic cells.

    PubMed

    Diebold, Sandra S; Plank, Christian; Cotten, Matt; Wagner, Ernst; Zenke, Martin

    2002-11-01

    Dendritic cells are professional antigen presenting cells and are unique in their ability to prime naïve T cells. Gene modification of dendritic cells is of particular interest for immunotherapy of diseases where the immune system has failed or is aberrantly regulated, such as in cancer or autoimmune disease, respectively. Dendritic cells abundantly express mannose receptor and mannose receptor-related receptors, and receptor-mediated gene transfer via mannose receptor offers a versatile tool for targeted gene delivery into these cells. Accordingly, mannose polyethylenimine DNA transfer complexes were generated and used for gene delivery into dendritic cells. Mannose receptor belongs to the group of scavenger receptors that allow dendritic cells to take up pathogenic material, which is directed for degradation and MHC class II presentation. Therefore, a limiting step of transgene expression by mannose receptor-mediated gene delivery is endosomal degradation of DNA. Several strategies have been explored to overcome this limitation including the addition of endosomolytic components to DNA transfer complexes like adenovirus particles and influenza peptides. Here, we review the current understanding of mannose receptor-mediated gene delivery into dendritic cells and discuss strategies to identify appropriate endosomolytic agents to improve DNA transfer efficacy.

  15. Different receptors mediate the electrophysiological and growth cone responses of an identified neuron to applied dopamine.

    PubMed

    Dobson, K S; Dmetrichuk, J M; Spencer, G E

    2006-09-15

    Neurotransmitters are among the many cues that may guide developing axons toward appropriate targets in the developing nervous system. We have previously shown in the mollusk Lymnaea stagnalis that dopamine, released from an identified pre-synaptic cell, differentially affects growth cone behavior of its target and non-target cells in vitro. Here, we describe a group of non-target cells that also produce an inhibitory electrophysiological response to applied dopamine. We first determined, using pharmacological blockers, which receptors mediate this physiological response. We demonstrated that the dopaminergic electrophysiological responses of non-target cells were sensitive to a D2 receptor antagonist, as are known target cell responses. However, the non-target cell receptors were linked to different G-proteins and intracellular signaling pathways than the target cell receptors. Despite the presence of a D2-like receptor at the soma, the growth cone collapse of these non-target cells was mediated by D1-like receptors. This study shows that different dopamine receptor sub-types mediated the inhibitory physiological and growth cone responses of an identified cell type. We therefore not only provide further evidence that D2- and D1-like receptors can be present on the same neuron in invertebrates, but also show that these receptors are likely involved in very different cellular functions.

  16. IgM-Dependent Phagocytosis in Microglia Is Mediated by Complement Receptor 3, Not Fcα/μ Receptor.

    PubMed

    Weinstein, Jonathan R; Quan, Yi; Hanson, Josiah F; Colonna, Lucrezia; Iorga, Michael; Honda, Shin-ichiro; Shibuya, Kazuko; Shibuya, Akira; Elkon, Keith B; Möller, Thomas

    2015-12-01

    Microglia play an important role in receptor-mediated phagocytosis in the CNS. In brain abscess and other CNS infections, invading bacteria undergo opsonization with Igs or complement. Microglia recognize these opsonized pathogens by Fc or complement receptors triggering phagocytosis. In this study, we investigated the role of Fcα/μR, the less-studied receptor for IgM and IgA, in microglial phagocytosis. We showed that primary microglia, as well as N9 microglial cells, express Fcα/μR. We also showed that anti-Staphylococcus aureus IgM markedly increased the rate of microglial S. aureus phagocytosis. To unequivocally test the role of Fcα/μR in IgM-mediated phagocytosis, we performed experiments in microglia from Fcα/μR(-/-) mice. Surprisingly, we found that IgM-dependent phagocytosis of S. aureus was similar in microglia derived from wild-type or Fcα/μR(-/-) mice. We hypothesized that IgM-dependent activation of complement receptors might contribute to the IgM-mediated increase in phagocytosis. To test this, we used immunologic and genetic inactivation of complement receptor 3 components (CD11b and CD18) as well as C3. IgM-, but not IgG-mediated phagocytosis of S. aureus was reduced in wild-type microglia and macrophages following preincubation with an anti-CD11b blocking Ab. IgM-dependent phagocytosis of S. aureus was also reduced in microglia derived from CD18(-/-) and C3(-/-) mice. Taken together, our findings implicate complement receptor 3 and C3, but not Fcα/μR, in IgM-mediated phagocytosis of S. aureus by microglia.

  17. Menthol enhances phasic and tonic GABAA receptor-mediated currents in midbrain periaqueductal grey neurons

    PubMed Central

    Lau, Benjamin K; Karim, Shafinaz; Goodchild, Ann K; Vaughan, Christopher W; Drew, Geoffrey M

    2014-01-01

    Background and Purpose Menthol, a naturally occurring compound in the essential oil of mint leaves, is used for its medicinal, sensory and fragrant properties. Menthol acts via transient receptor potential (TRPM8 and TRPA1) channels and as a positive allosteric modulator of recombinant GABAA receptors. Here, we examined the actions of menthol on GABAA receptor-mediated currents in intact midbrain slices. Experimental Approach Whole-cell voltage-clamp recordings were made from periaqueductal grey (PAG) neurons in midbrain slices from rats to determine the effects of menthol on GABAA receptor-mediated phasic IPSCs and tonic currents. Key Results Menthol (150–750 μM) produced a concentration-dependent prolongation of spontaneous GABAA receptor-mediated IPSCs, but not non-NMDA receptor-mediated EPSCs throughout the PAG. Menthol actions were unaffected by TRPM8 and TRPA1 antagonists, tetrodotoxin and the benzodiazepine antagonist, flumazenil. Menthol also enhanced a tonic current, which was sensitive to the GABAA receptor antagonists, picrotoxin (100 μM), bicuculline (30 μM) and Zn2+ (100 μM), but unaffected by gabazine (10 μM) and a GABAC receptor antagonist, 1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid hydrate (TPMPA; 50 μM). In addition, menthol potentiated currents induced by the extrasynaptic GABAA receptor agonist THIP/gaboxadol (10 μM). Conclusions and Implications These results suggest that menthol positively modulates both synaptic and extrasynaptic populations of GABAA receptors in native PAG neurons. The development of agents that potentiate GABAA-mediated tonic currents and phasic IPSCs in a manner similar to menthol could provide a basis for novel GABAA-related pharmacotherapies. PMID:24460753

  18. Receptor-mediated endocytosis for drug delivery in African trypanosomes: fulfilling Paul Ehrlich's vision of chemotherapy.

    PubMed

    Alsford, Sam; Field, Mark C; Horn, David

    2013-05-01

    Bloodstream-form cells of Trypanosoma brucei exhibit massively increased endocytic activity relative to the insect midgut stage, enabling rapid recycling of variant surface glycoprotein and antibody clearance from the surface. In addition, recent advances have identified a role for receptor-mediated endocytosis in the uptake of the antitrypanosomal drug, suramin, via invariant surface glycoprotein 75, and in the uptake of trypanosome lytic factor 1 via haptoglobin-haemoglobin receptor. Here, we argue that receptor-mediated endocytosis represents both a validated drug target and a promising route for the delivery of novel therapeutics into trypanosomes.

  19. Cinnamaldehyde suppresses toll-like receptor 4 activation mediated through the inhibition of receptor oligomerization.

    PubMed

    Youn, Hyung S; Lee, Jun K; Choi, Yong J; Saitoh, Shin I; Miyake, Kensuke; Hwang, Daniel H; Lee, Joo Y

    2008-01-15

    Toll-like receptors (TLRs) play a critical role in induction of innate immune and inflammatory responses by recognizing invading pathogens or non-microbial endogenous molecules. TLRs have two major downstream signaling pathways, MyD88- and TRIF-dependent pathways leading to the activation of NFkappaB and IRF3 and the expression of inflammatory mediators. Deregulation of TLR activation is known to be closely linked to the increased risk of many chronic diseases. Cinnamaldehyde (3-phenyl-2-propenal) has been reported to inhibit NFkappaB activation induced by pro-inflammatory stimuli and to exert anti-inflammatory and anti-bacterial effects. However, the underlying mechanism has not been clearly identified. Our results showed that cinnamaldehyde suppressed the activation of NFkappaB and IRF3 induced by LPS, a TLR4 agonist, leading to the decreased expression of target genes such as COX-2 and IFNbeta in macrophages (RAW264.7). Cinnamaldehyde did not inhibit the activation of NFkappaB or IRF3 induced by MyD88-dependent (MyD88, IKKbeta) or TRIF-dependent (TRIF, TBK1) downstream signaling components. However, oligomerization of TLR4 induced by LPS was suppressed by cinnamaldehyde resulting in the downregulation of NFkappaB activation. Further, cinnamaldehyde inhibited ligand-independent NFkappaB activation induced by constitutively active TLR4 or wild-type TLR4. Our results demonstrated that the molecular target of cinnamaldehyde in TLR4 signaling is oligomerization process of receptor, but not downstream signaling molecules suggesting a novel mechanism for anti-inflammatory activity of cinnamaldehyde.

  20. Cannabinoid Receptor Activation Modifies NMDA Receptor Mediated Release of Intracellular Calcium: Implications for Endocannabinoid Control of Hippocampal Neural Plasticity

    PubMed Central

    Hampson, Robert E.; Miller, Frances; Palchik, Guillermo; Deadwyler, Sam A.

    2011-01-01

    Chronic activation or inhibition of cannabinoid receptors (CB1) leads to continuous suppression of neuronal plasticity in hippocampus and other brain regions, suggesting that endocannabinoids may have a functional role in synaptic processes that produce state-dependent transient modulation of hippocampal cell activity. In support of this, it has previously been shown in vitro that cannabinoid CB1 receptors modulate second messenger systems in hippocampal neurons that can modulate intracellular ion channels, including channels which release calcium from intracellular stores. Here we demonstrate in hippocampal slices a similar endocannabinoid action on excitatory glutamatergic synapses via modulation of NMDA-receptor mediated intracellular calcium levels in confocal imaged neurons. Calcium entry through glutamatergic NMDA-mediated ion channels increases intracellular calcium concentrations via modulation of release from ryanodine-sensitive channels in endoplasmic reticulum. The studies reported here show that NMDA-elicited increases in Calcium Green fluorescence are enhanced by CB1 receptor antagonists (i.e. rimonabant), and inhibited by CB1 agonists (i.e. WIN 55,212-2). Suppression of endocannabinoid breakdown by either reuptake inhibition (AM404) or fatty-acid amide hydrolase inhibition (URB597) produced suppression of NMDA elicited calcium increases comparable to WIN 55,212-2, while enhancement of calcium release provoked by endocannabinoid receptor antagonists (Rimonabant) was shown to depend on the blockade of CB1 receptor mediated de-phosphorylation of Ryanodine receptors. Such CB1 receptor modulation of NMDA elicited increases in intracellular calcium may account for the respective disruption and enhancement by CB1 agents of trial-specific hippocampal neuron ensemble firing patterns during performance of a short-term memory task, reported previously from this laboratory. PMID:21288475

  1. Mechanisms of Biased β-Arrestin-Mediated Signaling Downstream from the Cannabinoid 1 Receptor

    PubMed Central

    Delgado-Peraza, Francheska; Ahn, Kwang H.; Nogueras-Ortiz, Carlos; Mungrue, Imran N.; Mackie, Ken; Kendall, Debra A.

    2016-01-01

    Activation of G protein-coupled receptors results in multiple waves of signaling that are mediated by heterotrimeric G proteins and the scaffolding proteins β-arrestin 1/2. Ligands can elicit full or subsets of cellular responses, a concept defined as ligand bias or functional selectivity. However, our current understanding of β-arrestin-mediated signaling is still very limited. Here we provide a comprehensive view of β-arrestin-mediated signaling from the cannabinoid 1 receptor (CB1R). By using a signaling biased receptor, we define the cascades, specific receptor kinases, and molecular mechanism underlying β-arrestin-mediated signaling: We identify the interaction kinetics of CB1R and β-arrestin 1 during their endocytic trafficking as directly proportional to its efficacy. Finally, we demonstrate that signaling results in the control of genes clustered around prosurvival and proapoptotic functions among others. Together, these studies constitute a comprehensive description of β-arrestin-mediated signaling from CB1Rs and suggest modulation of receptor endocytic trafficking as a therapeutic approach to control β-arrestin-mediated signaling. PMID:27009233

  2. Ethanol inhibits epileptiform activity and NMDA receptor-mediated synaptic transmission in rat amygdaloid slices

    SciTech Connect

    Gean, P.W. )

    1992-02-26

    The effect of ethanol on the epileptiform activity induced by Mg{sup ++}-free solution was studied in rat amygdalar slices using intracellular recording techniques. The spontaneous and evoked epileptiform discharges consisting of an initial burst followed by afterdischarges were observed 20-30 min after switching to Mg{sup ++}-free medium. Superfusion with ethanol reversibly reduced the duration of spontaneous and evoked bursting discharges in a concentration-dependent manner. Synaptic response mediated by N-methyl-D-aspartate (NMDA) receptor activation was isolated by application of a solution containing the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and either in Mg{sup ++}-free solution or in the presence of 50 {mu}M bicuculline. Application of ethanol reversibly suppressed the duration of NMDA receptor-mediated synaptic response. These results suggest that intoxicating concentrations of ethanol possess anticonvulsant activity through blocking the NMDA receptor-mediated synaptic excitation.

  3. The Role of Estrogen Related Receptor in Modulating Estrogen Receptor Mediated Transcription in Breast Cancer Cells

    DTIC Science & Technology

    2005-04-01

    receptors ) by demonstrating that mitochondrial biogenesis and fatty acid P- oxidation , processes ERRa is known to regulate , are robustly...gluconeogenesis, and fatty acid oxidation (Lin 2003; Puigserver 1998; Wu 1999; Yoon 2001). In addition to its activity on a number of nuclear receptors , this...in target cells. They were generated by replacing the receptor interaction domains in peroxisome proliferator activated receptor

  4. USP17 is required for clathrin mediated endocytosis of epidermal growth factor receptor

    PubMed Central

    Jaworski, Jakub; de la Vega, Michelle; Fletcher, Sarah J.; McFarlane, Cheryl; Greene, Michelle K.; Smyth, Andrew W.; Van Schaeybroeck, Sandra; Johnston, James A.; Scott, Christopher J.; Rappoport, Joshua Z.; Burrows, James F.

    2014-01-01

    Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of ‘CaaX’ motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis. PMID:25026282

  5. Site of action of the general anesthetic propofol in muscarinic M1 receptor-mediated signal transduction.

    PubMed

    Murasaki, Osamu; Kaibara, Muneshige; Nagase, Yoshihisa; Mitarai, Sayaka; Doi, Yoshiyuki; Sumikawa, Koji; Taniyama, Kohtaro

    2003-12-01

    Although a potential target site of general anesthetics is primarily the GABA A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein alpha subunits (Gqalpha). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC50 = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) into oocytes overexpressing Gqalpha was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gqalpha-mediated signal transduction with the intracellular injection of GTPgammaS. We also studied effects of propofol on l-[N-methyl-3H]scopolamine methyl chloride ([3H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [3H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the beta2 adrenoceptor (beta2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K(+) channels). Neither beta2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 microM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

  6. IL-10 mediates sigma 1 receptor-dependent suppression of antitumor immunity.

    PubMed

    Zhu, Li X; Sharma, Sherven; Gardner, Brian; Escuadro, Brian; Atianzar, Kimberly; Tashkin, Donald P; Dubinett, Steven M

    2003-04-01

    Sigma receptors are unique endoplasmic reticulum proteins that mediate signaling for a variety of drugs. We determined the effect of sigma(1) receptor agonists on immune responses in a syngeneic lung cancer model. Sigma(1) receptor agonists, including cocaine, up-regulated splenocyte IL-10 mRNA and protein production in vitro in a sigma receptor-dependent, pertussis toxin-sensitive manner. In vivo, sigma(1) receptor agonists promoted tumor growth and induced IL-10 at the tumor site. Increased tumor growth was prevented by administration of specific Abs to IL-10 or by administration of specific sigma(1) receptor antagonists. We report that sigma(1) receptor ligands, including cocaine, augment tumor growth through an IL-10 dependent mechanism.

  7. Histamine H4 receptor mediates eosinophil chemotaxis with cell shape change and adhesion molecule upregulation

    PubMed Central

    Ling, Ping; Ngo, Karen; Nguyen, Steven; Thurmond, Robin L; Edwards, James P; Karlsson, Lars; Fung-Leung, Wai-Ping

    2004-01-01

    During mast cell degranulation, histamine is released in large quantities. Human eosinophils were found to express histamine H4 but not H3 receptors. The possible effects of histamine on eosinophils and the receptor mediating these effects were investigated in our studies. Histamine (0.01–30 μM) induced a rapid and transient cell shape change in human eosinophils, but had no effects on neutrophils. The maximal shape change was at 0.3 μM histamine with EC50 at 19 nM. After 60 min incubation with 1 μM histamine, eosinophils were desensitized and were refractory to shape change response upon histamine restimulation. Histamine (0.01–1 μM) also enhanced the eosinophil shape change induced by other chemokines. Histamine-induced eosinophil shape change was mediated by the H4 receptor. This effect was completely inhibited by H4 receptor-specific antagonist JNJ 7777120 (IC50 0.3 μM) and H3/H4 receptor antagonist thioperamide (IC50 1.4 μM), but not by selective H1, H2 or H3 receptor antagonists. H4 receptor agonists imetit (EC50 25 nM) and clobenpropit (EC50 72 nM) could mimic histamine effect in inducing eosinophil shape change. Histamine (0.01–100 μM) induced upregulation of adhesion molecules CD11b/CD18 (Mac-1) and CD54 (ICAM-1) on eosinophils. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 and thioperamide. Histamine (0.01–10 μM) induced eosinophil chemotaxis with an EC50 of 83 nM. This effect was mediated by the H4 receptor and could be blocked by H4 receptor antagonists JNJ 7777120 (IC50 86 nM) and thioperamide (IC50 519 nM). Histamine (0.5 μM) also enhanced the eosinophil shape change induced by other chemokines. In conclusion, we have demonstrated a new mechanism of eosinophil recruitment driven by mast cells via the release of histamine. Using specific histamine receptor ligands, we have provided a definitive proof that the H4 receptor mediates eosinophil chemotaxis, cell shape change and

  8. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  9. Fluid shear stress sensitizes cancer cells to receptor-mediated apoptosis via trimeric death receptors

    NASA Astrophysics Data System (ADS)

    Mitchell, Michael J.; King, Michael R.

    2013-01-01

    Cancer metastasis, the process of cancer cell migration from a primary to distal location, typically leads to a poor patient prognosis. Hematogenous metastasis is initiated by intravasation of circulating tumor cells (CTCs) into the bloodstream, which are then believed to adhere to the luminal surface of the endothelium and extravasate into distal locations. Apoptotic agents such as tumor necrosis factor apoptosis-inducing ligand (TRAIL), whether in soluble ligand form or expressed on the surface of natural killer cells, have shown promise in treating CTCs to reduce the probability of metastasis. The role of hemodynamic shear forces in altering the cancer cell response to apoptotic agents has not been previously investigated. Here, we report that human colon cancer COLO 205 and prostate cancer PC-3 cells exposed to a uniform fluid shear stress in a cone-and-plate viscometer become sensitized to TRAIL-induced apoptosis. Shear-induced sensitization directly correlates with the application of fluid shear stress, and TRAIL-induced apoptosis increases in a fluid shear stress force- and time-dependent manner. In contrast, TRAIL-induced necrosis is not affected by the application fluid shear stress. Interestingly, fluid shear stress does not sensitize cancer cells to apoptosis when treated with doxorubicin, which also induces apoptosis in cancer cells. Caspase inhibition experiments reveal that shear stress-induced sensitization to TRAIL occurs via caspase-dependent apoptosis. These results suggest that physiological fluid shear forces can modulate receptor-mediated apoptosis of cancer cells in the presence of apoptotic agents.

  10. Drosophila Lipophorin Receptors Recruit the Lipoprotein LTP to the Plasma Membrane to Mediate Lipid Uptake

    PubMed Central

    Rodríguez-Vázquez, Míriam; Mejía-Morales, John E.; Culi, Joaquim

    2015-01-01

    Lipophorin, the main Drosophila lipoprotein, circulates in the hemolymph transporting lipids between organs following routes that must adapt to changing physiological requirements. Lipophorin receptors expressed in developmentally dynamic patterns in tissues such as imaginal discs, oenocytes and ovaries control the timing and tissular distribution of lipid uptake. Using an affinity purification strategy, we identified a novel ligand for the lipophorin receptors, the circulating lipoprotein Lipid Transfer Particle (LTP). We show that specific isoforms of the lipophorin receptors mediate the extracellular accumulation of LTP in imaginal discs and ovaries. The interaction requires the LA-1 module in the lipophorin receptors and is strengthened by a contiguous region of 16 conserved amino acids. Lipophorin receptor variants that do not interact with LTP cannot mediate lipid uptake, revealing an essential role of LTP in the process. In addition, we show that lipophorin associates with the lipophorin receptors and with the extracellular matrix through weak interactions. However, during lipophorin receptor-mediated lipid uptake, LTP is required for a transient stabilization of lipophorin in the basolateral plasma membrane of imaginal disc cells. Together, our data suggests a molecular mechanism by which the lipophorin receptors tether LTP to the plasma membrane in lipid acceptor tissues. LTP would interact with lipophorin particles adsorbed to the extracellular matrix and with the plasma membrane, catalyzing the exchange of lipids between them. PMID:26121667

  11. Cellular mechanisms of the 5-HT7 receptor-mediated signaling

    PubMed Central

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes. PMID:25324743

  12. Cellular mechanisms of the 5-HT7 receptor-mediated signaling.

    PubMed

    Guseva, Daria; Wirth, Alexander; Ponimaskin, Evgeni

    2014-01-01

    Serotonin (5-hydroxytryptamine or 5-HT) is an important neurotransmitter regulating a wide range of physiological and pathological functions via activation of heterogeneously expressed 5-HT receptors. The 5-HT7 receptor is one of the most recently described members of the 5-HT receptor family. Functionally, 5-HT7 receptor is associated with a number of physiological and pathological responses, including serotonin-induced phase shifting of the circadian rhythm, control of memory as well as locomotor and exploratory activity. A large body of evidence indicates involvement of the 5-HT7 receptor in anxiety and depression, and recent studies suggest that 5-HT7 receptor can be highly relevant for the treatment of major depressive disorders. The 5-HT7 receptor is coupled to the stimulatory Gs-protein, and receptor stimulation results in activation of adenylyl cyclase (AC) leading to a rise of cAMP concentration. In addition, this receptor is coupled to the G12-protein to activate small GTPases of the Rho family. This review focuses on molecular mechanisms responsible for the 5-HT7 receptor-mediated signaling. We provide detailed overview of signaling cascades controlled and regulated by the 5-HT7 receptor and discuss the functional impact of 5-HT7 receptor for the regulation of different cellular and subcellular processes.

  13. Two PTP receptors mediate CSPG inhibition by convergent and divergent signaling pathways in neurons

    PubMed Central

    Ohtake, Yosuke; Wong, Daniella; Abdul-Muneer, P. M.; Selzer, Michael E.; Li, Shuxin

    2016-01-01

    Receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member LAR act as transmembrane receptors that mediate growth inhibition of chondroitin sulfate proteoglycans (CSPGs). Inhibition of either receptor increases axon growth into and beyond scar tissues after CNS injury. However, it is unclear why neurons express two similar CSPG receptors, nor whether they use the same or different intracellular pathways. We have now studied the signaling pathways of these two receptors using N2A cells and primary neurons derived from knockout mice. We demonstrate that both receptors share certain signaling pathways (RhoA, Akt and Erk), but also use distinct signals to mediate CSPG actions. Activation of PTPσ by CSPGs selectively inactivated CRMP2, APC, S6 kinase and CREB. By contrast LAR activation inactivated PKCζ, cofilin and LKB1. For the first time, we propose a model of the signaling pathways downstream of these two CSPG receptors. We also demonstrate that deleting both receptors exhibits additive enhancement of axon growth in adult neuronal cultures in vitro. Our findings elucidate the novel downstream pathways of CSPGs and suggest potential synergy of blocking their two PTP receptors. PMID:27849007

  14. G protein-coupled receptors: extranuclear mediators for the non-genomic actions of steroids.

    PubMed

    Wang, Chen; Liu, Yi; Cao, Ji-Min

    2014-09-01

    Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs) are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30) for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A) and zinc transporter member 9 (ZIP9) for androgen, and trace amine associated receptor 1 (TAAR1) for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid), the structural basis of steroids and GPCRs' interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  15. Muscarinic receptor-mediated excitation of rat intracardiac ganglion neurons.

    PubMed

    Hirayama, Michiko; Ogata, Masanori; Kawamata, Tomoyuki; Ishibashi, Hitoshi

    2015-08-01

    Modulation of the membrane excitability of rat parasympathetic intracardiac ganglion neurons by muscarinic receptors was studied using an amphotericin B-perforated patch-clamp recording configuration. Activation of muscarinic receptors by oxotremorine-M (OxoM) depolarized the membrane, accompanied by repetitive action potentials. OxoM evoked inward currents under voltage-clamp conditions at a holding potential of -60 mV. Removal of extracellular Ca(2+) markedly increased the OxoM-induced current (IOxoM). The inward IOxoM in the absence of extracellular Ca(2+) was fully inhibited by removal of extracellular Na(+), indicating the involvement of non-selective cation channels. The IOxoM was inhibited by organic cation channel antagonists including SKF-96365 and ML-204. The IOxoM was antagonized by muscarinic receptor antagonists with the following potency: 4-DAMP > pirenzepine = darifenacin > methoctramine. Muscarinic toxin 7 (MT-7), a highly selective inhibitor for M1 receptor, produced partial inhibition of the IOxoM. In the presence of MT-7, concentration-inhibition curve of the M3-preferring antagonist darifenacin was shifted to the left. These results suggest the contribution of M1 and M3 receptors to the OxoM response. The IOxoM was inhibited by U-73122, a phospholipase C inhibitor. The membrane-permeable IP3 receptor blocker xestospongin C also inhibited the IOxoM. Furthermore, pretreatment with thapsigargin and BAPTA-AM inhibited the IOxoM, while KN-62, a blocker of Ca(2+)/calmodulin-dependent protein kinase II, had no effect. These results suggest that the activation mechanism involves a PLC pathway, release of Ca(2+) from intracellular Ca(2+) stores and calmodulin. The cation channels activated by muscarinic receptors may play an important role in neuronal membrane depolarization in rat intracardiac ganglion neurons.

  16. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na/sup +/ channel interaction

    SciTech Connect

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-12

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na/sup +/ channels is a coupled event mediated by guanine nucleotide binding protein(s) (G-protein(s)). These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of (/sup 3/H) acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of (/sup 3/H)batrachotoxin to Na/sup +/ channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced /sup 22/Na/sup +/ uptake in the presence and absence of tetrodotoxin, which blocks Na/sup +/ channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na/sup +/channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na/sup +/ channel-is such that at resting potential the muscarinic receptor induces opening of Na/sup +/ channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues.

  17. Dual effects of anandamide on NMDA receptor-mediated responses and neurotransmission.

    PubMed

    Hampson, A J; Bornheim, L M; Scanziani, M; Yost, C S; Gray, A T; Hansen, B M; Leonoudakis, D J; Bickler, P E

    1998-02-01

    Anandamide is an endogenous ligand of cannabinoid receptors that induces pharmacological responses in animals similar to those of cannabinoids such as delta9-tetrahydrocannabinol (THC). Typical pharmacological effects of cannabinoids include disruption of pain, memory formation, and motor coordination, systems that all depend on NMDA receptor mediated neurotransmission. We investigated whether anandamide can influence NMDA receptor activity by examining NMDA-induced calcium flux (deltaCa2+NMDA) in rat brain slices. The presence of anandamide reduced deltaCa2+NMDA and the inhibition was disrupted by cannabinoid receptor antagonist, pertussis toxin treatment, and agatoxin (a calcium channel inhibitor). Whereas these treatments prevented anandamide inhibiting deltaCa2+NMDA, they also revealed another, underlying mechanism by which anandamide influences deltaCa2+NMDA. In the presence of cannabinoid receptor antagonist, anandamide potentiated deltaCa2+NMDA in cortical, cerebellar, and hippocampal slices. Anandamide (but not THC) also augmented NMDA-stimulated currents in Xenopus oocytes expressing cloned NMDA receptors, suggesting a capacity to directly modulate NMDA receptor activity. In a similar manner, anandamide enhanced neurotransmission across NMDA receptor-dependent synapses in hippocampus in a manner that was not mimicked by THC and was unaffected by cannabinoid receptor antagonist. These data demonstrate that anandamide can modulate NMDA receptor activity in addition to its role as a cannabinoid receptor ligand.

  18. Agonist-mediated assembly of the crustacean methyl farnesoate receptor

    PubMed Central

    Kakaley, Elizabeth K. Medlock; Wang, Helen Y.; LeBlanc, Gerald A.

    2017-01-01

    The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly. PMID:28322350

  19. In silico modelling of prostacyclin and other lipid mediators to nuclear receptors reveal novel thyroid hormone receptor antagonist properties.

    PubMed

    Perez Diaz, Noelia; Zloh, Mire; Patel, Pryank; Mackenzie, Louise S

    2016-01-01

    Prostacyclin (PGI2) is a key mediator involved in cardiovascular homeostasis, acting predominantly on two receptor types; cell surface IP receptor and cytosolic peroxisome proliferator activated receptor (PPAR) β/δ. Having a very short half-life, direct methods to determine its long term effects on cells is difficult, and little is known of its interactions with nuclear receptors. Here we used computational chemistry methods to investigate the potential for PGI2, beraprost (IP receptor agonist), and GW0742 (PPARβ/δ agonist), to bind to nuclear receptors, confirmed with pharmacological methods. In silico screening predicted that PGI2, beraprost, and GW0742 have the potential to bind to different nuclear receptors, in particular thyroid hormone β receptor (TRβ) and thyroid hormone α receptor (TRα). Docking analysis predicts a binding profile to residues thought to have allosteric control on the TR ligand binding site. Luciferase reporter assays confirmed that beraprost and GW0742 display TRβ and TRα antagonistic properties; beraprost IC50 6.3 × 10(-5)mol/L and GW0742 IC50 4.9 × 10(-6) mol/L. Changes to triiodothyronine (T3) induced vasodilation of rat mesenteric arteries measured on the wire myograph were measured in the presence of the TR antagonist MLS000389544 (10(-5) mol/L), beraprost (10(-5) mol/L) and GW0742 (10(-5) mol/L); all significantly inhibited T3 induced vasodilation compared to controls. We have shown that both beraprost and GW0742 exhibit TRβ and TRα antagonist behaviour, and suggests that PGI2 has the ability to affect the long term function of cells through binding to and inactivating thyroid hormone receptors.

  20. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  1. The endocytic receptor protein LRP also mediates neuronal calcium signaling via N-methyl-d-aspartate receptors

    PubMed Central

    Bacskai, B. J.; Xia, M. Q.; Strickland, D. K.; Rebeck, G. W.; Hyman, B. T.

    2000-01-01

    The low density lipoprotein receptor-related protein (LRP) is an endocytic receptor that is a member of the low density lipoprotein receptor family. We report that the LRP ligand, activated α2-macroglobulin (α2M*), induces robust calcium influx in cultured primary neurons, but not in nonneuronal LRP-containing cells in the same culture. The calcium influx is mediated through N-methyl-d-aspartate receptor channels, which explains the neuron specificity of the response. Microapplication of α2M* leads to a localized response at the site of application that dissipates rapidly, suggesting that the calcium signal is temporally and spatially discrete. Calcium influx to α2M* is blocked by the physiological LRP inhibitor, receptor-associated protein. Bivalent antibodies to the extracellular domain of LRP, but not Fab fragments of the same antibody, cause calcium influx, indicating that the response is specific to LRP and may require dimerization of the receptor. Thus, LRP is an endocytic receptor with a novel signaling role. PMID:11016955

  2. Sperm Epidermal Growth Factor Receptor (EGFR) Mediates α7 Acetylcholine Receptor (AChR) Activation to Promote Fertilization

    PubMed Central

    Jaldety, Yael; Glick, Yair; Orr-Urtreger, Avi; Ickowicz, Debby; Gerber, Doron; Breitbart, Haim

    2012-01-01

    To attain fertilization the spermatozoon binds to the egg zona pellucida (ZP) via sperm receptor(s) and undergoes an acrosome reaction (AR). Several sperm receptors have been described in the literature; however, the identity of this receptor is not yet certain. In this study, we suggest that the α7 nicotinic acetylcholine receptor (α7nAChR) might be a sperm receptor activated by ZP to induce epidermal growth factor receptor (EGFR)-mediated AR. We found that isolated ZP or α7 agonists induced the AR in sperm from WT but not α7-null spermatozoa, and the induced AR was inhibited by α7 or EGFR antagonists. Moreover, α7-null sperm showed very little binding to the egg, and microfluidic affinity in vitro assay clearly showed that α7nAChR, as well as EGFR, interacted with ZP3. Induction of EGFR activation and the AR by an α7 agonist was inhibited by a Src family kinase (SFK) inhibitor. In conclusion we suggest that activation of α7 by ZP leads to SFK-dependent EGFR activation, Ca2+ influx, and the acrosome reaction. PMID:22577141

  3. Cannabinoid receptor type 1- and 2-mediated increase in cyclic AMP inhibits T cell receptor-triggered signaling.

    PubMed

    Börner, Christine; Smida, Michal; Höllt, Volker; Schraven, Burkhart; Kraus, Jürgen

    2009-12-18

    The aim of this study was to characterize inhibitory mechanisms on T cell receptor signaling mediated by the cannabinoid receptors CB1 and CB2. Both receptors are coupled to G(i/o) proteins, which are associated with inhibition of cyclic AMP formation. In human primary and Jurkat T lymphocytes, activation of CB1 by R(+)-methanandamide, CB2 by JWH015, and both by Delta9-tetrahydrocannabinol induced a short decrease in cyclic AMP lasting less than 1 h. However, this decrease was followed by a massive (up to 10-fold) and sustained (at least up to 48 h) increase in cyclic AMP. Mediated by the cyclic AMP-activated protein kinase A and C-terminal Src kinase, the cannabinoids induced a stable phosphorylation of the inhibitory Tyr-505 of the leukocyte-specific protein tyrosine kinase (Lck). By thus arresting Lck in its inhibited form, the cannabinoids prevented the dephosphorylation of Lck at Tyr-505 in response to T cell receptor activation, which is necessary for the subsequent initiation of T cell receptor signaling. In this way the cannabinoids inhibited the T cell receptor-triggered signaling, i.e. the activation of the zeta-chain-associated protein kinase of 70 kDa, the linker for activation of T cells, MAPK, the induction of interleukin-2, and T cell proliferation. All of the effects of the cannabinoids were blocked by the CB1 and CB2 antagonists AM281 and AM630. These findings help to better understand the immunosuppressive effects of cannabinoids and explain the beneficial effects of these drugs in the treatment of T cell-mediated autoimmune disorders like multiple sclerosis.

  4. Androgen receptor serine 81 mediates Pin1 interaction and activity

    PubMed Central

    La Montagna, Raffaele; Caligiuri, Isabella; Maranta, Pasquale; Lucchetti, Chiara; Esposito, Luca; Paggi, Marco G.; Toffoli, Giuseppe; Rizzolio, Flavio; Giordano, Antonio

    2012-01-01

    Hormone-dependent tumors are characterized by deregulated activity of specific steroid receptors, allowing aberrant expression of many genes involved in cancer initiation, progression and metastasis. In prostate cancer, the androgen receptor (AR) protein has pivotal functions, and over the years it has been the target of different drugs. AR is a nuclear receptor whose activity is regulated by a phosphorylation mechanism controlled by hormone and growth factors. Following phosphorylation, AR interacts with many cofactors that closely control its function. Among such cofactors, Pin1 is a peptidyl-prolyl isomerase that is involved in the control of protein phosphorylation and has a prognostic value in prostate cancer. In the present study, we demonstrate that ARSer81 is involved in the interaction with Pin1, and that this interaction is important for the transcriptional activity of AR. Since Pin1 expression positively correlates with tumor grade, our results suggest that Pin1 can participate in this process by modulating AR function. PMID:22894932

  5. The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila.

    PubMed

    Ni, Lina; Klein, Mason; Svec, Kathryn V; Budelli, Gonzalo; Chang, Elaine C; Ferrer, Anggie J; Benton, Richard; Samuel, Aravinthan Dt; Garrity, Paul A

    2016-04-29

    Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity.

  6. The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila

    PubMed Central

    Ni, Lina; Klein, Mason; Svec, Kathryn V; Budelli, Gonzalo; Chang, Elaine C; Ferrer, Anggie J; Benton, Richard; Samuel, Aravinthan DT; Garrity, Paul A

    2016-01-01

    Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer cool-responsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity. DOI: http://dx.doi.org/10.7554/eLife.13254.001 PMID:27126188

  7. Nucleus accumbens μ-opioid receptors mediate social reward.

    PubMed

    Trezza, Viviana; Damsteegt, Ruth; Achterberg, E J Marijke; Vanderschuren, Louk J M J

    2011-04-27

    Positive social interactions are essential for emotional well-being and proper behavioral development of young individuals. Here, we studied the neural underpinnings of social reward by investigating the involvement of opioid neurotransmission in the nucleus accumbens (NAc) in social play behavior, a highly rewarding social interaction in adolescent rats. Intra-NAc infusion of morphine (0.05-0.1 μg) increased pinning and pouncing, characteristic elements of social play behavior in rats, and blockade of NAc opioid receptors with naloxone (0.5 μg) prevented the play-enhancing effects of systemic morphine (1 mg/kg, s.c.) administration. Thus, stimulation of opioid receptors in the NAc was necessary and sufficient for morphine to increase social play. Intra-NAc treatment with the selective μ-opioid receptor agonist [D-Ala(2),N-MePhe(4),Gly(5)-ol]enkephalin (DAMGO) (0.1-10 ng) and the μ-opioid receptor antagonist Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) (0.3-3 μg) increased and decreased social play, respectively. The δ-opioid receptor agonist DPDPE ([D-Pen(2),D-Pen(5)]-enkephalin) (0.3-3 μg) had no effects, whereas the κ-opioid receptor agonist U69593 (N-methyl-2-phenyl-N-[(5R,7S,8S)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]dec-8-yl]acetamide) (0.01-1 μg) decreased social play. Intra-NAc treatment with β-endorphin (0.01-1 μg) increased social play, but met-enkephalin (0.1-5 μg) and the enkephalinase inhibitor thiorphan (0.1-1 μg) were ineffective. DAMGO (0.1-10 ng) increased social play after infusion into both the shell and core subregions of the NAc. Last, intra-NAc infusion of CTAP (3 μg) prevented the development of social play-induced conditioned place preference. These findings identify NAc μ-opioid receptor stimulation as an important neural mechanism for the attribution of positive value to social interactions in adolescent rats. Altered NAc μ-opioid receptor function may underlie social impairments in psychiatric disorders such as autism

  8. Excitatory Synaptic Responses Mediated by GABA_A Receptors in the Hippocampus

    NASA Astrophysics Data System (ADS)

    Michelson, Hillary B.; Wong, Robert K. S.

    1991-09-01

    Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the cortex. Activation of postsynaptic GABA_A receptors hyperpolarizes cells and inhibits neuronal activity. Synaptic responses mediated by GABA_A receptors also strongly excited hippocampal neurons. This excitatory response was recorded in morphologically identified interneurons in the presence of 4-aminopyridine or after elevation of extracellular potassium concentrations. The synaptic excitation sustained by GABA_A receptors synchronized the activity of inhibitory interneurons. This synchronized discharge of interneurons in turn elicited large-amplitude inhibitory postsynaptic potentials in pyramidal and granule cells. Excitatory synaptic responses mediated by GABA_A receptors may thus provide a mechanism for the recruitment of GABAergic interneurons through their recurrent connections.

  9. Cloned M1 muscarinic receptors mediate both adenylate cyclase inhibition and phosphoinositide turnover.

    PubMed Central

    Stein, R; Pinkas-Kramarski, R; Sokolovsky, M

    1988-01-01

    The rat M1 muscarinic receptor gene was cloned and expressed in a rat cell line lacking endogenous muscarinic receptors. Assignment of the cloned receptors to the M1 class was pharmacologically confirmed by their high affinity for the M1-selective muscarinic antagonist pirenzepine and low affinity for the M2-selective antagonist AF-DX-116. Guanylyl imidodiphosphate [Gpp(NH)p] converted agonist binding sites on the receptor, from high-affinity to the low-affinity state, thus indicating that the cloned receptors couple to endogenous G-proteins. The cloned receptors mediated both adenylate cyclase inhibition and phosphoinositide hydrolysis, but by different mechanisms. Pertussis toxin blocked the inhibition of adenylate cyclase (indicating coupling of the receptor to inhibitory G-protein), but did not affect phosphoinositide turnover. Furthermore, the stimulation of phosphoinositide hydrolysis was less efficient than the inhibition of adenylate cyclase. These findings demonstrate that cloned M1 receptors are capable of mediating multiple responses in the cell by coupling to different effectors, possibly to different G-proteins. Images PMID:2846274

  10. 5-HT(1A) and 5-HT(7) receptors differently modulate AMPA receptor-mediated hippocampal synaptic transmission.

    PubMed

    Costa, L; Trovato, C; Musumeci, S A; Catania, M V; Ciranna, L

    2012-04-01

    We have studied the effects of 5-HT(1A) and 5-HT(7) serotonin receptor activation in hippocampal CA3-CA1 synaptic transmission using patch clamp on mouse brain slices. Application of either 5-HT or 8-OH DPAT, a mixed 5-HT(1A)/5-HT(7) receptor agonist, inhibited AMPA receptor-mediated excitatory post synaptic currents (EPSCs); this effect was mimicked by the 5-HT(1A) receptor agonist 8-OH PIPAT and blocked by the 5-HT(1A) antagonist NAN-190. 8-OH DPAT increased paired-pulse facilitation and reduced the frequency of mEPSCs, indicating a presynaptic reduction of glutamate release probability. In another group of neurons, 8-OH DPAT enhanced EPSC amplitude but did not alter paired-pulse facilitation, suggesting a postsynaptic action; this effect persisted in the presence of NAN-190 and was blocked by the 5-HT(7) receptor antagonist SB-269970. To confirm that EPSC enhancement was mediated by 5-HT(7) receptors, we used the compound LP-44, which is considered a selective 5-HT(7) agonist. However, LP-44 reduced EPSC amplitude in most cells and instead increased EPSC amplitude in a subset of neurons, similarly to 8-OH DPAT. These effects were respectively antagonized by NAN-190 and by SB-269970, indicating that under our experimental condition LP-44 behaved as a mixed agonist. 8-OH DPAT also modulated the current evoked by exogenously applied AMPA, inducing either a reduction or an increase of amplitude in distinct neurons; these effects were respectively blocked by 5-HT(1A) and 5-HT(7) receptor antagonists, indicating that both receptors exert a postsynaptic action. Our results show that 5-HT(1A) receptors inhibit CA3-CA1 synaptic transmission acting both pre- and postsynaptically, whereas 5-HT(7) receptors enhance CA3-CA1 synaptic transmission acting exclusively at a postsynaptic site. We suggest that a selective pharmacological targeting of either subtype may be envisaged in pathological loss of hippocampal-dependent cognitive functions. In this respect, we underline the

  11. Extranuclear Signaling Effects Mediated by the Estrogen Receptor

    DTIC Science & Technology

    2008-03-01

    inhibition of Pyk2 with salicylate disrupted the ability of E2 to induce the phosphorylation of ERK1/2 without affecting αCaMKII activity (data not...Identification of a phosphorylation site for calcium/calmodulin-dependent protein kinase II in the 13 NR2B subunit of the N- methyl -D-aspartate receptor. J Biol

  12. Ultrasound-Mediated Therapies Using Receptor-Targeted Nanodroplets

    NASA Astrophysics Data System (ADS)

    Schumann, P.; LaBell, R.; Penrose, K.; Kerschen, A.; Unger, E.; Matsunaga, T.; Zutshi, R.

    2006-05-01

    Angiogenic vessels at tumor sites are leaky and allow particles less than 400 nm to pass through. We have developed targeted nanodroplets that are less than 400 nm in size and are capable of encapsulating drug molecules which can be released at the tumor site upon insonation. These nanodroplets are targeted to the α6β1 receptor, which is upregulated in prostate cancer.

  13. Regulation of the androgen receptor by SET9-mediated methylation.

    PubMed

    Gaughan, Luke; Stockley, Jacqueline; Wang, Nan; McCracken, Stuart R C; Treumann, Achim; Armstrong, Kelly; Shaheen, Fadhel; Watt, Kate; McEwan, Iain J; Wang, Chenguang; Pestell, Richard G; Robson, Craig N

    2011-03-01

    The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

  14. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    SciTech Connect

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-09-09

    Highlights: {yields} A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. {yields} The second zinc finger of LIM domain 1 of testin is critical for interaction. {yields} Testin bound to a region of the receptor tail important for cell signalling. {yields} Testin and receptor interaction was confirmed in mammalian (HEK293) cells. {yields} Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  15. CB1 cannabinoid receptor-mediated neurite remodeling in mouse neuroblastoma N1E-115 cells.

    PubMed

    Zhou, D; Song, Z H

    2001-08-15

    The morphological remodeling of neuronal cells influences neurogenesis and brain functions. We hypothesize that psychoactive and neurotoxic effects of cannabinoids may be mediated, at least in part, by their morphoregulatory activities. In the present study, mouse neuroblastoma N1E-115 cells were used as an in vitro model to investigate cannabinoid-induced neurite remodeling effects and to identify the involvement of cannabinoid receptors in this neurite remodeling process. Using reverse transcription-polymerase chain reaction and immunofluorescence microscopy, the endogenously expressed CB1, but not CB2, cannabinoid receptors were detected in morphologically differentiated N1E-115 cells. Activation of these natively expressed CB1 cannabinoid receptors by cannabinoid agonist HU-210 led to a concentration-dependent inhibition of adenylate cyclase activity. Importantly, HU-210 treatment induced neurite retraction in a concentration-dependent manner. Pretreatment of N1E-115 cells with a CB1 antisense oligodeoxynucleotide (ODN) suppressed HU-210-induced inhibition of forskolin-stimulated cAMP accumulation, indicating that the knocking down of functional CB1 cannabinoid receptor expression was achieved. Antisense ODN pretreatment also abolished HU-210-induced neurite retraction, demonstrating the involvement of CB1 cannabinoid receptors in mediating the neurite remodeling effects of HU-210. In addition, reversing HU-210-induced intracellular cAMP declination by 8-Br-cAMP partially prevented HU-210-induced neurite retraction, indicating the involvement of cAMP-dependent signaling pathways in mediating the neurite remodeling function of CB1 cannabinoid receptors in N1E-115 cells. These data demonstrate that neurite remodeling is a newly discovered function of CB1 cannabinoid receptors. This morphoregulatory function of CB1 cannabinoid receptors might be a new mechanism that mediates the psychoactive and neurotoxic effects of cannabinoids in developing and adult brain.

  16. Crosstalk between Leptin Receptor and Igf-Ir in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2008-04-01

    AD_________________ Award Number: W81XWH-06-1-0452 TITLE: Crosstalk between Leptin Receptor and Igf...NUMBER Crosstalk between Leptin Receptor and Igf-Ir in Breast Cancer: A Potential Mediator of Chemoresistance 5b. GRANT NUMBER W81XWH-06-1...cancer, and is associated with reduced treatment response and reduced overall survival. The obesity-associated hormones IGF-I and leptin and their

  17. Crosstalk Between Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance

    DTIC Science & Technology

    2009-04-01

    5). Leptin , a product of the obese (ob) gene, is an adipocytokine that regulates appetite , bone formation, reproduction, cellular proliferation, and...AD_________________ Award Number: W81XWH-06-1-0452 TITLE: Crosstalk Between Leptin Receptor and... Leptin Receptor and IGF-IR in Breast Cancer: A Potential Mediator of Chemoresistance 5b. GRANT NUMBER W81XWH-06-1-0452 5c. PROGRAM ELEMENT NUMBER

  18. Studies of the cAMP mediated aggregation in Dictyostelium discoideum: receptor mediated activation of the adenylate cyclase

    SciTech Connect

    Theibert, W.E.A.B.

    1985-01-01

    Dictyostelium discoideum, a eukaryotic amoeba of the cellular slime mold family, provides an interesting paradigm in developmental biology. During development, hundreds of thousands of cells aggregate to form a multicellular aggregate. Aggregation is mediated by chemotaxis and chemical signaling. Waves of adenosine 3'-5' cyclic monophosphate (cAMP) propagate through the monolayer and provide transient gradients for chemotaxis. The author has used a reversible inhibitor of the cAMP signaling response to demonstrate that adaptation to cAMP is independent of the activation of the adenylate cyclase and therefore is not caused by the rise in intracellular cAMP. Next, it is shown that adenosine inhibits the cAMP signaling response. Inhibition is rapid, reversible, and depends on the cAMP stimulus concentration. Then the specificity of the cAMP receptors which mediates signaling is determined and compared with the receptors which mediate chemotaxis, the cGMP response, and cAMP binding antagonism. The cAMP surface receptor has been identified by photoaffinity labeling intact cells with (/sup 32/P)-8-N/sub 3/-cAMP using an ammonium sulfate binding stabilization technique. The photoactivated ligand specifically labels a polypeptide, localized to the membrane fraction, which migrates as a closely spaced doublet on SDS Page.

  19. Glucocorticoid receptor-mediated induction of glutamine synthetase in skeletal muscle cells in vitro

    NASA Technical Reports Server (NTRS)

    Max, Stephen R.; Thomas, John W.; Banner, Carl; Vitkovic, Ljubisa; Konagaya, Masaaki

    1987-01-01

    The regulation by glucocorticoids of glutamine synthetase in L6 muscle cells in culture is studied. Glutamine synthetase activity was strikingly enhanced by dexamethasone. The dexamethasone-mediated induction of glutamine synthetase activity was blocked by RU38486, a glucocorticoid antagonist, indicating the involvement of intracellular glucocorticoid receptors in the induction process. RU38486 alone was without effect. Northern blot analysis revealed that dexamethasone-mediated enhancement of glutamine synthetase activity involves increased levels of glutamine synthetase mRNA. Glucocorticoids regulate the expression of glutamine synthetase mRNA in cultured muscle cells via interaction with intracellular receptors. Such regulation may be relevant to control of glutamine production by muscle.

  20. Orchestration of salivary secretion mediated by two different dopamine receptors in the blacklegged tick Ixodes scapularis

    PubMed Central

    Kim, Donghun; Šimo, Ladislav; Park, Yoonseong

    2014-01-01

    Salivary secretion is crucial for successful tick feeding, and it is the mediator of pathogen transmission. Salivation functions to inhibit various components of the host immune system and remove excess water and ions during the ingestion of large blood meals. Control of salivary glands involves autocrine/paracrine dopamine, which is the most potent inducer of tick salivation. Previously, we reported the presence of two dopamine receptors in the salivary glands of the blacklegged tick (Ixodes scapularis): dopamine receptor (D1) and invertebrate specific D1-like dopamine receptor (InvD1L). Here, we investigated the different physiological roles of the dopamine receptors in tick salivary glands by using pharmacological tools that discriminate between the two distinct receptors. Heterologous expressions followed by reporter assays of the dopamine receptors identified receptor-specific antagonists and agonists. These pharmacological tools were further used to discriminate the physiological role of each receptor by using in vitro assays: measuring salivary secretions of isolated salivary glands and monitoring dynamic changes in the size of individual salivary gland acini. We propose that the D1 receptor acts on salivary gland acini epithelial cells for inward fluid transport. InvD1L controls (or modulates) each acinus for expelling saliva from the acini to the salivary ducts, presumably through the actions of myoepithelial cells and valves for pumping/gating. We conclude that dopamine acts on the D1 and the InvD1L receptors and leads different physiological actions to orchestrate tick salivary secretion. PMID:25320269

  1. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling.

    PubMed

    Mattila, Pieta K; Batista, Facundo D; Treanor, Bebhinn

    2016-02-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival.

  2. Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

    PubMed

    Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

    2014-03-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters.

  3. Fc receptor-mediated, antibody-dependent enhancement of bacteriophage lambda-mediated gene transfer in mammalian cells.

    PubMed

    Sapinoro, Ramil; Volcy, Ketna; Rodrigo, W W Shanaka I; Schlesinger, Jacob J; Dewhurst, Stephen

    2008-04-10

    Lambda phage vectors mediate gene transfer in cultured mammalian cells and in live mice, and in vivo phage-mediated gene expression is increased when mice are pre-immunized with bacteriophage lambda. We now show that, like eukaryotic viruses, bacteriophage vectors are subject to Fc receptor-mediated, antibody-dependent enhancement of infection in mammalian cells. Antibody-dependent enhancement of phage gene transfer required FcgammaRI, but not its associated gamma-chain, and was not supported by other FcgammaR family members (FcgammaRIIA, FcgammaRIIB, and FcgammaRIII). Studies using chlorpromazine and latrunculin A revealed an important role for clathrin-mediated endocytosis (chlorpromazine) and actin filaments (latrunculin A) in antibody-enhanced phage gene transfer. This was confirmed by experiments using inhibitors of endosomal acidification (bafilomycin A1, monensin) and by immunocytochemical colocalization of internalized phage particles with early endosome-associated protein-1 (EAA1). In contrast, microtubule-targeting agents (nocodazole, taxol) increased the efficiency of antibody-enhanced phage gene transfer. These results reveal an unexpected antibody-dependent, FcgammaRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer.

  4. P2 receptor-mediated signaling in mast cell biology.

    PubMed

    Bulanova, Elena; Bulfone-Paus, Silvia

    2010-03-01

    Mast cells are widely recognized as effector cells of allergic inflammatory reactions. They contribute to the pathogenesis of different chronic inflammatory diseases, wound healing, fibrosis, thrombosis/fibrinolysis, and anti-tumor immune responses. In this paper, we summarized the role of P2X and P2Y receptors in mast cell activation and effector functions. Mast cells are an abundant source of ATP which is stored in their granules and secreted upon activation. We discuss the contribution of mast cells to the extracellular ATP release and to the maintenance of extracellular nucleotides pool. Recent publications highlight the importance of purinergic signaling for the pathogenesis of chronic airway inflammation. Therefore, the role of ATP and P2 receptors in allergic inflammation with focus on mast cells was analyzed. Finally, ATP functions as mast cell autocrine/paracrine factor and as messenger in intercellular communication between mast cells, nerves, and glia in the central nervous system.

  5. Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

    PubMed Central

    Gonzalez-Rodriguez, David; Barakat, Abdul I.

    2015-01-01

    Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

  6. Visualizing clathrin-mediated IgE receptor internalization by electron and atomic force microscopy.

    PubMed

    Burns, Alan R; Oliver, Janet M; Pfeiffer, Janet R; Wilson, Bridget S

    2008-01-01

    A significant step in the immunoglobulin E (IgE) receptor signaling pathway in mast cell membranes is receptor internalization by clathrin-coated vesicles. Visualization in native membrane sheets of the emerging clathrin lattice structures containing the IgE receptor and associated signaling partners has been accomplished with high-resolution transmission electron microscopy (TEM). More recently, membrane sheets with labeled clathrin have also been characterized with atomic force microscopy (AFM) in combination with fluorescence imaging. We discuss here the procedure for creating fixed, native cell membrane sheets, labeling with immunogold or fluorescent labels, and utilization for TEM or AFM/fluorescence imaging of clathrin-mediated IgE internalization.

  7. An Fcγ receptor-dependent mechanism drives antibody-mediated target-receptor signaling in cancer cells.

    PubMed

    Wilson, Nicholas S; Yang, Becky; Yang, Annie; Loeser, Stefanie; Marsters, Scot; Lawrence, David; Li, Yun; Pitti, Robert; Totpal, Klara; Yee, Sharon; Ross, Sarajane; Vernes, Jean-Michel; Lu, Yanmei; Adams, Cam; Offringa, Rienk; Kelley, Bob; Hymowitz, Sarah; Daniel, Dylan; Meng, Gloria; Ashkenazi, Avi

    2011-01-18

    Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.

  8. An histidine covalent receptor/butenolide complex mediates strigolactone perception

    PubMed Central

    Badet-Denisot, Marie-Ange; Pillot, Jean-Paul; Cornu, David; Le Caer, Jean-Pierre; Burger, Marco; Pelissier, Frank; Retailleau, Pascal; Turnbull, Colin; Bonhomme, Sandrine; Chory, Joanne; Rameau, Catherine; Boyer, François-Didier

    2016-01-01

    Strigolactone plant hormones control plant architecture and are key players in both symbiotic and parasitic interactions. They contain an ABC tricyclic lactone connected to a butenolide group, the D-ring. The DWARF14 (D14) strigolactone receptor belongs to the superfamily of α/β-hydrolases and is known to hydrolyze the bond between the ABC lactone and the D-ring. Here we characterize the binding and catalytic functions of RAMOSUS3 (RMS3), the pea (Pisum sativum) ortholog of rice (Oryza sativa) D14 strigolactone receptor. Using novel profluorescent probes with strigolactone-like bioactivity, we show that RMS3 acts as a single-turnover enzyme that explains its apparent low enzymatic rate. We further demonstrate the formation of a covalent RMS3/D-ring complex, essential for bioactivity, in which the D-ring is attached to Histidine 247 of the catalytic triad. These results reveal an undescribed mechanism of plant hormone reception where the receptor performs an irreversible enzymatic reaction to generate its own ligand. PMID:27479744

  9. Taste Receptors Mediate Sinonasal Immunity and Respiratory Disease

    PubMed Central

    Douglas, Jennifer E.; Cohen, Noam A.

    2017-01-01

    The bitter taste receptor T2R38 has been shown to play a role in the pathogenesis of chronic rhinosinusitis (CRS), where the receptor functions to enhance upper respiratory innate immunity through a triad of beneficial immune responses. Individuals with a functional version of T2R38 are tasters for the bitter compound phenylthiocarbamide (PTC) and exhibit an anti-microbial response in the upper airway to certain invading pathogens, while those individuals with a non-functional version of the receptor are PTC non-tasters and lack this beneficial response. The clinical ramifications are significant, with the non-taster genotype being an independent risk factor for CRS requiring surgery, poor quality-of-life (QOL) improvements post-operatively, and decreased rhinologic QOL in patients with cystic fibrosis. Furthermore, indirect evidence suggests that non-tasters also have a larger burden of biofilm formation. This new data may influence the clinical management of patients with infectious conditions affecting the upper respiratory tract and possibly at other mucosal sites throughout the body. PMID:28218655

  10. Glucocorticoid hormone resistance during primate evolution: receptor-mediated mechanisms.

    PubMed

    Chrousos, G P; Renquist, D; Brandon, D; Eil, C; Pugeat, M; Vigersky, R; Cutler, G B; Loriaux, D L; Lipsett, M B

    1982-03-01

    The concentrations of total and protein-unbound plasma cortisol of New World monkeys are higher than those of Old World primates and prosimians. The urinary free-cortisol excretion also is increased markedly. However, there is no physiologic evidence of increased cortisol effect. These findings suggest end-organ resistance to glucocorticoids. This was confirmed by showing that the hypothalamic-pituitary adrenal axis is resistant to suppression by dexamethasone. To study this phenomenon, glucocorticoid receptors were examined in circulating mononuclear leukocytes and cultured skin fibroblasts from both New and Old World species. The receptor content is the same in all species, but the New World monkeys have a markedly decreased binding affinity for dexamethasone. Thus, the resistance of these species to the action of cortisol is due to the decreased binding affinity of the glucocorticoid receptor. This presumed mutation must have occurred after the bifurcation of Old and New World primates (approximately 60 x 10(6) yr ago) and before the diversion of the New World primates from each other (approximately 15 x 10(6) yr ago).

  11. Azadirachtin interacts with retinoic acid receptors and inhibits retinoic acid-mediated biological responses.

    PubMed

    Thoh, Maikho; Babajan, Banaganapalli; Raghavendra, Pongali B; Sureshkumar, Chitta; Manna, Sunil K

    2011-02-11

    Considering the role of retinoids in regulation of more than 500 genes involved in cell cycle and growth arrest, a detailed understanding of the mechanism and its regulation is useful for therapy. The extract of the medicinal plant Neem (Azadirachta indica) is used against several ailments especially for anti-inflammatory, anti-itching, spermicidal, anticancer, and insecticidal activities. In this report we prove the detailed mechanism on the regulation of retinoic acid-mediated cell signaling by azadirachtin, active components of neem extract. Azadirachtin repressed all trans-retinoic acid (ATRA)-mediated nuclear transcription factor κB (NF-κB) activation, not the DNA binding but the NF-κB-dependent gene expression. It did not inhibit IκBα degradation, IκBα kinase activity, or p65 phosphorylation and its nuclear translocation but inhibited NF-κB-dependent reporter gene expression. Azadirachtin inhibited TRAF6-mediated, but not TRAF2-mediated NF-κB activation. It inhibited ATRA-induced Sp1 and CREB (cAMP-response element-binding protein) DNA binding. Azadirachtin inhibited ATRA binding with retinoid receptors, which is supported by biochemical and in silico evidences. Azadirachtin showed strong interaction with retinoid receptors. It suppressed ATRA-mediated removal of retinoid receptors, bound with DNA by inhibiting ATRA binding to its receptors. Overall, our data suggest that azadirachtin interacts with retinoic acid receptors and suppresses ATRA binding, inhibits falling off the receptors, and activates transcription factors like CREB, Sp1, NF-κB, etc. Thus, azadirachtin exerts anti-inflammatory and anti-metastatic responses by a novel pathway that would be beneficial for further anti-inflammatory and anti-cancer therapies.

  12. Receptors, Mediators, and Mechanisms Involved in Bacterial Sepsis and Septic Shock

    PubMed Central

    Van Amersfoort, Edwin S.; Van Berkel, Theo J. C.; Kuiper, Johan

    2003-01-01

    Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins. PMID:12857774

  13. Optimization of stress response through the nuclear receptor-mediated cortisol signalling network

    PubMed Central

    Kolodkin, Alexey; Sahin, Nilgun; Phillips, Anna; Hood, Steve R.; Bruggeman, Frank J.; Westerhoff, Hans V.; Plant, Nick

    2013-01-01

    It is an accepted paradigm that extended stress predisposes an individual to pathophysiology. However, the biological adaptations to minimize this risk are poorly understood. Using a computational model based upon realistic kinetic parameters we are able to reproduce the interaction of the stress hormone cortisol with its two nuclear receptors, the high-affinity glucocorticoid receptor and the low-affinity pregnane X-receptor. We demonstrate that regulatory signals between these two nuclear receptors are necessary to optimize the body’s response to stress episodes, attenuating both the magnitude and duration of the biological response. In addition, we predict that the activation of pregnane X-receptor by multiple, low-affinity endobiotic ligands is necessary for the significant pregnane X-receptor-mediated transcriptional response observed following stress episodes. This integration allows responses mediated through both the high and low-affinity nuclear receptors, which we predict is an important strategy to minimize the risk of disease from chronic stress. PMID:23653204

  14. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  15. Neuroprotection Mediated by P2Y13 Nucleotide Receptors in Neurons

    PubMed Central

    Pérez-Sen, Raquel; Queipo, Mª José; Morente, Verónica; Ortega, Felipe; Delicado, Esmerilda G.; Miras-Portugal, Mª Teresa

    2015-01-01

    ADP-specific P2Y13 receptor constitutes one of the most recently identified nucleotide receptor and the understanding of their physiological role is currently under investigation. Cerebellar astrocytes and granule neurons provide excellent models to study P2Y13 expression and function since the first identification of ADP-evoked calcium responses not attributable to the related P2Y1 receptor was performed in these cell populations. In this regard, all responses induced by ADP analogues in astrocytes resulted to be Gi-coupled activities mediated by P2Y13 instead of P2Y1 receptors. Similarly, both glycogen synthase kinase-3 (GSK3) and ERK1/2 signaling triggered by 2MeSADP in cerebellar granule neurons were also dependent on Gi-coupled receptors, and mediated by PI3K activity. In granule neurons, P2Y13 receptor was specifically coupled to the main neuronal survival PI3K/Akt-cascade targeting GSK3 phosphorylation. GSK3 inhibition led to nuclear translocation of transcriptional targets, including β-catenin and Nrf2. The activation of the Nrf2/heme oxygenase-1 (HO-1) axis was responsible for the prosurvival effect against oxidative stress. In addition, P2Y13-mediated ERK1/2 signaling in granule neurons also triggered activation of transcription factors, such as CREB, which underlined the antiapoptotic action against glutamate-induced excitotoxicity. Finally, a novel signaling mechanism has been recently described for a P2Y13 receptor in granule neurons that involved the expression of a dual protein phosphatase, DUSP2. This activity contributed to regulate MAPK activation after genotoxic stress. In conclusion, P2Y13 receptors harbored in cerebellar astrocytes and granule neurons exhibit specific signaling properties that link them to specialized functions at the level of neuroprotection and trophic activity in both cerebellar cell populations. PMID:25750704

  16. Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

    PubMed Central

    Knutson, Todd P.; Lange, Carol A.

    2014-01-01

    Ovarian steroid hormones contribute to breast cancer initiation and progression primarily through the actions of their nuclear transcription factors, the estrogen receptor alpha (ERα) and progesterone receptors (PRs). These receptors are important drivers of the luminal A and B subtypes of breast cancer, where estrogen-blocking drugs have been effective endocrine therapies for patients with these tumors. However, many patients do not respond, or become resistant to treatment. When endocrine therapies fail, the luminal subtypes of breast cancer are more difficult to treat because these subtypes are among the most heterogeneous in terms of mutation diversity and gene expression profiles. Recent evidence suggests that progestin and PR actions may be important drivers of luminal breast cancers. Clinical trial data has demonstrated that hormone replacement therapy with progestins drives invasive breast cancer and results in greater mortality. PR transcriptional activity is dependent upon cross-talk with growth factor signaling pathways that alter PR phosphorylation, acetylation, or SUMOylation as mechanisms for regulating PR target gene selection required for increased cell proliferation and survival. Site-specific PR phosphorylation is the primary driver of gene-selective PR transcriptional activity. However, PR phosphorylation and heightened transcriptional activity is coupled to rapid PR protein degradation; the range of active PR detected in tumors is likely to be dynamic. Thus, PR target gene signatures may provide a more accurate means of tracking PR’s contribution to tumor progression rather than standard clinical protein-based (IHC) assays. Further development of antiprogestin therapies should be considered along side antiestrogens and aromatase inhibitors. PMID:24291072

  17. Scavenger receptor-mediated endocytosis by sinusoidal cells in rat bone marrow

    SciTech Connect

    Geoffroy, J.S.

    1987-01-01

    Endocytosis of serum albumin by sinusoidal endothelial cells in rat bone marrow was investigated initially at the ultrastructural level with subsequent biochemical investigation of the specificity mediating this event. Bovine serum albumin adsorbed to 20nm colloidal gold particles (AuBSA) was chosen as the electron microscopic probe. Morphological data strongly suggested that a receptor was involved in uptake of AuBSA. Confirmation of receptor involvement in the uptake of AuBSA by marrow sinusoidal endothelial cells was achieved utilizing an in situ isolated hind limb perfusion protocol in conjunction with unlabeled, radiolabeled, and radio-/colloidal gold labeled probes. The major findings of competition and saturation experiments were: (1) endocytosis of AuBSA was mediated by a receptor for modified/treated serum albumin; (2) endocytosis of formaldehyde-treated serum albumin was mediated by a binding site which may be the same or closely related to the site responsible for the uptake of AuBSA; and (3) endocytosis of native untreated albumin was not mediated by receptor and probably represents fluid-phase pinocitosis.

  18. Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family.

    PubMed

    Andjelic, S; Liou, H C

    1998-02-01

    An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.

  19. Asialoglycoprotein receptor-magnetic dual targeting nanoparticles for delivery of RASSF1A to hepatocellular carcinoma

    PubMed Central

    Xue, Wan-Jiang; Feng, Ying; Wang, Fei; Guo, Yi-Bing; Li, Peng; Wang, Lei; Liu, Yi-Fei; Wang, Zhi-Wei; Yang, Yu-Min; Mao, Qin-Sheng

    2016-01-01

    We developed a nanovector with double targeting properties for efficiently delivering the tumor suppressor gene RASSF1A specifically into hepatocellular carcinoma (HCC) cells by preparing galactosylated-carboxymethyl chitosan-magnetic iron oxide nanoparticles (Gal-CMCS-Fe3O4-NPs). After conjugating galactose and CMCS to the surface of Fe3O4-NPs, we observed that Gal-CMCS-Fe3O4-NPs were round with a relatively stable zeta potential of +6.5 mV and an mean hydrodynamic size of 40.1 ± 5.3 nm. Gal-CMCS-Fe3O4-NPs had strong DNA condensing power in pH 7 solution and were largely nontoxic. In vitro experiments demonstrated that Gal-CMCS-Fe3O4-NPs were highly selective for HCC cells and liver cells. In vivo experiments showed the specific accumulation of Gal-CMCS-Fe3O4-NPs in HCC tissue, especially with the aid of an external magnetic field. Nude mice with orthotopically transplanted HCC received an intravenous injection of the Gal-CMCS-Fe3O4-NPs/pcDNA3.1(+)RASSF1A compound and intraperitoneal injection of mitomycin and had an external magnetic field applied to the tumor area. These mice had the smallest tumors, largest percentage of TUNEL-positive cells, and highest caspase-3 expression levels in tumor tissue compared to other groups of treated mice. These results suggest the potential application of Gal-CMCS-Fe3O4-NPs for RASSF1A gene delivery for the treatment of HCC. PMID:26915683

  20. Asialoglycoprotein receptor-magnetic dual targeting nanoparticles for delivery of RASSF1A to hepatocellular carcinoma

    NASA Astrophysics Data System (ADS)

    Xue, Wan-Jiang; Feng, Ying; Wang, Fei; Guo, Yi-Bing; Li, Peng; Wang, Lei; Liu, Yi-Fei; Wang, Zhi-Wei; Yang, Yu-Min; Mao, Qin-Sheng

    2016-02-01

    We developed a nanovector with double targeting properties for efficiently delivering the tumor suppressor gene RASSF1A specifically into hepatocellular carcinoma (HCC) cells by preparing galactosylated-carboxymethyl chitosan-magnetic iron oxide nanoparticles (Gal-CMCS-Fe3O4-NPs). After conjugating galactose and CMCS to the surface of Fe3O4-NPs, we observed that Gal-CMCS-Fe3O4-NPs were round with a relatively stable zeta potential of +6.5 mV and an mean hydrodynamic size of 40.1 ± 5.3 nm. Gal-CMCS-Fe3O4-NPs had strong DNA condensing power in pH 7 solution and were largely nontoxic. In vitro experiments demonstrated that Gal-CMCS-Fe3O4-NPs were highly selective for HCC cells and liver cells. In vivo experiments showed the specific accumulation of Gal-CMCS-Fe3O4-NPs in HCC tissue, especially with the aid of an external magnetic field. Nude mice with orthotopically transplanted HCC received an intravenous injection of the Gal-CMCS-Fe3O4-NPs/pcDNA3.1(+)RASSF1A compound and intraperitoneal injection of mitomycin and had an external magnetic field applied to the tumor area. These mice had the smallest tumors, largest percentage of TUNEL-positive cells, and highest caspase-3 expression levels in tumor tissue compared to other groups of treated mice. These results suggest the potential application of Gal-CMCS-Fe3O4-NPs for RASSF1A gene delivery for the treatment of HCC.

  1. Lysosphingolipid receptor-mediated diuresis and natriuresis in anaesthetized rats.

    PubMed

    Bischoff, A; Meyer Zu Heringdorf, D; Jakobs, K H; Michel, M C

    2001-04-01

    Lysosphingolipids such as sphingosine-1-phosphate (SPP) and sphingosylphosphorylcholine (SPPC) can act on specific G-protein-coupled receptors. Since SPP and SPPC cause renal vasoconstriction, we have investigated their effects on urine and electrolyte excretion in anaesthetized rats. Infusion of SPP (1 - 30 microg kg(-1) min(-1)) for up to 120 min dose-dependently but transiently (peak after 15 min, disappearance after 60 min) reduced renal blood flow without altering endogenous creatinine clearance. Nevertheless, infusion of SPP increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. These tubular lysosphingolipid effects developed more slowly (maximum after 60 - 90 min) and also abated more slowly upon lysosphingolipid washout than the renovascular effects. Infusion of SPPC, sphingosine and glucopsychosine (3 - 30 microg kg(-1) min(-1) each) caused little if any alterations in renal blood flow but also increased diuresis, natriuresis and calciuresis and, to a lesser extent, kaliuresis. Pretreatment with pertussis toxin (10 microg kg(-1) 3 days before the acute experiment) abolished the renovascular and tubular effects of 30 microg kg(-1) min(-1) SPP. These findings suggest that lysosphingolipids are a hitherto unrecognized class of endogenous modulators of renal function. SPP affects renovascular tone and tubular function via receptors coupled to G(i)-type G-proteins. SPPC, sphingosine and glucopsychosine mimic only the tubular effects of SPP, and hence may act on distinct sites.

  2. Toll-Like Receptor-9-Mediated Invasion in Breast Cancer

    DTIC Science & Technology

    2011-07-01

    and purify TLR9 in-house, recombinant hTLR9 is now available for purchase from BioClone, Inc., San Diego, CA (http://www.bioclone.us/CD- Marker -cDNA...structure dominates at lower pH, even in the presence of salt. Correlation of DNA structure and stabilit y with TLR9-mediated cancer cell in vasion...TTAGGG)3], was examined. This sequence is found at the ends of human chromosomes and due to the nature of the repetitive GGGs forms G-tetrads that

  3. Mediators, Receptors, and Signalling Pathways in the Anti-Inflammatory and Antihyperalgesic Effects of Acupuncture

    PubMed Central

    McDonald, John L.; Cripps, Allan W.; Smith, Peter K.

    2015-01-01

    Acupuncture has been used for millennia to treat allergic diseases including both intermittent rhinitis and persistent rhinitis. Besides the research on the efficacy and safety of acupuncture treatment for allergic rhinitis, research has also investigated how acupuncture might modulate immune function to exert anti-inflammatory effects. A proposed model has previously hypothesized that acupuncture might downregulate proinflammatory neuropeptides, proinflammatory cytokines, and neurotrophins, modulating transient receptor potential vallinoid (TRPV1), a G-protein coupled receptor which plays a central role in allergic rhinitis. Recent research has been largely supportive of this model. New advances in research include the discovery of a novel cholinergic anti-inflammatory pathway activated by acupuncture. A chemokine-mediated proliferation of opioid-containing macrophages in inflamed tissues, in response to acupuncture, has also been demonstrated for the first time. Further research on the complex cross talk between receptors during inflammation is also helping to elucidate the mediators and signalling pathways activated by acupuncture. PMID:26339274

  4. Pharmacological characterization of prostanoid receptors mediating vasoconstriction in human umbilical vein

    PubMed Central

    Daray, Federico Manuel; Minvielle, Ana Itatí; Puppo, Soledad; Rothlin, Rodolfo Pedro

    2003-01-01

    This study was undertaken to characterize pharmacologically the prostanoid receptor subtypes mediating contraction in human umbilical vein (HUV).HUV rings were mounted in organ baths and concentration–response curves to U-46619 (TXA2 mimetic) were constructed in the absence or presence of SQ-29548 or ICI-192,605 (TP receptor antagonists). U-46619 was a potent constrictor (pEC50: 8.03). SQ-29548 and ICI-192,605 competitively antagonized responses to U-46619 with pKB values of 7.96 and 9.07, respectively.Concentration–response curves to EP receptor agonists: PGE2, misoprostol and 17-phenyl-trinor-PGE2 gave pEC50 values of 5.06, 5.25 and 5.32, respectively. Neither pEC50 nor maximum of PGE2 and 17-phenyl-trinor-PGE2 concentration–response curves were modified by the DP/EP1/EP2 receptor antagonist AH 6809 (1 μM). However, ICI-192,605 produced a concentration-dependent antagonism of the responses to all the EP receptor agonists. The pA2 estimated for ICI-192,605 against PGE2 or misoprostol were 8.91 and 9.22, respectively.Concentration–response curves to FP receptor agonists: PGF2α and fluprostenol gave pEC50 values of 6.20 and 5.82, respectively. ICI-192,605 (100 nM) was completely ineffective against PGF2α or fluprostenol. In addition, lack of antagonistic effect of AH 6809 (1 μM) against PGF2α was observed.In conclusion, the findings obtained with TP-selective agonist and antagonists provide strong evidence of the involvement of TP receptors promoting vasoconstriction in HUV. Furthermore, the action of the natural and synthetic EP receptor agonists appears to be mediated via TP receptors. On the other hand, the results employing FP receptor agonists and antagonists of different prostanoid receptors suggest the presence of FP receptors mediating vasoconstriction in this vessel. PMID:12922927

  5. Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

    PubMed

    Celik, Melih Ö; Labuz, Dominika; Henning, Karen; Busch-Dienstfertig, Melanie; Gaveriaux-Ruff, Claire; Kieffer, Brigitte L; Zimmer, Andreas; Machelska, Halina

    2016-10-01

    Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, β-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, μ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gβγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gβγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the G

  6. Pathogen-mediated inflammatory atherosclerosis is mediated in part via Toll-like receptor 2-induced inflammatory responses.

    PubMed

    Hayashi, Chie; Madrigal, Andres G; Liu, Xinyan; Ukai, Takashi; Goswami, Sulip; Gudino, Cynthia V; Gibson, Frank C; Genco, Caroline A

    2010-01-01

    Studies in humans have established that polymorphisms in genes encoding the innate immune Toll-like receptors (TLRs) are associated with inflammatory atherosclerosis. In hyperlipidemic mice, TLR2 and TLR4 have been reported to contribute to atherosclerosis progression. Human and mouse studies support a role for the oral pathogen Porphyromonas gingivalis in atherosclerosis, although the mechanisms by which this pathogen stimulates inflammatory atherosclerosis via innate immune system activation is not known. Using a genetically defined apolipoprotein E-deficient (ApoE(-/-)) mouse model we demonstrate that pathogen-mediated inflammatory atherosclerosis occurs via both TLR2-dependent and TLR2-independent mechanisms. P. gingivalis infection in mice possessing functional TLR2 induced the accumulation of macrophages as well as inflammatory mediators including CD40, IFN-gamma and the pro-inflammatory cytokines IL-1 beta, IL-6 and tumor necrosis factor-alpha in atherosclerotic lesions. The expression of these inflammatory mediators was reduced in atherosclerotic lesions from P. gingivalis-infected TLR2-deficient (TLR2(-/-)) mice. These studies provide a mechanistic link between an innate immune receptor and pathogen-accelerated atherosclerosis by a clinically and biologically relevant bacterial pathogen.

  7. Evidence for inhibition mediated by coassembly of GABAA and GABAC receptor subunits in native central neurons.

    PubMed

    Milligan, Carol J; Buckley, Noel J; Garret, Maurice; Deuchars, Jim; Deuchars, Susan A

    2004-08-18

    Fast inhibition in the nervous system is commonly mediated by GABA(A) receptors comprised of 2alpha/2beta/1gamma subunits. In contrast, GABA(C) receptors containing only rho subunits (rho1-rho3) have been predominantly detected in the retina. However, here using reverse transcription-PCR and in situ hybridization we show that mRNA encoding the rho1 subunit is highly expressed in brainstem neurons. Immunohistochemistry localized the rho1 subunit to neurons at light and electron microscopic levels, where it was detected at synaptic junctions. Application of the GABA(C) receptor agonist cis-4-aminocrotonic acid (100-800 microM) requires the rho1 subunit to elicit responses, which surprisingly are blocked independently by antagonists to GABA(A) (bicuculline, 10 microM) and GABA(C) [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA); 40-160 microM] receptors. Responses to GABA(C) agonists were also enhanced by the GABA(A) receptor modulator pentobarbitone (300 microM). Spontaneous and evoked IPSPs were reduced in amplitude but never abolished by TPMPA, but were completely blocked by bicuculline. We therefore tested the hypothesis that GABA(A) and GABA(C) subunits formed a heteromeric receptor. Immunohistochemistry indicated that rho1 and alpha1 subunits were colocalized at light and electron microscopic levels. Electrophysiology revealed that responses to GABA(C) receptor agonists were enhanced by the GABA(A) receptor modulator zolpidem (500 nm), which acts on the alpha1 subunit when the gamma2 subunit is also present. Finally, coimmunoprecipitation indicated that the rho1 subunit formed complexes that also containedalpha1 and gamma2 subunits. Taken together these separate lines of evidence suggest that the effects of GABA in central neurons can be mediated by heteromeric complexes of GABA(A) and GABA(C) receptor subunits.

  8. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression

    PubMed Central

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-IL; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y.L.; Choi, Hueng-Sik

    2017-01-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ -binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism. PMID:26348907

  9. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression.

    PubMed

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-Il; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y L; Choi, Hueng-Sik

    2015-09-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism.

  10. Hypersensitivity Induced by Activation of Spinal Cord PAR2 Receptors Is Partially Mediated by TRPV1 Receptors

    PubMed Central

    Mrozkova, Petra; Spicarova, Diana; Palecek, Jiri

    2016-01-01

    Protease-activated receptors 2 (PAR2) and transient receptor potential vanilloid 1 (TRPV1) receptors in the peripheral nerve endings are implicated in the development of increased sensitivity to mechanical and thermal stimuli, especially during inflammatory states. Both PAR2 and TRPV1 receptors are co-expressed in nociceptive dorsal root ganglion (DRG) neurons on their peripheral endings and also on presynaptic endings in the spinal cord dorsal horn. However, the modulation of nociceptive synaptic transmission in the superficial dorsal horn after activation of PAR2 and their functional coupling with TRPV1 is not clear. To investigate the role of spinal PAR2 activation on nociceptive modulation, intrathecal drug application was used in behavioural experiments and patch-clamp recordings of spontaneous, miniature and dorsal root stimulation-evoked excitatory postsynaptic currents (sEPSCs, mEPSCs, eEPSCs) were performed on superficial dorsal horn neurons in acute rat spinal cord slices. Intrathecal application of PAR2 activating peptide SLIGKV-NH2 induced thermal hyperalgesia, which was prevented by pretreatment with TRPV1 antagonist SB 366791 and was reduced by protein kinases inhibitor staurosporine. Patch-clamp experiments revealed robust decrease of mEPSC frequency (62.8 ± 4.9%), increase of sEPSC frequency (127.0 ± 5.9%) and eEPSC amplitude (126.9 ± 12.0%) in dorsal horn neurons after acute SLIGKV-NH2 application. All these EPSC changes, induced by PAR2 activation, were prevented by SB 366791 and staurosporine pretreatment. Our results demonstrate an important role of spinal PAR2 receptors in modulation of nociceptive transmission in the spinal cord dorsal horn at least partially mediated by activation of presynaptic TRPV1 receptors. The functional coupling between the PAR2 and TRPV1 receptors on the central branches of DRG neurons may be important especially during different pathological states when it may enhance pain perception. PMID:27755539

  11. Neurobiology of microglial action in CNS injuries: receptor-mediated signaling mechanisms and functional roles

    PubMed Central

    Hu, Xiaoming; Liou, Anthony K.F.; Leak, Rehana K.; Xu, Mingyue; An, Chengrui; Suenaga, Jun; Shi, Yejie; Gao, Yanqin; Zheng, Ping; Chen, Jun

    2014-01-01

    Microglia are the first line of immune defense against central nervous system (CNS) injuries and disorders. These highly plastic cells play dualistic roles in neuronal injury and recovery and are known for their ability to assume diverse phenotypes. A broad range of surface receptors are expressed on microglia and mediate microglial ‘On’ or ‘Off’ responses to signals from other host cells as well as invading microorganisms. The integrated actions of these receptors result in tightly regulated biological functions, including cell mobility, phagocytosis, the induction of acquired immunity, and trophic factor/inflammatory mediator release. Over the last few years, significant advances have been made towards deciphering the signaling mechanisms related to these receptors and their specific cellular functions. In this review, we describe the current state of knowledge of the surface receptors involved in microglial activation, with an emphasis on their engagement of distinct functional programs and their roles in CNS injuries. It will become evident from this review that microglial homeostasis is carefully maintained by multiple counterbalanced strategies, including, but not limited to, ‘On’ and ‘Off’ receptor signaling. Specific regulation of theses microglial receptors may be a promising therapeutic strategy against CNS injuries. PMID:24923657

  12. A slow excitatory postsynaptic current mediated by a novel metabotropic glutamate receptor in CA1 pyramidal neurons.

    PubMed

    Sheng, Nengyin; Yang, Jing; Silm, Katlin; Edwards, Robert H; Nicoll, Roger A

    2017-03-15

    Slow excitatory postsynaptic currents (EPSCs) mediated by metabotropic glutamate receptors (mGlu receptors) have been reported in several neuronal subtypes, but their presence in hippocampal pyramidal neurons remains elusive. Here we find that in CA1 pyramidal neurons a slow EPSC is induced by repetitive stimulation while ionotropic glutamate receptors and glutamate-uptake are blocked whereas it is absent in the VGLUT1 knockout mouse in which presynaptic glutamate is lost, suggesting the slow EPSC is mediated by glutamate activating mGlu receptors. However, it is not inhibited by known mGlu receptor antagonists. These findings suggest that this slow EPSC is mediated by a novel mGlu receptor, and that it may be involved in neurological diseases associated with abnormal high-concentration of extracellular glutamate. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

  13. Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

    PubMed

    Wu, Zhuang; Li, Linlang; Zheng, Long-Tai; Xu, Zhihong; Guo, Lin; Zhen, Xuechu

    2015-09-01

    Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS).

  14. Pre-synaptic adenosine A2A receptors control cannabinoid CB1 receptor-mediated inhibition of striatal glutamatergic neurotransmission.

    PubMed

    Martire, Alberto; Tebano, Maria Teresa; Chiodi, Valentina; Ferreira, Samira G; Cunha, Rodrigo A; Köfalvi, Attila; Popoli, Patrizia

    2011-01-01

    An interaction between adenosine A(2A) receptors (A(2A) Rs) and cannabinoid CB(1) receptors (CB(1) Rs) has been consistently reported to occur in the striatum, although the precise mechanisms are not completely understood. As both receptors control striatal glutamatergic transmission, we now probed the putative interaction between pre-synaptic CB(1) R and A(2A) R in the striatum. In extracellular field potentials recordings in corticostriatal slices from Wistar rats, A(2A) R activation by CGS21680 inhibited CB(1) R-mediated effects (depression of synaptic response and increase in paired-pulse facilitation). Moreover, in superfused rat striatal nerve terminals, A(2A) R activation prevented, while A(2A) R inhibition facilitated, the CB(1) R-mediated inhibition of 4-aminopyridine-evoked glutamate release. In summary, the present study provides converging neurochemical and electrophysiological support for the occurrence of a tight control of CB(1) R function by A(2A) Rs in glutamatergic terminals of the striatum. In view of the key role of glutamate to trigger the recruitment of striatal circuits, this pre-synaptic interaction between CB(1) R and A(2A) R may be of relevance for the pathogenesis and the treatment of neuropsychiatric disorders affecting the basal ganglia.

  15. GSG1L suppresses AMPA receptor-mediated synaptic transmission and uniquely modulates AMPA receptor kinetics in hippocampal neurons

    PubMed Central

    Gu, Xinglong; Mao, Xia; Lussier, Marc P.; Hutchison, Mary Anne; Zhou, Liang; Hamra, F. Kent; Roche, Katherine W.; Lu, Wei

    2016-01-01

    Regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is a key mechanism for synaptic plasticity. In the brain, AMPARs assemble with a number of auxiliary subunits, including TARPs, CNIHs and CKAMP44, which are important for AMPAR forward trafficking to synapses. Here we report that the membrane protein GSG1L negatively regulates AMPAR-mediated synaptic transmission. Overexpression of GSG1L strongly suppresses, and GSG1L knockout (KO) enhances, AMPAR-mediated synaptic transmission. GSG1L-dependent regulation of AMPAR synaptic transmission relies on the first extracellular loop domain and its carboxyl-terminus. GSG1L also speeds up AMPAR deactivation and desensitization in hippocampal CA1 neurons, in contrast to the effects of TARPs and CNIHs. Furthermore, GSG1L association with AMPARs inhibits CNIH2-induced slowing of the receptors in heterologous cells. Finally, GSG1L KO rats have deficits in LTP and show behavioural abnormalities in object recognition tests. These data demonstrate that GSG1L represents a new class of auxiliary subunit with distinct functional properties for AMPARs. PMID:26932439

  16. Muscarinic receptor binding and muscarinic receptor-mediated inhibition of adenylate cyclase in rat brain myelin

    SciTech Connect

    Larocca, J.N.; Ledeen, R.W.; Dvorkin, B.; Makman, M.H.

    1987-12-01

    High-affinity muscarinic cholinergic receptors were detected in myelin purified from rat brain stem with use of the radioligands /sup 3/H-N-methylscopolamine (/sup 3/H-NMS), /sup 3/H-quinuclidinyl benzilate (/sup 3/H-QNB), and /sup 3/H-pirenzepine. /sup 3/H-NMS binding was also present in myelin isolated from corpus callosum. In contrast, several other receptor types, including alpha 1- and alpha 2-adrenergic receptors, present in the starting brain stem, were not detected in myelin. Based on Bmax values from Scatchard analyses, /sup 3/H-pirenzepine, a putative M1 selective ligand, bound to about 25% of the sites in myelin labeled by /sup 3/H-NMS, a nonselective ligand that binds to both M1 and M2 receptor subtypes. Agonist affinity for /sup 3/H-NMS binding sites in myelin was markedly decreased by Gpp(NH)p, indicating that a major portion of these receptors may be linked to a second messenger system via a guanine-nucleotide regulatory protein. Purified myelin also contained adenylate cyclase activity; this activity was stimulated several fold by forskolin and to small but significant extents by prostaglandin E1 and the beta-adrenergic agonist isoproterenol. Myelin adenylate cyclase activity was inhibited by carbachol and other muscarinic agonists; this inhibition was blocked by the antagonist atropine. Levels in myelin of muscarinic receptors were 20-25% and those of forskolin-stimulated adenylate cyclase 10% of the values for total particulate fraction of whole brain stem. These levels in myelin are appreciably greater than would be predicted on the basis of contamination. Also, additional receptors and adenylate cyclase, added by mixing nonmyelin tissue with whole brain stem, were quantitatively removed during the purification procedure.

  17. D2-like dopamine receptors mediate the response to amphetamine in a mouse model of ADHD

    PubMed Central

    Fan, Xueliang; Hess, Ellen J.

    2007-01-01

    The mechanisms underlying the effects of psychostimulants in attention deficit hyperactivity disorder (ADHD) are not well understood, but indirect evidence implicates D2 dopamine receptors. Here we dissect the components of dopaminergic neurotransmission in the hyperactive mouse mutant coloboma to identify pre- and postsynaptic elements essential for the effects of amphetamine in these mice. Amphetamine treatment reduced locomotor activity in coloboma mice, but induced a robust increase in dopamine overflow suggesting that abnormal regulation of dopamine efflux does not account for the behavioral effect. However, the D2-like dopamine receptor antagonists haloperidol and raclopride, but not the D1-like dopamine receptor antagonist SCH23390, blocked the amphetamine-induced reduction in locomotor activity in coloboma mice, providing direct evidence that D2-like dopamine receptors mediate the effect of amphetamine in these mice. With the precedent established that it is possible to directly antagonize this response, this strategy should prove useful for identifying novel therapeutics in ADHD. PMID:17291774

  18. Oseltamivir blocks human neuronal nicotinic acetylcholine receptor-mediated currents.

    PubMed

    Muraki, Katsuhiko; Hatano, Noriyuki; Suzuki, Hiroka; Muraki, Yukiko; Iwajima, Yui; Maeda, Yasuhiro; Ono, Hideki

    2015-02-01

    The effects of oseltamivir, a neuraminidase inhibitor, were tested on the function of neuronal nicotinic acetylcholine receptors (nAChRs) in a neuroblastoma cell line IMR32 derived from human peripheral neurons and on recombinant human α3β4 nAChRs expressed in HEK cells. IMR32 cells predominately express α3β4 nAChRs. Nicotine (nic, 30 μm)-evoked currents recorded at -90 mV in IMR32 cells using the whole-cell patch clamp technique were reversibly blocked by oseltamivir in a concentration-dependent manner. In contrast, an active metabolite of oseltamivir, oseltamivir carboxylate (OC) at 30 μm had little effect on the nic-evoked currents. Oseltamivir also blocked nic-evoked currents derived from HEK cells with recombinant α3β4 nAChRs. This blockade was voltage-dependent with 10, 30 and 100 μm oseltamivir inhibiting ~50% at -100, -60 and -40 mV, respectively. Non-inactivating currents in IMR32 cells and in HEK cells with α3β4 nAChRs, which were evoked by an endogenous nicotinic agonist, ACh (5 μm), were reversibly blocked by oseltamivir. These data demonstrate that oseltamivir blocks nAChRs, presumably via binding to a site in the channel pore.

  19. The mannose receptor mediates dengue virus infection of macrophages.

    PubMed

    Miller, Joanna L; de Wet, Barend J M; deWet, Barend J M; Martinez-Pomares, Luisa; Radcliffe, Catherine M; Dwek, Raymond A; Rudd, Pauline M; Gordon, Siamon

    2008-02-08

    Macrophages (MØ) and mononuclear phagocytes are major targets of infection by dengue virus (DV), a mosquito-borne flavivirus that can cause haemorrhagic fever in humans. To our knowledge, we show for the first time that the MØ mannose receptor (MR) binds to all four serotypes of DV and specifically to the envelope glycoprotein. Glycan analysis, ELISA, and blot overlay assays demonstrate that MR binds via its carbohydrate recognition domains to mosquito and human cell-produced DV antigen. This binding is abrogated by deglycosylation of the DV envelope glycoprotein. Surface expression of recombinant MR on NIH3T3 cells confers DV binding. Furthermore, DV infection of primary human MØ can be blocked by anti-MR antibodies. MR is a prototypic marker of alternatively activated MØ, and pre-treatment of human monocytes or MØ with type 2 cytokines (IL-4 or IL-13) enhances their susceptibility to productive DV infection. Our findings indicate a new functional role for the MR in DV infection.

  20. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release*

    PubMed Central

    Woodier, Jason; Rainbow, Richard D.; Stewart, Alan J.; Pitt, Samantha J.

    2015-01-01

    Aberrant Zn2+ homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn2+ modulates cardiac ryanodine receptor gating and Ca2+ dynamics in isolated cardiomyocytes. We reveal that Zn2+ is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn2+ concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca2+. At concentrations of free Zn2+ >1 nm, Zn2+ is the main activating ligand, and the dependence on Ca2+ is removed. Zn2+ is therefore a higher affinity activator of RyR2 than Ca2+. Millimolar levels of free Zn2+ were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn2+ modulates both the frequency and amplitude of Ca2+ waves in a concentration-dependent manner and that physiological levels of Zn2+ elicit Ca2+ release in the absence of activating levels of cytosolic Ca2+. This highlights a new role for intracellular Zn2+ in shaping Ca2+ dynamics in cardiomyocytes through modulation of RyR2 gating. PMID:26041778

  1. Intracellular Zinc Modulates Cardiac Ryanodine Receptor-mediated Calcium Release.

    PubMed

    Woodier, Jason; Rainbow, Richard D; Stewart, Alan J; Pitt, Samantha J

    2015-07-10

    Aberrant Zn(2+) homeostasis is a hallmark of certain cardiomyopathies associated with altered contractile force. In this study, we addressed whether Zn(2+) modulates cardiac ryanodine receptor gating and Ca(2+) dynamics in isolated cardiomyocytes. We reveal that Zn(2+) is a high affinity regulator of RyR2 displaying three modes of operation. Picomolar free Zn(2+) concentrations potentiate RyR2 responses, but channel activation is still dependent on the presence of cytosolic Ca(2+). At concentrations of free Zn(2+) >1 nm, Zn(2+) is the main activating ligand, and the dependence on Ca(2+) is removed. Zn(2+) is therefore a higher affinity activator of RyR2 than Ca(2+). Millimolar levels of free Zn(2+) were found to inhibit channel openings. In cardiomyocytes, consistent with our single channel results, we show that Zn(2+) modulates both the frequency and amplitude of Ca(2+) waves in a concentration-dependent manner and that physiological levels of Zn(2+) elicit Ca(2+) release in the absence of activating levels of cytosolic Ca(2+). This highlights a new role for intracellular Zn(2+) in shaping Ca(2+) dynamics in cardiomyocytes through modulation of RyR2 gating.

  2. Ryanodine receptor-mediated arrhythmias and sudden cardiac death

    PubMed Central

    Blayney, Lynda M.; Lai, F. Anthony

    2009-01-01

    The cardiac ryanodine receptor-Ca2+ release channel (RyR2) is an essential sarcoplasmic reticulum (SR) transmembrane protein that plays a central role in excitation–contraction coupling (ECC) in cardiomyocytes. Aberrant spontaneous, diastolic Ca2+ leak from the SR due to dysfunctional RyR2 contributes to the formation of delayed after-depolarisations, which are thought to underlie the fatal arrhythmia that occurs in both heart failure (HF) and in catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is an inherited disorder associated with mutations in either the RyR2 or a SR luminal protein, calsequestrin. RyR2 shows normal function at rest in CPVT but the RyR2 dysfunction is unmasked by physical exercise or emotional stress, suggesting abnormal RyR2 activation as an underlying mechanism. Several potential mechanisms have been advanced to explain the dysfunctional RyR2 observed in HF and CPVT, including enhanced RyR2 phosphorylation status, altered RyR2 regulation at luminal/cytoplasmic sites and perturbed RyR2 intra/inter-molecular interactions. This review considers RyR2 dysfunction in the context of the structural and functional modulation of the channel, and potential therapeutic strategies to stabilise RyR2 function in cardiac pathology. PMID:19345240

  3. EP2 receptors mediate airway relaxation to substance P, ATP, and PGE2.

    PubMed

    Fortner, C N; Breyer, R M; Paul, R J

    2001-08-01

    Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2 from airway epithelium in other species, suggesting PGE2 as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2 receptor [EP2(-/-)], one of the PGE2 receptors, we tested the hypothesis that PGE2 is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(-/-) mice. There were no differences between EP2(-/-) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(-/-) and wild-type tracheas in basal or stimulated PGE2 production. Exogenous PGE2 produced significantly less relaxation in EP2(-/-) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2 receptors are of primary importance in airway relaxation to PGE2 and relaxation to SP or ATP is mediated through PGE2 acting on EP2 receptors.

  4. Sphingosine 1-phosphate receptors are essential mediators of eyelid closure during embryonic development.

    PubMed

    Herr, Deron R; Lee, Chang-Wook; Wang, Wei; Ware, Adam; Rivera, Richard; Chun, Jerold

    2013-10-11

    The fetal development of the mammalian eyelid involves the expansion of the epithelium over the developing cornea, fusion into a continuous sheet covering the eye, and a splitting event several weeks later that results in the formation of the upper and lower eyelids. Recent studies have revealed a significant number of molecular signaling components that are essential mediators of eyelid development. Receptor-mediated sphingosine 1-phosphate (S1P) signaling is known to influence diverse biological processes, but its involvement in eyelid development has not been reported. Here, we show that two S1P receptors, S1P2 and S1P3, are collectively essential mediators of eyelid closure during murine development. Homozygous deletion of the gene encoding either receptor has no apparent effect on eyelid development, but double-null embryos are born with an "eyes open at birth" defect due to a delay in epithelial sheet extension. Both receptors are expressed in the advancing epithelial sheet during the critical period of extension. Fibroblasts derived from double-null embryos have a deficient response to epidermal growth factor, suggesting that S1P2 and S1P3 modulate this essential signaling pathway during eyelid closure.

  5. syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

    PubMed Central

    Bonnerot, C; Briken, V; Brachet, V; Lankar, D; Cassard, S; Jabri, B; Amigorena, S

    1998-01-01

    B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes. PMID:9707420

  6. The Wedelolactone Derivative Inhibits Estrogen Receptor-Mediated Breast, Endometrial, and Ovarian Cancer Cells Growth

    PubMed Central

    Xu, Defeng; Lin, Tzu-Hua; Cheng, Max A.; Chen, Lu-Min; Chang, Chawnshang; Yeh, Shuyuan

    2014-01-01

    Estrogen and estrogen receptor (ER)-mediated signaling pathways play important roles in the etiology and progression of human breast, endometrial, and ovarian cancers. Attenuating ER activities by natural products and their derivatives is a relatively practical strategy to control and reduce breast, endometrial, and ovarian cancer risk. Here, we found 3-butoxy-1,8,9-trihydroxy-6H-benzofuro[3,2-c]benzopyran-6-one (BTB), a new derivative of wedelolactone, could effectively inhibit the 17-estradiol (E2)-induced ER transactivation and suppress the growth of breast cancer as well as endometrial and ovarian cancer cells. Our results indicate that 2.5 μM BTB effectively suppresses ER-positive, but not ER-negative, breast, endometrial, and ovarian cancer cells. Furthermore, our data indicate that BTB can modulate ER transactivation and suppress the expression of E2-mediated ER target genes (Cyclin D1, E2F1, and TERT) in the ER-positive MCF-7, Ishikawa, and SKOV-3 cells. Importantly, this BTB mediated inhibition of ER activity is selective since BTB does not suppress the activities of other nuclear receptors, including glucocorticoid receptor and progesterone receptor, suggesting that BTB functions as a selective ER signaling inhibitor with the potential to treat breast, endometrial, and ovarian cancers. PMID:25221777

  7. Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated calcium release.

    PubMed

    Takeda, Atsushi; Fuke, Sayuri; Minami, Akira; Oku, Naoto

    2007-05-01

    The uptake of free zinc into CA3 pyramidal cells and its significance was examined in rat hippocampal slices with ZnAF-2DA, a membrane-permeable zinc indicator. Intracellular ZnAF-2 signal in the CA3 pyramidal cell layer was increased during delivery of tetanic stimuli to the dentate granule cell layer. This increase was completely blocked in the presence of CNQX, an AMPA/kainate receptor antagonist. These results suggest that free zinc is taken up into CA3 pyramidal cells via activation of AMPA/kainate receptors. The effect of free zinc levels in the CA3 pyramidal cells on the increase in intracellular calcium via Group I metabotropic glutamate receptors was examined by regional delivery of tADA, a Group I metabotropic glutamate receptor agonist, to the stratum lucidum after blockade of AMPA/kainate receptor-mediated calcium and zinc influx. Intracellular calcium orange signal in the CA3 pyramidal cell layer was increased by tADA, whereas intracellular ZnAF-2 signal was not increased even in the presence of 100 muM zinc, suggesting that tADA induces calcium release from internal stores in CA3 pyramidal cells and is not involved in zinc uptake. The increase in calcium orange signal by tADA was enhanced by perfusion with pyrithione, a zinc ionophore that decreased basal ZnAF-2 signal in the CA3 pyramidal cell layer. It was blocked by perfusion with pyrithione and zinc that increased basal ZnAF-2 signal. The present study indicates that the increase in free calcium levels via the metabotropic glutamate receptor pathway is inversely related to free zinc levels in CA3 pyramidal cells.

  8. Analgesic effect of dimethyl trisulfide in mice is mediated by TRPA1 and sst4 receptors.

    PubMed

    Pozsgai, Gábor; Payrits, Maja; Sághy, Éva; Sebestyén-Bátai, Réka; Steen, Elise; Szőke, Éva; Sándor, Zoltán; Solymár, Margit; Garami, András; Orvos, Péter; Tálosi, László; Helyes, Zsuzsanna; Pintér, Erika

    2017-05-01

    TRPA1 receptors are calcium-permeable ligand-gated channels expressed in primary sensory neurons and involved in inflammation and pain. Activation of these neurons might have analgesic effect. Suggested mechanism of analgesic effect mediated by TRPA1 activation is the release of somatostatin (SOM) and its action on sst4 receptors. In the present study analgesic effect of TRPA1 activation on primary sensory neurons by organic trisulfide compound dimethyl trisulfide (DMTS) presumably leading to SOM release was investigated. Opening of TRPA1 by DMTS in CHO cells was examined by patch-clamp and fluorescent Ca(2+) detection. Ca(2+) influx upon DMTS administration in trigeminal ganglion (TRG) neurons of TRPA1 receptor wild-type (WT) and knockout (KO) mice was detected by ratiometric Ca(2+) imaging. SOM release from sensory nerves of murine skin was assessed by radioimmunoassay. Analgesic effect of DMTS in mild heat injury-induced mechanical hyperalgesia was examined by dynamic plantar aesthesiometry. Regulatory role of DMTS on deep body temperature (Tb) was measured by thermocouple thermometry with respirometry and by telemetric thermometry. DMTS produced TRPA1-mediated currents and elevated [Ca(2+)]i in CHO cells. Similar data were obtained in TRG neurons. DMTS released SOM from murine sensory neurons TRPA1-dependently. DMTS exerted analgesic effect mediated by TRPA1 and sst4 receptors. DMTS-evoked hypothermia and hypokinesis were attenuated in freely-moving TRPA1 KO animals. Our study has presented original evidence regarding analgesic action of DMTS which might be due to TRPA1-mediated SOM release from sensory neurons and activation of sst4 receptors. DMTS could be a novel analgesic drug candidate.

  9. NOP Receptor Mediates Anti-analgesia Induced by Agonist-Antagonist Opioids

    PubMed Central

    Gear, Robert W.; Bogen, Oliver; Ferrari, Luiz F.; Green, Paul G.; Levine, Jon D.

    2014-01-01

    Clinical studies have shown that agonist-antagonist opioid analgesics that produce their analgesic effect via action on the kappa-opioid receptor, produce a delayed-onset anti-analgesia in men but not women, an effect blocked by co-administration of a low dose of naloxone. We now report the same time-dependent anti-analgesia and its underlying mechanism in an animal model. Using the Randall-Selitto paw-withdrawal assay in male rats, we found that nalbuphine, pentazocine, and butorphanol each produced analgesia during the first hour followed by anti-analgesia starting at ~90 minutes after administration in males but not females, closely mimicking its clinical effects. As observed in humans, co-administration of nalbuphine with naloxone in a dose ratio of 12.5:1 blocked anti-analgesia but not analgesia. Administration of the highly selective kappa-opioid receptor agonist U69,593 produced analgesia without subsequent anti-analgesia, and confirmed by the failure of the selective kappa antagonist nor-binaltorphimine to block nalbuphine-induced anti-analgesia, indicating that anti-analgesia is not mediated by kappa-opioid receptors. We therefore tested the role of other receptors in nalbuphine anti-analgesia. Nociceptin/orphanin FQ (NOP) and sigma-1 and sigma-2 receptors were chosen on the basis of their known anti-analgesic effects and receptor binding studies. The selective NOP receptor antagonists, JTC801, and J113397, but not the sigma receptor antagonist, BD 1047, antagonized nalbuphine anti-analgesia. Furthermore, the NOP receptor agonist NNC 63-0532 produced anti-analgesia with the same delay in onset observed with the three agonist-antagonists, but without producing preceding analgesia and this anti-analgesia was also blocked by naloxone. These results strongly support the suggestion that clinically used agonist-antagonists act at the NOP receptor to produce anti-analgesia. PMID:24188792

  10. Only high-affinity receptors for interleukin 2 mediate internalization of ligand

    SciTech Connect

    Weissman, A.M.; Harford, J.B.; Svetlik, P.B.; Leonard, W.L.; Depper, J.M.; Waldmann, T.A.; Greene, W.C.; Klausner, R.D.

    1986-03-01

    Interleukin 2 (IL-2) receptors are expressed on activated T cells and in select T-cell leukemias. Recently, it has been demonstrated that at least two classes of receptor for IL-2 exist with markedly different affinities for ligand. All known biological actions of IL-2 have been correlated with occupancy of high-affinity sites; the function of the low-affinity sites remains unknown. Receptor-mediated endocytosis is the primary means of internalization of cell-surface receptors and their ligands. The internalization of IL-2 bound to high- and low-affinity receptor sites was studied in a human T-cell lymphotrophic virus type 1 (HTLV-1)-infected human T-cell leukemia cell line and in a cloned murine cytotoxic T-cell line (CTLL). Internalization of IL-2 occurred only when bound to high-affinity sites. In addition, an anti-receptor antibody (anti-Tac), which binds equally well to high- and low-affinity sites, demonstrated no detectable internalization. The implications of these findings as they relate to IL-2 receptor structure and function are discussed.

  11. Norepinephrine-mediated regulation of 5HT1 receptor functioning in human platelets.

    PubMed

    Trincavelli, M L; Cuboni, S; Montali, M; Santaguida, S; Lucacchini, A; Martini, C

    2008-07-01

    Adaptive changes in serotonergic 5HT1 receptor signalling are believed to underlie the therapeutic effectiveness of antidepressant drugs. Since cells are continuously exposed to neurotransmitters/neuromodulators, spatially and temporally integrated, the responsiveness of a receptor system is dependent upon the physio-pathological state of the cell and the interaction between different neurotransmitters. In the present work, we investigated heterologous regulation of 5HT1 receptors induced by norepinephrine (NE) in human platelets. NE platelet treatment induced a time and concentration dependent 5HT1 receptor desensitisation mediated by both alpha and beta receptors through activation of intracellular protein kinases. In particular NE, through PKC activation, regulated 5HT1 receptor phosphorylation on threonine residues, causing in turn serotonin receptor-G protein uncoupling and functional responsiveness drop. These results suggest that high NE levels (released i.e. during stress disorders) may play an important role in regulating the 5HT1 responsiveness and in controlling effectiveness of drugs acting on these neurotransmitter systems.

  12. Sex mediates dopamine and adrenergic receptor expression in adult rats exposed prenatally to cocaine

    PubMed Central

    Ferris, Mark J.; Mactutus, Charles F.; Silvers, Janelle M.; Hasselrot, Ulla; Strupp, Barbara J.; Booze, Rosemarie M.

    2010-01-01

    The extent of catecholaminergic receptor and respective behavioral alterations associated with prenatal cocaine exposure varies according to exogenous factors such as the amount, frequency, and route of maternal exposure, as well as endogenous factors such as specific brain regions under consideration and sex of the species. The goal of the current study was to use autoradiography to delineate possible moderators of dopaminergic and adrenergic receptor expression in adult rat offspring exposed to cocaine in utero. The current study demonstrated sex-dependent D1 receptor, α2, and noradrenergic transporter binding alterations in prelimbic, hippocampus, and anterior cingulate regions of adult rat brains exposed to cocaine during gestational days 8–21. Of further interest was the lack of alterations in the nucleus accumbens for nearly all receptors/transporters investigated, as well as the lack of alterations in D3 receptor binding in nearly all of the regions investigated (nucleus accumbens, prelimbic region, hippocampus, and cingulate gyrus). Thus, the current investigation demonstrated persistent receptor and transporter alterations that extend well into adulthood as a result of cocaine exposure in utero. Furthermore, the demonstration that sex played a mediating role in prenatal cocaine-induced, aberrant receptor/transporter expression is of primary importance for future studies that seek to control for sex in either design or analysis. PMID:17933484

  13. MICAL-like1 mediates epidermal growth factor receptor endocytosis

    PubMed Central

    Abou-Zeid, Nancy; Pandjaitan, Rudy; Sengmanivong, Lucie; David, Violaine; Le Pavec, Gwenaelle; Salamero, Jean; Zahraoui, Ahmed

    2011-01-01

    Small GTPase Rabs are required for membrane protein sorting/delivery to precise membrane domains. Rab13 regulates epithelial tight junction assembly and polarized membrane transport. Here we report that Molecule Interacting with CasL (MICAL)-like1 (MICAL-L1) interacts with GTP-Rab13 and shares a similar domain organization with MICAL. MICAL-L1 has a calponin homology (CH), LIM, proline rich and coiled-coil domains. It is associated with late endosomes. Time-lapse video microscopy shows that green fluorescent protein–Rab7 and mcherry-MICAL-L1 are present within vesicles that move rapidly in the cytoplasm. Depletion of MICAL-L1 by short hairpin RNA does not alter the distribution of a late endosome/lysosome-associated protein but affects the trafficking of epidermal growth factor receptor (EGFR). Overexpression of MICAL-L1 leads to the accumulation of EGFR in the late endosomal compartment. In contrast, knocking down MICAL-L1 results in the distribution of internalized EGFR in vesicles spread throughout the cytoplasm and promotes its degradation. Our data suggest that the N-terminal CH domain associates with the C-terminal Rab13 binding domain (RBD) of MICAL-L1. The binding of Rab13 to RBD disrupts the CH/RBD interaction, and may induce a conformational change in MICAL-L1, promoting its activation. Our results provide novel insights into the MICAL-L1/Rab protein complex that can regulate EGFR trafficking at late endocytic pathways. PMID:21795389

  14. Dual role of dopamine D(2)-like receptors in the mediation of conditioned and unconditioned fear.

    PubMed

    Brandão, Marcus Lira; de Oliveira, Amanda Ribeiro; Muthuraju, Sangu; Colombo, Ana Caroline; Saito, Viviane Mitsuko; Talbot, Teddy

    2015-11-14

    A reduction of dopamine release or D2 receptor blockade in the terminal fields of the mesolimbic system, particularly the amygdala, clearly reduces conditioned fear. Similar D2 receptor antagonism in the neural substrates of fear in the midbrain tectum attenuates the processing of unconditioned aversive information. However, the implications of the interplay between opposing actions of dopamine in the rostral and caudal segments of the dopaminergic system are still unclear. Previous studies from this laboratory have reported the effects of dopaminergic drugs on behavior in rats in the elevated plus maze, auditory-evoked potentials (AEPs) recorded from the midbrain tectum, fear-potentiated startle, and conditioned freezing. These findings led to an interesting framework on the functional roles of dopamine in both anxiety and fear states. Dopamine D2 receptor inhibition in the terminal fields of the mesolimbic dopamine system generally causes anxiolytic-like effects, whereas the activity of midbrain substrates of unconditioned fear are enhanced by D2 receptor antagonists, suggesting that D2 receptor-mediated mechanisms play opposing roles in fear/anxiety processes, depending on the brain region under study. Dopamine appears to mediate conditioned fear by acting at rostral levels of the brain and regulate unconditioned fear at the midbrain level, likely by reducing the sensorimotor gating of aversive events.

  15. 5-HT2 receptors mediate functional modulation of GABAa receptors and inhibitory synaptic transmissions in human iPS-derived neurons

    PubMed Central

    Wang, Haitao; Hu, Lingli; Liu, Chunhua; Su, Zhenghui; Wang, Lihui; Pan, Guangjin; Guo, Yiping; He, Jufang

    2016-01-01

    Neural progenitors differentiated from induced pluripotent stem cells (iPS) hold potentials for treating neurological diseases. Serotonin has potent effects on neuronal functions through multiple receptors, underlying a variety of neural disorders. Glutamate and GABA receptors have been proven functional in neurons differentiated from iPS, however, little is known about 5-HT receptor-mediated modulation in such neuronal networks. In the present study, human iPS were differentiated into cells possessing featured physiological properties of cortical neurons. Whole-cell patch-clamp recording was used to examine the involvement of 5-HT2 receptors in functional modulation of GABAergic synaptic transmission. We found that serotonin and DOI (a selective agonist of 5-HT2A/C receptor) reversibly reduced GABA-activated currents, and this 5-HT2A/C receptor mediated inhibition required G protein, PLC, PKC, and Ca2+ signaling. Serotonin increased the frequency of miniature inhibitory postsynaptic currents (mIPSCs), which could be mimicked by α-methylserotonin, a 5-HT2 receptor agonist. In contrast, DOI reduced both frequency and amplitude of mIPSCs. These findings suggested that in iPS-derived human neurons serotonin postsynaptically reduced GABAa receptor function through 5-HT2A/C receptors, but presynaptically other 5-HT2 receptors counteracted the action of 5-HT2A/C receptors. Functional expression of serotonin receptors in human iPS-derived neurons provides a pre-requisite for their normal behaviors after grafting. PMID:26837719

  16. Investigations of receptor-mediated phagocytosis by hormone-induced (imprinted) Tetrahymena pyriformis.

    PubMed

    Kovács, P; Sundermann, C A; Csaba, G

    1996-08-15

    Receptor-mediated endocytosis by Tetrahvmena pyriformis was studied using tetramethylrhodamine isothiocyanate-labeled concanavalin A (TRITC-Con A) with fluorescence and confocal microscopy. In the presence of insulin, or 24 h after insulin pretreatment (hormonal imprinting), the binding and uptake of TRITC-Con A increased when compared to controls, owing to the binding of TRITC-Con A to sugar oligomers of insulin receptors. Mannose inhibited the binding of Con A, thus demonstrating the specificity of binding. Histamine, a phagocytosis-promoting factor in mammals and Tetrahymena, and galactose, did not influence the uptake of TRITC-Con A.

  17. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  18. Scavenger Receptor C-Type Lectin Binds to the Leukocyte Cell Surface Glycan Lewis By a Novel Mechanism

    SciTech Connect

    Feinberg, H.; Taylor, M.E.; Weis, W.I.; /Stanford U., Med. School /Imperial Coll., London

    2007-07-10

    The scavenger receptor C-type lectin (SRCL) is unique in the family of class A scavenger receptors, because in addition to binding sites for oxidized lipoproteins it also contains a C-type carbohydrate-recognition domain (CRD) that interacts with specific glycans. Both human and mouse SRCL are highly specific for the Lewis(x) trisaccharide, which is commonly found on the surfaces of leukocytes and some tumor cells. Structural analysis of the CRD of mouse SRCL in complex with Lewis(x) and mutagenesis show the basis for this specificity. The interaction between mouse SRCL and Lewis(x) is analogous to the way that selectins and DC-SIGN bind to related fucosylated glycans, but the mechanism of the interaction is novel, because it is based on a primary galactose-binding site similar to the binding site in the asialoglycoprotein receptor. Crystals of the human receptor lacking bound calcium ions reveal an alternative conformation in which a glycan ligand would be released during receptor-mediated endocytosis.

  19. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies.

    PubMed

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-07-14

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects.

  20. Roles of OA1 octopamine receptor and Dop1 dopamine receptor in mediating appetitive and aversive reinforcement revealed by RNAi studies

    PubMed Central

    Awata, Hiroko; Wakuda, Ryo; Ishimaru, Yoshiyasu; Matsuoka, Yuji; Terao, Kanta; Katata, Satomi; Matsumoto, Yukihisa; Hamanaka, Yoshitaka; Noji, Sumihare; Mito, Taro; Mizunami, Makoto

    2016-01-01

    Revealing reinforcing mechanisms in associative learning is important for elucidation of brain mechanisms of behavior. In mammals, dopamine neurons are thought to mediate both appetitive and aversive reinforcement signals. Studies using transgenic fruit-flies suggested that dopamine neurons mediate both appetitive and aversive reinforcements, through the Dop1 dopamine receptor, but our studies using octopamine and dopamine receptor antagonists and using Dop1 knockout crickets suggested that octopamine neurons mediate appetitive reinforcement and dopamine neurons mediate aversive reinforcement in associative learning in crickets. To fully resolve this issue, we examined the effects of silencing of expression of genes that code the OA1 octopamine receptor and Dop1 and Dop2 dopamine receptors by RNAi in crickets. OA1-silenced crickets exhibited impairment in appetitive learning with water but not in aversive learning with sodium chloride solution, while Dop1-silenced crickets exhibited impairment in aversive learning but not in appetitive learning. Dop2-silenced crickets showed normal scores in both appetitive learning and aversive learning. The results indicate that octopamine neurons mediate appetitive reinforcement via OA1 and that dopamine neurons mediate aversive reinforcement via Dop1 in crickets, providing decisive evidence that neurotransmitters and receptors that mediate appetitive reinforcement indeed differ among different species of insects. PMID:27412401

  1. Receptor-mediated control of regulatory volume decrease (RVD) and apoptotic volume decrease (AVD)

    PubMed Central

    Okada, Yasunobu; Maeno, Emi; Shimizu, Takahiro; Dezaki, Katsuya; Wang, Jun; Morishima, Shigeru

    2001-01-01

    A fundamental property of animal cells is the ability to regulate their own cell volume. Even under hypotonic stress imposed by either decreased extracellular or increased intracellular osmolarity, the cells can re-adjust their volume after transient osmotic swelling by a mechanism known as regulatory volume decrease (RVD). In most cell types, RVD is accomplished mainly by KCl efflux induced by parallel activation of K+ and Cl− channels. We have studied the molecular mechanism of RVD in a human epithelial cell line (Intestine 407). Osmotic swelling results in a significant increase in the cytosolic Ca2+ concentration and thereby activates intermediate-conductance Ca2+-dependent K+ (IK) channels. Osmotic swelling also induces ATP release from the cells to the extracellular compartment. Released ATP stimulates purinergic ATP (P2Y2) receptors, thereby inducing phospholipase C-mediated Ca2+ mobilization. Thus, RVD is facilitated by stimulation of P2Y2 receptors due to augmentation of IK channels. In contrast, stimulation of another G protein-coupled Ca2+-sensing receptor (CaR) enhances the activity of volume-sensitive outwardly rectifying Cl− channels, thereby facilitating RVD. Therefore, it is possible that Ca2+ efflux stimulated by swelling-induced and P2Y2 receptor-mediated intracellular Ca2+ mobilization activates the CaR, thereby secondarily upregulating the volume-regulatory Cl− conductance. On the other hand, the initial process towards apoptotic cell death is coupled to normotonic cell shrinkage, called apoptotic volume decrease (AVD). Stimulation of death receptors, such as TNFα receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma × rat glioma hybrid (NG108-15) and rat phaeochromocytoma (PC12) cells. In those cells exhibiting AVD, facilitation of RVD is always observed. Both AVD induction and RVD facilitation as well as succeeding apoptotic events can be

  2. Activation of the repulsive receptor Roundabout inhibits N-cadherin-mediated cell adhesion.

    PubMed

    Rhee, Jinseol; Mahfooz, Najmus S; Arregui, Carlos; Lilien, Jack; Balsamo, Janne; VanBerkum, Mark F A

    2002-10-01

    The formation of axon trajectories requires integration of local adhesive interactions with directional information from attractive and repulsive cues. Here, we show that these two types of information are functionally integrated; activation of the transmembrane receptor Roundabout (Robo) by its ligand, the secreted repulsive guidance cue Slit, inactivates N-cadherin-mediated adhesion. Loss of N-cadherin-mediated adhesion is accompanied by tyrosine phosphorylation of beta-catenin and its loss from the N-cadherin complex, concomitant with the formation of a supramolecular complex containing Robo, Abelson (Abl) kinase and N-cadherin. Local formation of such a receptor complex is an ideal mechanism to steer the growth cone while still allowing adhesion and growth in other directions.

  3. Bradykinin as a pain mediator: receptors are localized to sensory neurons, and antagonists have analgesic actions

    SciTech Connect

    Steranka, L.R.; Manning, D.C.; DeHaas, C.J.; Ferkany, J.W.; Borosky, S.A.; Connor, J.R.; Vavrek, R.J.; Stewart, J.M.; Snyder, S.H.

    1988-05-01

    Autoradiographic studies localize (/sup 3/H)bradykinin receptor binding sites to the substantia gelatinosa, dorsal root, and a subset of small cells in both the dorsal root and trigeminal ganglia of the guinea pig. (/sup 3/H)Bradykinin labeling is also observed over myocardinal/coronary visceral afferent fibers. The localization of (/sup 3/H)bradykinin receptors to nociceptive pathways supports a role for bradykinin in pain mediation. Several bradkykinin antagonists block bradykinin-induced acute vascular pain in the rat. The bradykinin antagonists also relieve bradykinin- and urate-induced hyperalgesia in the rat paw. These results indicate that bradykinin is a physiologic mediator of pain and that bradykinin antagonists have analgesic activity in both acute and chronic pain models.

  4. H2 receptor-mediated facilitation and H3 receptor-mediated inhibition of noradrenaline release in the guinea-pig brain.

    PubMed

    Timm, J; Marr, I; Werthwein, S; Elz, S; Schunack, W; Schlicker, E

    1998-03-01

    , hippocampal or hypothalamic slices were used instead of cortical slices. The Ca2+-induced tritium overflow in guinea-pig cortex slices was inhibited by histamine (in the presence of ranitidine); this effect was abolished by clobenpropit. In slices superfused in the presence of clobenpropit, impromidine failed to facilitate the Ca2+-evoked tritium overflow. The electrically evoked tritium overflow in mouse brain cortex slices was inhibited by histamine by about 60% (both in the absence or presence of ranitidine). The inhibitory effect of histamine was abolished (but not reversed) by clobenpropit. In conclusion, noradrenaline release in the guinea-pig brain cortex is inhibited via presynaptic H3 receptors and facilitated via H2 receptors not located presynaptically. In the mouse brain cortex, only inhibitory H3 receptors occur. The extent of the H3 receptor-mediated effect is more marked in the mouse than in the guinea-pig brain cortex.

  5. Chloride transporters and receptor-mediated endocytosis in the renal proximal tubule

    PubMed Central

    Devuyst, Olivier; Luciani, Alessandro

    2015-01-01

    Abstract The epithelial cells lining the proximal tubules of the kidney reabsorb a large amount of filtered ions and solutes owing to receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Dedifferentiation of proximal tubule cells and dysfunction of receptor-mediated endocytosis characterize Dent’s disease, a rare disorder caused by inactivating mutations in the CLCN5 gene that encodes the endosomal chloride–proton exchanger, ClC-5. The disease is characterized by a massive urinary loss of solutes (renal Fanconi syndrome), with severe metabolic complications and progressive renal failure. Investigations of mutations affecting the gating of ClC-5 revealed that the proximal tubule dysfunction may occur despite normal endosomal acidification. In addition to defective endocytosis, proximal tubule cells lacking ClC-5 show a trafficking defect in apical receptors and transporters, as well as lysosomal dysfunction and typical features of dedifferentiation, proliferation and oxidative stress. A similar but milder defect is observed in mouse models with defective CFTR, a chloride channel that is also expressed in the endosomes of proximal tubule cells. These data suggest a major role for endosomal chloride transport in the maintenance of epithelial differentiation and reabsorption capacity of the renal proximal tubule. Key points The reabsorptive activity of renal proximal tubule cells is mediated by receptor-mediated endocytosis and polarized transport systems that reflect final cell differentiation. Loss-of-function mutations of the endosomal chloride–proton exchanger ClC-5 (Dent’s disease) cause a major trafficking defect in proximal tubule cells, associated with lysosomal dysfunction, oxidative stress and dedifferentiation/proliferation. A similar but milder defect is associated with mutations in CFTR (cystic fibrosis transmembrane conductance regulator). Vesicular chloride transport appears to be important for

  6. Metabotropic glutamate receptor-mediated signaling dampens the HPA axis response to restraint stress.

    PubMed

    Evanson, Nathan K; Herman, James P

    2015-10-15

    Glutamate is an important neurotransmitter in the regulation of the neural portion of hypothalamus-pituitary-adrenal (HPA) axis activity, and signals through ionotropic and metabotropic receptors. In the current studies we investigated the role of hypothalamic paraventricular group I metabotropic glutamate receptors in the regulation of the HPA axis response to restraint stress in rats. Direct injection of the group I metabotropic glutamate receptor agonist 3,5-dihydroxyphenylglycine (DHPG) into the PVN prior to restraint leads to blunting of the HPA axis response in awake animals. Consistent with this result, infusion of the group I receptor antagonist hexyl-homoibotenic acid (HIBO) potentiates the HPA axis response to restraint. The excitatory effect of blocking paraventricular group I metabotropic glutamate signaling is blocked by co-administration of dexamethasone into the PVN. However, the inhibitory effect of DHPG is not affected by co-administration of the cannabinoid CB1 receptor antagonist AM-251 into the PVN. Together, these results suggest that paraventricular group I metabotropic glutamate receptor signaling acts to dampen HPA axis reactivity. This effect appears to be similar to the rapid inhibitory effect of glucocorticoids at the PVN, but is not mediated by endocannabinoid signaling.

  7. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.

    PubMed Central

    Walczak, H; Degli-Esposti, M A; Johnson, R S; Smolak, P J; Waugh, J Y; Boiani, N; Timour, M S; Gerhart, M J; Schooley, K A; Smith, C A; Goodwin, R G; Rauch, C T

    1997-01-01

    TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines and induces apoptosis in a wide variety of cells. Based on homology searching of a private database, a receptor for TRAIL (DR4 or TRAIL-R1) was recently identified. Here we report the identification of a distinct receptor for TRAIL, TRAIL-R2, by ligand-based affinity purification and subsequent molecular cloning. TRAIL-R2 was purified independently as the only receptor for TRAIL detectable on the surface of two different human cell lines that undergo apoptosis upon stimulation with TRAIL. TRAIL-R2 contains two extracellular cysteine-rich repeats, typical for TNF receptor (TNFR) family members, and a cytoplasmic death domain. TRAIL binds to recombinant cell-surface-expressed TRAIL-R2, and TRAIL-induced apoptosis is inhibited by a TRAIL-R2-Fc fusion protein. TRAIL-R2 mRNA is widely expressed and the gene encoding TRAIL-R2 is located on human chromosome 8p22-21. Like TRAIL-R1, TRAIL-R2 engages a caspase-dependent apoptotic pathway but, in contrast to TRAIL-R1, TRAIL-R2 mediates apoptosis via the intracellular adaptor molecule FADD/MORT1. The existence of two distinct receptors for the same ligand suggests an unexpected complexity to TRAIL biology, reminiscent of dual receptors for TNF, the canonical member of this family. PMID:9311998

  8. Distinct combinations of variant ionotropic glutamate receptors mediate thermosensation and hygrosensation in Drosophila

    PubMed Central

    Knecht, Zachary A; Silbering, Ana F; Ni, Lina; Klein, Mason; Budelli, Gonzalo; Bell, Rati; Abuin, Liliane; Ferrer, Anggie J; Samuel, Aravinthan DT; Benton, Richard; Garrity, Paul A

    2016-01-01

    Ionotropic Receptors (IRs) are a large subfamily of variant ionotropic glutamate receptors present across Protostomia. While these receptors are most extensively studied for their roles in chemosensory detection, recent work has implicated two family members, IR21a and IR25a, in thermosensation in Drosophila. Here we characterize one of the most evolutionarily deeply conserved receptors, IR93a, and show that it is co-expressed and functions with IR21a and IR25a to mediate physiological and behavioral responses to cool temperatures. IR93a is also co-expressed with IR25a and a distinct receptor, IR40a, in a discrete population of sensory neurons in the sacculus, a multi-chambered pocket within the antenna. We demonstrate that this combination of receptors is required for neuronal responses to dry air and behavioral discrimination of humidity differences. Our results identify IR93a as a common component of molecularly and cellularly distinct IR pathways important for thermosensation and hygrosensation in insects. DOI: http://dx.doi.org/10.7554/eLife.17879.001 PMID:27656904

  9. New Insights into VacA Intoxication Mediated through Its Cell Surface Receptors

    PubMed Central

    Yahiro, Kinnosuke; Hirayama, Toshiya; Moss, Joel; Noda, Masatoshi

    2016-01-01

    Helicobacter pylori (H. pylori), a major cause of gastroduodenal diseases, produces VacA, a vacuolating cytotoxin associated with gastric inflammation and ulceration. The C-terminal domain of VacA plays a crucial role in receptor recognition on target cells. We have previously identified three proteins (i.e., RPTPα, RPTPβ, and LRP1) that serve as VacA receptors. These receptors contribute to the internalization of VacA into epithelial cells, activate signal transduction pathways, and contribute to cell death and gastric ulceration. In addition, other factors (e.g., CD18, sphingomyelin) have also been identified as cell-surface, VacA-binding proteins. Since we believe that, following interactions with its host cell receptors, VacA participates in events leading to disease, a better understanding of the cellular function of VacA receptors may provide valuable information regarding the mechanisms underlying the pleiotropic actions of VacA and the pathogenesis of H. pylori-mediated disease. In this review, we focus on VacA receptors and their role in events leading to cell damage. PMID:27187473

  10. Voltage-dependent inhibition of recombinant NMDA receptor-mediated currents by 5-hydroxytryptamine

    PubMed Central

    Kloda, Anna; Adams, David J

    2005-01-01

    The effect of 5-HT and related indolealkylamines on heteromeric recombinant NMDA receptors expressed in Xenopus oocytes was investigated using the two-electrode voltage-clamp recording technique. In the absence of external Mg2+ ions, 5-HT inhibited NMDA receptor-mediated currents in a concentration-dependent manner. The inhibitory effect of 5-HT was independent of the NR1a and NR2 subunit combination. The inhibition of glutamate-evoked currents by 5-HT was use- and voltage-dependent. The voltage sensitivity of inhibition for NR1a+NR2 subunit combinations by 5-HT was similar, exhibiting an e-fold change per ∼20 mV, indicating that 5-HT binds to a site deep within the membrane electric field. The inhibition of the open NMDA receptor by external Mg2+ and 5-HT was not additive, suggesting competition between Mg2+ and 5-HT for a binding site in the NMDA receptor channel. The concentration-dependence curves for 5-HT and 5-methoxytryptamine (5-MeOT) inhibition of NMDA receptor-mediated currents are shifted to the right in the presence of external Mg2+. The related indolealkylamines inhibited glutamate-evoked currents with the following order of inhibitory potency: 5-MeOT=5-methyltryptamine>tryptamine>7-methyltryptamine>5-HT≫tryptophan=melatonin. Taken together, these data suggest that 5-HT and related compounds can attenuate glutamate-mediated excitatory synaptic responses and may provide a basis for drug treatment of excitoxic neurodegeneration. PMID:15655527

  11. Structural insight into the MC4R conformational changes via different agonist-mediated receptor signaling.

    PubMed

    Yang, Yingkui; Chen, Min; Dimmitt, Reed; Harmon, Carroll M

    2014-11-18

    The melanocortin-4 receptor (MC4R) plays a key role in the regulation of food intake and body weight. Previous studies indicate that α-melanocyte stimulating hormone (α-MSH) binds to MC4R and activates three signal pathways (cAMP, calcium, and mitogen-activated protein kinase pathways), whereas MC4R synthetic agonist THIQ can activate only the cAMP pathway. The molecular basis of the MC4R responsible for different ligand-mediated signaling is unknown. We hypothesize that different MC4R agonists can stabilize different MC4R conformations and result in ligand-mediated signal transduction. In this study, we examined the effect of the MC4R conformational change in cAMP signaling pathways mediated by different agonists by cross-linking two transmembrane helices (TM3 and TM6). We generated and tested 11 single and 8 double mutations that are located at the end of TM3 and beginning of TM6 in MC4R. Our results indicate that (1) single or double mutations of the MC4R did not significantly alter cAMP production induced by NDP-MSH compared to that of wild-type MC4R except single mutation 243H (the mutation 243H significantly decreased cAMP production mediated by NDP-MSH or THIQ due to a low level of receptor expression at the cell surface), (2) the mutation 247H significantly decreased THIQ-mediated cAMP production but not NDP-MSH, and (3) the receptor cAMP signaling pathway activation by THIQ is blocked in the presence of Zn(2+) with the double mutation I150/242H but activation by NDP-MSH is not, suggesting that the activated conformation of MC4R mediated by NDP-MSH and THIQ is different. This study provides insight into the molecular basis of MC4R responsible for receptor signaling mediated by different agonists.

  12. The miR-199-dynamin regulatory axis controls receptor-mediated endocytosis.

    PubMed

    Aranda, Juan F; Canfrán-Duque, Alberto; Goedeke, Leigh; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-09-01

    Small non-coding RNAs (microRNAs) are important regulators of gene expression that modulate many physiological processes; however, their role in regulating intracellular transport remains largely unknown. Intriguingly, we found that the dynamin (DNM) genes, a GTPase family of proteins responsible for endocytosis in eukaryotic cells, encode the conserved miR-199a and miR-199b family of miRNAs within their intronic sequences. Here, we demonstrate that miR-199a and miR-199b regulate endocytic transport by controlling the expression of important mediators of endocytosis such as clathrin heavy chain (CLTC), Rab5A, low-density lipoprotein receptor (LDLR) and caveolin-1 (Cav-1). Importantly, miR-199a-5p and miR-199b-5p overexpression markedly inhibits CLTC, Rab5A, LDLR and Cav-1 expression, thus preventing receptor-mediated endocytosis in human cell lines (Huh7 and HeLa). Of note, miR-199a-5p inhibition increases target gene expression and receptor-mediated endocytosis. Taken together, our work identifies a new mechanism by which microRNAs regulate intracellular trafficking. In particular, we demonstrate that the DNM, miR-199a-5p and miR-199b-5p genes act as a bifunctional locus that regulates endocytosis, thus adding an unexpected layer of complexity in the regulation of intracellular trafficking.

  13. Nanoscale imaging and mechanical analysis of Fc receptor-mediated macrophage phagocytosis against cancer cells.

    PubMed

    Li, Mi; Liu, Lianqing; Xi, Ning; Wang, Yuechao; Xiao, Xiubin; Zhang, Weijing

    2014-02-18

    Fc receptor-mediated macrophage phagocytosis against cancer cells is an important mechanism in the immune therapy of cancers. Traditional research about macrophage phagocytosis was based on optical microscopy, which cannot reveal detailed information because of the 200-nm-resolution limit. Quantitatively investigating the macrophage phagocytosis at micro- and nanoscale levels is still scarce. The advent of atomic force microscopy (AFM) offers an excellent analytical instrument for quantitatively investigating the biological processes at single-cell and single-molecule levels under native conditions. In this work, we combined AFM and fluorescence microscopy to visualize and quantify the detailed changes in cell morphology and mechanical properties during the process of Fc receptor-mediated macrophage phagocytosis against cancer cells. Lymphoma cells were discernible by fluorescence staining. Then, the dynamic process of phagocytosis was observed by time-lapse optical microscopy. Next, AFM was applied to investigate the detailed cellular behaviors during macrophage phagocytosis under the guidance of fluorescence recognition. AFM imaging revealed the distinct features in cellular ultramicrostructures for the different steps of macrophage phagocytosis. AFM cell mechanical property measurements indicated that the binding of cancer cells to macrophages could make macrophages become stiffer. The experimental results provide novel insights in understanding the Fc-receptor-mediated macrophage phagocytosis.

  14. MAGI-1 acts as a scaffolding molecule for NGF receptor-mediated signaling pathway.

    PubMed

    Ito, Hidenori; Morishita, Rika; Iwamoto, Ikuko; Mizuno, Makoto; Nagata, Koh-ichi

    2013-10-01

    We have recently found that the membrane-associated guanylate kinase with inverted organization-1 (MAGI-1) was enriched in rat nervous tissues such as the glomeruli in olfactory bulb of adult rats and dorsal root entry zone in spinal cord of embryonic rats. In addition, we revealed the localization of MAGI-1 in the growth cone of the primary cultured rat dorsal root ganglion cells. These results point out the possibility that MAGI-1 is involved in the regulation of neurite extension or guidance. In this study, we attempted to reveal the physiological role(s) of MAGI-1 in neurite extension. We found that RNA interference (RNAi)-mediated knockdown of MAGI-1 caused inhibition of nerve growth factor (NGF)-induced neurite outgrowth in PC12 rat pheochromocytoma cells. To clarify the involvement of MAGI-1 in NGF-mediated signal pathway, we tried to identify binding partners for MAGI-1 and identified p75 neurotrophin receptor (p75NTR), a low affinity NGF receptor, and Shc, a phosphotyrosine-binding adaptor. These three proteins formed an immunocomplex in PC12 cells. Knockdown as well as overexpression of MAGI-1 caused suppression of NGF-stimulated activation of the Shc-ERK pathway, which is supposed to play important roles in neurite outgrowth of PC12 cells. These results indicate that MAGI-1 may act as a scaffolding molecule for NGF receptor-mediated signaling pathway.

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

    PubMed

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

    2005-01-01

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

  16. Adenosine A2B-receptor-mediated cyclic AMP accumulation in primary rat astrocytes.

    PubMed Central

    Peakman, M. C.; Hill, S. J.

    1994-01-01

    1. The effects of adenosine receptor agonists and antagonists on the accumulation of cyclic AMP have been investigated in primary cultures of rat astrocytes. 2. Adenosine A2-receptor stimulation caused a concentration-dependent increase in the accumulation of [3H]-cyclic AMP in cells prelabelled with [3H]-adenine. The rank order of agonist potencies was 5'-N-ethylcarboxamidoadenosine (NECA; EC50 = 1 microM) > adenosine (EC50 = 5 microM) > 2-chloroadenosine (EC50 = 20 microM) >> CGS 21680 (EC50 > 10 microM). The presence of 0.5 microM dipyridamole, an adenosine uptake blocker, had no effect on the potency of adenosine. 3. The response to 10 microM NECA was antagonized in a concentration-dependent manner by the non-selective adenosine receptor antagonists, xanthine amine congener (apparent KD = 12 nM), PD 115,199 (apparent KD = 134 nM) and 8-phenyltheophylline (apparent KD = 126 nM). However, the A1-receptor-selective antagonist, 8-cyclopentyl-1,3-dipropylxanthine, had no significant effect on the responses to NECA or 2-chloroadenosine at concentrations up to 1 microM. 4. Stimulation of A1-receptors with the selective agonist, N6-cyclopentyladenosine, did not alter the basal accumulation of [3H]-cyclic AMP but inhibited a forskolin-mediated elevation of [3H]-cyclic AMP accumulation by a maximal value of 42%. This inhibition was fully reversed in the presence of 0.1 microM, 8-cyclopentyl-1,3-dipropylxanthine. 5. The time course for NECA-mediated [3H]-cyclic AMP accumulation was investigated. The results suggest that there is a substantial efflux of cyclic AMP from the cells in addition to the rapid and sustained elevation of intracellular cyclic AMP (5 fold over basal) which was also observed. 6. These data indicate that rat astrocytes in primary culture express an A2B-adenosine receptor coupled positively to adenylyl cyclase. Furthermore, the presence of A1-receptors negatively coupled to adenylyl cyclase appears to have no significant effect on the A2B-receptor-mediated

  17. The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases

    PubMed Central

    Liang, Jing; Wu, Yan-Ling; Chen, Bing-Jia; Zhang, Wen; Tanaka, Yoshimasa; Sugiyama, Hiroshi

    2013-01-01

    As an important member of tyrosine kinase family, c-kit receptor causes specific expression of certain genes, regulates cell differentiation and proliferation, resists cell apoptosis, and plays a key role in tumor occurrence, development, migration and recurrence through activating the downstream signaling molecules following interaction with stem cell factor (SCF). The abnormality of SCF/c-kit signaling pathway is closely related to some certain tumors. The discovery of c-kit receptor-targeted drugs has promoted clinical-related cancer's diagnosis and treatment. In this paper, we review recent research progress on c-kit receptor-mediated signal transduction and its potential therapeutic application as a target in tumor-related diseases. PMID:23678293

  18. Bicarbonate contributes to GABAA receptor-mediated neuronal excitation in surgically resected human hypothalamic hamartomas.

    PubMed

    Kim, Do-Young; Fenoglio, Kristina A; Kerrigan, John F; Rho, Jong M

    2009-01-01

    The role of bicarbonate (HCO(3)(-)) in GABA(A) receptor-mediated depolarization of human hypothalamic hamartoma (HH) neurons was investigated using cellular electrophysiological and calcium imaging techniques. Activation of GABA(A) receptors with muscimol (30 microM) provoked neuronal excitation in over 70% of large (18-22 microM) HH neurons in HCO(3)(-) buffer. Subsequent perfusion of HCO(3)(-)-free HEPES buffer produced partial suppression of muscimol-induced excitation. Additionally, 53% of large HH neurons under HCO(3)(-)-free conditions exhibited reduced intracellular calcium accumulation by muscimol. These results suggest that HCO(3)(-) efflux through GABA(A) receptors on a subpopulation of large HH neurons may contribute to membrane depolarization and subsequent activation of L-type calcium channels.

  19. STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis

    PubMed Central

    Chevalier, David; Batoux, Martine; Fulton, Lynette; Pfister, Karen; Yadav, Ram Kishor; Schellenberg, Maja; Schneitz, Kay

    2005-01-01

    An open question remains as to what coordinates cell behavior during organogenesis, permitting organs to reach their appropriate size and shape. The Arabidopsis gene STRUBBELIG (SUB) defines a receptor-mediated signaling pathway in plants. SUB encodes a putative leucine-rich repeat transmembrane receptor-like kinase. The mutant sub phenotype suggests that SUB affects the formation and shape of several organs by influencing cell morphogenesis, the orientation of the division plane, and cell proliferation. Mutational analysis suggests that the kinase domain is important for SUB function. Biochemical assays using bacterially expressed fusion proteins indicate that the SUB kinase domain lacks enzymatic phosphotransfer activity. Furthermore, transgenes encoding WT and different mutant variants of SUB were tested for their ability to rescue the mutant sub phenotype. These genetic data also indicate that SUB carries a catalytically inactive kinase domain. The SUB receptor-like kinase may therefore signal in an atypical fashion. PMID:15951420

  20. Cocaine exposure impairs multineage hematopoiesis of human hematopoietic progenitor cells mediated by the sigma-1 receptor

    PubMed Central

    Nixon, Christopher C.; Schwartz, Brandon H.; Dixit, Dhaval; Zack, Jerome A.; Vatakis, Dimitrios N.

    2015-01-01

    Prenatal exposure to cocaine is a significant source of fetal and neonatal developmental defects. While cocaine associated neurological and cardiac pathologies are well-documented, it is apparent that cocaine use has far more diverse physiological effects. It is known that in some cell types, the sigma-1 receptor mediates many of cocaine's cellular effects. Here we present a novel and concise investigation into the mechanism that underlies cocaine associated hematopoietic pathology. Indeed, this is the first examination of the effects of cocaine on hematopoiesis. We show that cocaine impairs multilineage hematopoiesis from human progenitors from multiple donors and tissue types. We go on to present the first demonstration of the expression of the sigma-1 receptor in human CD34 + human hematopoietic stem/progenitor cells. Furthermore, we demonstrate that these cocaine-induced hematopoietic defects can be reversed through sigma-1 receptor blockade. PMID:25728014

  1. IL-6 Overexpression in ERG-Positive Prostate Cancer Is Mediated by Prostaglandin Receptor EP2.

    PubMed

    Merz, Constanze; von Mässenhausen, Anne; Queisser, Angela; Vogel, Wenzel; Andrén, Ove; Kirfel, Jutta; Duensing, Stefan; Perner, Sven; Nowak, Michael

    2016-04-01

    Prostate cancer is the most diagnosed cancer in men and multiple risk factors and genetic alterations have been described. The TMPRSS2-ERG fusion event and the overexpression of the transcription factor ERG are present in approximately 50% of all prostate cancer patients, however, the clinical outcome is still controversial. Prostate tumors produce various soluble factors, including the pleiotropic cytokine IL-6, regulating cellular processes such as proliferation and metastatic segregation. Here, we used prostatectomy samples in a tissue microarray format and analyzed the co-expression and the clinicopathologic data of ERG and IL-6 using immunohistochemical double staining and correlated the read-out with clinicopathologic data. Expression of ERG and IL-6 correlated strongly in prostate tissue samples. Forced expression of ERG in prostate tumor cell lines resulted in significantly increased secretion of IL-6, whereas the down-regulation of ERG decreased IL-6 secretion. By dissecting the underlying mechanism in prostate tumor cell lines we show the ERG-mediated up-regulation of the prostanoid receptors EP2 and EP3. The prostanoid receptor EP2 was overexpressed in human prostate cancer tissue. Furthermore, the proliferation rate and IL-6 secretion in DU145 cells was reduced after treatment with EP2-receptor antagonist. Collectively, our study shows that the expression of ERG in prostate cancer is linked to the expression of IL-6 mediated by the prostanoid receptor EP2.

  2. Evidence that the anorexia induced by lipopolysaccharide is mediated by the 5-HT2C receptor.

    PubMed

    von Meyenburg, Claudia; Langhans, Wolfgang; Hrupka, Brian J

    2003-01-01

    Rats consistently reduce their food intake following injections of bacterial lipopolysaccharides (LPS). Because inhibition of serotonergic (5-HT) activity by 8-OH-DPAT (5-HT(1A) activation) attenuates LPS-induced anorexia, we conducted a series of studies to examine whether other 5-HT-receptors are involved in the mediation of peripheral LPS-induced anorexia. In all experiments, rats were injected with LPS (100 microg/kg body weight [BW] ip) at lights out (hour 0). Antagonists were administered peripherally at hour 4, shortly after the onset of anorexia, which presumably follows the enhanced cytokine production after LPS. Food intake was then recorded during the subsequent 2 h or longer. 5-HT receptor antagonists cyanopindolol and SB 224289 (5-HT(1B)), ketanserin (5-HT(2A)), RS-102221 (5-HT(2C)), and metoclopramide (5-HT(3)) failed to attenuate LPS-induced anorexia. In contrast, both ritanserin (5-HT(2A/C)-receptor antagonist) (0.5 mg/kg BW) and SB 242084 (5-HT(2C)) (0.3 mg/kg BW) attenuated LPS-induced anorexia at doses that did not alter food intake in non-LPS-treated rats (all P<.01). Our results suggest that at least part of the anorexia following peripheral LPS administration is mediated through an enhanced 5-HT-ergic activity and the 5-HT(2C) receptor.

  3. Angiotensin receptor subtype mediated physiologies and behaviors: New discoveries and clinical targets

    PubMed Central

    Wright, John W.; Yamamoto, Brent J.; Harding, Joseph W.

    2008-01-01

    The renin–angiotensin system (RAS) mediates several classic physiologies including body water and electrolyte homeostasis, blood pressure, cyclicity of reproductive hormones and sexual behaviors, and the regulation of pituitary gland hormones. These functions appear to be mediated by the angiotensin II (AngII)/AT1 receptor subtype system. More recently, the angiotensin IV (AngIV)/AT4 receptor subtype system has been implicated in cognitive processing, cerebroprotection, local blood flow, stress, anxiety and depression. There is accumulating evidence to suggest an inhibitory influence by AngII acting at the AT1 subtype, and a facilitory role by AngIV acting at the AT4 subtype, on neuronal firing rate, long-term potentiation, associative and spatial learning, and memory. This review initially describes the biochemical pathways that permit synthesis and degradation of active angiotensin peptides and three receptor subtypes (AT1, AT2 and AT4) thus far characterized. There is vigorous debate concerning the identity of the most recently discovered receptor subtype, AT4. Descriptions of classic and novel physiologies and behaviors controlled by the RAS are presented. This review concludes with a consideration of the emerging therapeutic applications suggested by these newly discovered functions of the RAS. PMID:18160199

  4. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  5. Role of the hinge region of glucocorticoid receptor for HEXIM1-mediated transcriptional repression

    SciTech Connect

    Yoshikawa, Noritada; Shimizu, Noriaki; Sano, Motoaki; Ohnuma, Kei; Iwata, Satoshi; Hosono, Osamu; Fukuda, Keiichi; Morimoto, Chikao

    2008-06-20

    We previously reported that HEXIM1 (hexamethylene bisacetamide-inducible protein 1), which suppresses transcription elongation via sequestration of positive transcription elongation factor b (P-TEFb) using 7SK RNA as a scaffold, directly associates with glucocorticoid receptor (GR) to suppress glucocorticoid-inducible gene activation. Here, we revealed that the hinge region of GR is essential for its interaction with HEXIM1, and that oxosteroid receptors including GR show sequence homology in their hinge region and interact with HEXIM1, whereas the other members of nuclear receptors do not. We also showed that HEXIM1 suppresses GR-mediated transcription in two ways: sequestration of P-TEFb by HEXIM1 and direct interaction between GR and HEXIM1. In contrast, peroxisome proliferator-activated receptor {gamma}-dependent gene expression is negatively modulated by HEXIM1 solely via sequestration of P-TEFb. We, therefore, conclude that HEXIM1 may act as a gene-selective transcriptional regulator via direct interaction with certain transcriptional regulators including GR and contribute to fine-tuning of, for example, glucocorticoid-mediated biological responses.

  6. Strategies for differential sensory responses mediated through the same transmembrane receptor.

    PubMed Central

    Yaghmai, R; Hazelbauer, G L

    1993-01-01

    Trg mediates chemotaxis of Escherichia coli to galactose and ribose by recognition of respective, sugar-occupied binding proteins. Although both attractants act through one transmembrane receptor, maximal response is approximately 50% greater to ribose. This phenomenon was investigated by mutational analysis of a 20-residue segment of Trg implicated in ligand interaction and signalling. Among 17 defective receptors, responses to the two chemoattractants were reduced equivalently for seven and differentially for 10, in some cases reversing the preference order. Mutational substitutions with equivalent effects occurred throughout the segment, but those with a greater effect on galactose or ribose response were segregated to the amino-terminal two-thirds or the carboxy-terminal one-third, respectively, a segregation corresponding in large part to a functional division based on signalling phenotypes. A model for binding protein-mediated recognition revealed two strategies for differential responses. The wild-type preference for ribose probably reflects a balance of receptor affinities and a limiting supply of binding proteins. Mutants with reversed preference probably have differentially reduced receptor affinities and those with an accentuated ribose preference probably have altered signalling abilities. Two-step recognition of ligand allows functional separation of detection and response. Images PMID:8491182

  7. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling.

    PubMed

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-03-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GPCR-mediated signaling, leading to an intricate network of transduction pathways. In this regard, it should be mentioned that diverse GPCR family members contribute to the adaptive cell responses to low oxygen tension, which is a distinguishing feature of several illnesses like neoplastic and cardiovascular diseases. For instance, the G protein estrogen receptor, namely G protein estrogen receptor (GPER)/GPR30, has been shown to contribute to relevant biological effects induced by hypoxia via the hypoxia-inducible factor (HIF)-1α in diverse cell contexts, including cancer. Likewise, GPER has been found to modulate the biological outcome of hypoxic/ischemic stress in both cardiovascular and central nervous systems. Here, we describe the role exerted by GPCR-mediated signaling in low oxygen conditions, discussing, in particular, the involvement of GPER by a hypoxic microenvironment.

  8. Mechanism of HSV infection through soluble adapter-mediated virus bridging to the EGF receptor

    SciTech Connect

    Nakano, Kenji; Kobayashi, Masatoshi; Nakamura, Kei-ichiro; Nakanishi, Takeshi; Asano, Ryutaro; Kumagai, Izumi; Tahara, Hideaki; Kuwano, Michihiko; Cohen, Justus B.; Glorioso, Joseph C.

    2011-04-25

    Herpes simplex virus entry into cells requires the binding of envelope glycoprotein D (gD) to an entry receptor. Depending on the cell, entry occurs by different mechanisms, including fusion at the cell surface or endocytosis. Here we examined the entry mechanism through a non-HSV receptor mediated by a soluble bi-specific adapter protein composed of recognition elements for gD and the EGF receptor (EGFR). Virus entered into endosomes using either EGF or an EGFR-specific single chain antibody (scFv) for receptor recognition. Infection was less efficient with the EGF adapter which could be attributed to its weaker binding to a viral gD. Infection mediated by the scFv adapter was pH sensitive, indicating that gD-EGFR bridging alone was insufficient for capsid release from endosomes. We also show that the scFv adapter enhanced infection of EGFR-expressing tumor tissue in vivo. Our results indicate that adapters may retarget HSV infection without drastically changing the entry mechanism.

  9. The MYND domain-containing protein BRAM1 inhibits lymphotoxin beta receptor-mediated signaling through affecting receptor oligomerization.

    PubMed

    Liu, Hao-Ping; Chung, Pei-Jung; Liang, Chih-Lung; Chang, Yu-Sun

    2011-01-01

    MYND (myeloid-Nervy-DEAF-1) domains exist in a large number of proteins that are functionally important in development or associated with cancers. We have previously demonstrated that a MYND domain-containing protein, the bone morphogenesis protein receptor-associated molecule 1 (BRAM1), is able to interact with Epstein-Barr virus-encoded latent membrane protein 1 (LMP1), which acts as a constitutively activated tumor necrosis factor receptor (TNFR). Herein we further demonstrated that BRAM1 additionally associates with the TNFR-superfamily member, the lymphotoxin beta receptor (LTβR), and hence inhibits LTβR-mediated function. Using the yeast two-hybrid assay, we demonstrated that BRAM1 interacts with LTβR mainly through the self-association domain of LTβR (aa 336-398). The co-immunoprecipitation experiment further revealed that BRAM1 as well as MYND domain-containing proteins, MTG8 and DEAF-1, interacts with LTβR via their MYND domains. The BRAM1-LTβR interaction impedes the self-association of LTβR and the recruitment of TNFR-associated factors 2 and 3 (TRAF2 and TRAF3), leading to abolishment of LTβR-induced NF-κB signaling, JNK activation, and caspase-dependent cell death. In sum, our data demonstrate that the MYND-containing protein BRAM1 abrogates LTβR function through a protein-protein interaction. These findings may provide a direction for the treatment of dysregulation of LTβR-mediated signaling.

  10. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling.

    PubMed

    Magno, Aaron L; Ingley, Evan; Brown, Suzanne J; Conigrave, Arthur D; Ratajczak, Thomas; Ward, Bryan K

    2011-09-09

    The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  11. G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    PubMed

    Violin, Jonathan D; Dewire, Scott M; Barnes, William G; Lefkowitz, Robert J

    2006-11-24

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

  12. Contribution of the P2Y12 receptor-mediated pathway to platelet hyperreactivity in hypercholesterolemia

    PubMed Central

    Nagy, Béla; Jin, Jianguo; Ashby, Barrie; Reilly, Michael P.; Kunapuli, Satya P.

    2011-01-01

    Summary Background In hypercholesterolemia, platelets demonstrate increased reactivity and promote the development of cardiovascular disease. Objective This study was carried out to investigate the contribution of the ADP receptor P2Y12-mediated pathway in platelet hyperreactivity due to hypercholesterolemia. Methods Low-density lipoprotein receptor deficient mice and C57Bl/6 wild type mice were fed on normal chow and high-fat (Western or Paigen) diets for 8 weeks to generate differently elevated cholesterol levels. P2Y12 receptor induced functional responses via Gi signaling were studied ex vivo when washed murine platelets were activated by 2MeSADP and PAR4 agonist AYPGKF in the presence and absence of indomethacin. Platelet aggregation, secretion, αIIbβ3 receptor activation and the phosphorylation of extracellular signal-regulated protein kinase (ERK) and Akt were analyzed. Results Plasma cholesterol levels ranged from 69±10 to 1011±185 mg/dl depending on diet in mice with different genotypes. Agonist-dependent aggregation, dense and α-granule secretion and JON/A binding were gradually and significantly (P < 0.05) augmented at low agonist concentration in correlation with the increasing plasma cholesterol levels even if elevated thromboxane generation was blocked. These functional responses were induced via increased level of Gi mediated ERK and Akt phosphorylation in hypercholesterolemic mice versus normocholesterolemic animals. In addition, blocking of the P2Y12 receptor by AR-C69931MX (Cangrelor) resulted in strongly reduced platelet aggregation in mice with elevated cholesterol levels compared to normocholesterolemic controls. Conclusions These data revealed that the P2Y12 receptor pathway was substantially involved in platelet hyperreactivity associated with mild and severe hypercholesterolemia. PMID:21261805

  13. Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal.

    PubMed Central

    Indik, Z; Kelly, C; Chien, P; Levinson, A I; Schreiber, A D

    1991-01-01

    Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal. Images PMID:1834702

  14. Multiple receptor subtypes mediate the effects of serotonin on rat subfornical organ neurons

    NASA Technical Reports Server (NTRS)

    Scrogin, K. E.; Johnson, A. K.; Schmid, H. A.

    1998-01-01

    The subfornical organ (SFO) receives significant serotonergic innervation. However, few reports have examined the functional effects of serotonin on SFO neurons. This study characterized the effects of serotonin on spontaneously firing SFO neurons in the rat brain slice. Of 31 neurons tested, 80% responded to serotonin (1-100 microM) with either an increase (n = 15) or decrease (n = 10) in spontaneous activity. Responses to serotonin were dose dependent and persisted after synaptic blockade. Excitatory responses could also be mimicked by the 5-hydroxytryptamine (5-HT)2A/2C receptor agonist 2,5-dimethoxy-4-iodoamphetamine (DOI; 1-10 microM) and could be blocked by the 5-HT2A/2C-receptor antagonist LY-53,857 (10 microM). LY-53,857 unmasked inhibitory responses to serotonin in 56% of serotonin-excited cells tested. Serotonin-inhibited cells were also inhibited by the 5-HT1A-receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT; 1-10 microM; n = 7). The data indicate that SFO neurons are responsive to serotonin via postsynaptic activation of multiple receptor subtypes. The results suggest that excitatory responses to serotonin are mediated by 5-HT2A or 5-HT2C receptors and that inhibitory responses may be mediated by 5-HT1A receptors. In addition, similar percentages of serotonin-excited and -inhibited cells were also sensitive to ANG II. As such the functional relationship between serotonin and ANG II in the SFO remains unclear.

  15. Multiple Novel Signals Mediate Thyroid Hormone Receptor Nuclear Import and Export*

    PubMed Central

    Mavinakere, Manohara S.; Powers, Jeremy M.; Subramanian, Kelly S.; Roggero, Vincent R.; Allison, Lizabeth A.

    2012-01-01

    Thyroid hormone receptor (TR) is a member of the nuclear receptor superfamily that shuttles between the cytosol and nucleus. The fine balance between nuclear import and export of TR has emerged as a critical control point for modulating thyroid hormone-responsive gene expression; however, sequence motifs of TR that mediate shuttling are not fully defined. Here, we characterized multiple signals that direct TR shuttling. Along with the known nuclear localization signal in the hinge domain, we identified a novel nuclear localization signal in the A/B domain of thyroid hormone receptor α1 that is absent in thyroid hormone receptor β1 and inactive in the oncoprotein v-ErbA. Our prior studies showed that thyroid hormone receptor α1 exits the nucleus through two pathways, one dependent on the export factor CRM1 and the other CRM1-independent. Here, we identified three novel CRM1-independent nuclear export signal (NES) motifs in the ligand-binding domain as follows: a highly conserved NES in helix 12 (NES-H12) and two additional NES sequences spanning helix 3 and helix 6, respectively. Mutations predicted to disrupt the α-helical structure resulted in a significant decrease in NES-H12 activity. The high degree of conservation of helix 12 suggests that this region may function as a key NES in other nuclear receptors. Furthermore, our mutagenesis studies on NES-H12 suggest that altered shuttling of thyroid hormone receptor β1 may be a contributing factor in resistance to thyroid hormone syndrome. Taken together, our findings provide a detailed mechanistic understanding of the multiple signals that work together to regulate TR shuttling and transcriptional activity, and they provide important insights into nuclear receptor function in general. PMID:22815488

  16. Antidepressant-like Effects of Buprenorphine are Mediated by Kappa Opioid Receptors.

    PubMed

    Falcon, Edgardo; Browne, Caroline A; Leon, Rosa M; Fleites, Vanessa C; Sweeney, Rachel; Kirby, Lynn G; Lucki, Irwin

    2016-08-01

    Previous studies have identified potential antidepressant effects of buprenorphine (BPN), a drug with high affinity for mu opioid receptor (MORs) and kappa opioid receptors (KORs) and some affinity at delta opioid receptor (DOR) and opioid receptor-like 1 (ORL-1) receptors. Therefore, these studies examined which opioid receptors were involved in BPN's effects on animal behavior tests sensitive to antidepressant drugs. The acute effects of BPN were tested in the forced swim test (FST) using mice with genetic deletion of individual opioid receptors or after pharmacological blockade of receptors. For evaluating the effects of BPN on chronic stress, separate groups of mice were exposed to unpredictable chronic mild stress (UCMS) for 3 weeks and treated with BPN for at least 7 days before behavioral assessment and subsequent measurement of Oprk1, Oprm1, and Pdyn mRNA expression in multiple brain regions. BPN did not reduce immobility in mice with KOR deletion or after pretreatment with norbinaltorphimine, even though desipramine remained effective. In contrast, BPN reduced immobility in MOR and DOR knockout mice and in mice pretreated with the ORL-1 antagonist JTC-801. UCMS reduced sucrose preference, decreased time in the light side of the light/dark box, increased immobility in the FST and induced region-specific alterations in Oprk1, Oprm1, and PDYN mRNA expression in the frontal cortex and striatum. All of these changes were normalized following BPN treatment. The KOR was identified as a key player mediating the effects of BPN in tests sensitive to antidepressant drugs in mice. These studies support further development of BPN as a novel antidepressant.

  17. The supramammillary nucleus mediates primary reinforcement via GABA(A) receptors.

    PubMed

    Ikemoto, Satoshi

    2005-06-01

    The supramammillary nucleus (SUM), a dorsal layer of the mammillary body, has recently been implicated in positive reinforcement. The present study examined whether GABA(A) receptors in the SUM or adjacent regions are involved in primary reinforcement using intracranial self-administration procedures. Rats learned quickly to lever-press for infusions of the GABA(A) antagonist picrotoxin into the SUM. Although picrotoxin was also self-administered into the posterior hypothalamic nuclei and anterior ventral tegmental area, these regions were less responsive to lower doses of picrotoxin than the SUM. The finding that rats learned to respond selectively on the lever triggering drug infusions is consistent with picrotoxin's reinforcing effect. Coadministration of the GABA(A) agonist muscimol disrupted picrotoxin self-administration, and another GABA(A) antagonist, bicuculline, was also self-administered into the SUM; thus, the reinforcing effect of picrotoxin is mediated by GABA(A) receptors. Since rats did not self-administer the GABA(B) antagonist 2-hydroxysaclofen into the SUM, the role of GABA(B) receptors may be distinct from that of GABA(A) receptors. Pretreatment with the dopamine receptor antagonist SCH 23390 (0.05 mg/kg, i.p.) extinguished picrotoxin self-administration into the SUM, suggesting that the reinforcing effects of GABA(A) receptor blockade depend on normal dopamine transmission. In conclusion, the blockade of GABA(A) receptors in the SUM is reinforcing, and the brain 'reward' circuitry appears to be tonically inhibited via supramammillary GABA(A) receptors and more extensive than the meso-limbic dopamine system.

  18. Dysregulated Chemokine Receptor Expression and Chemokine-Mediated Cell Trafficking in Pediatric Patients with ESRD

    PubMed Central

    Sherry, Barbara; Dai, Wei Wei; Lesser, Martin L.; Trachtman, Howard

    2008-01-01

    Background and objectives: Children and adolescents with ESRD on dialysis are susceptible to serious bacterial infections (SBI). Chemokines and chemokine receptors play a critical role in modulating macrophage and neutrophil function. This study examined the hypothesis that expression and/or function of these molecules is dysregulated in patients with ESRD, contributing to leukocyte dysfunction. Design setting, participants, & measurements: Pediatric patients, age 6 mo to 18 yr, with ESRD treated with either hemodialysis or peritoneal dialysis were enrolled in this prospective, nontherapeutic study. Blood was collected for plasma chemokine levels, chemokine receptor profiling by flow cytometry, and functional chemotaxis studies on neutrophils and mononuclear cells. Results: ESRD in children was associated with reduced expression of the chemokine receptors CXCR1 and chemokine (C-C motif) receptor 2 (CCR2) on circulating neutrophils and monocytes, respectively. When ESRD patients were divided into two subgroups, those who were infection-free and those who had three or more SBI in the preceding year, the differences in chemokine receptor expression were statistically significant compared with control subjects only in those with recurrent infection. In addition to the effects of ESRD on baseline chemokine receptor expression, the hemodialysis procedure itself acutely lowered neutrophil CXCR1 and monocyte CCR2 expression. Furthermore, neutrophil and monocyte responsiveness to chemokine-mediated trafficking signals was impaired in all ESRD patients studied. This abnormality was independent of the level of chemokine receptor expression on the leukocytes. Conclusions: The data presented in this study suggest that chemokine receptor dysregulation contributes to leukocyte dysfunction in patients with ESRD. This alteration is especially prominent in ESRD patients with recurrent infection. PMID:18235145

  19. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  20. Effects of CYP7B1-mediated catalysis on estrogen receptor activation.

    PubMed

    Pettersson, Hanna; Lundqvist, Johan; Norlin, Maria

    2010-09-01

    Most of the many biological effects of estrogens are mediated via the estrogen receptors ERalpha and beta. The current study examines the role of CYP7B1-mediated catalysis for activation of ER. Several reports suggest that CYP7B1 may be important for hormonal action but previously published studies are contradictory concerning the manner in which CYP7B1 affects ERbeta-mediated response. In the current study, we examined effects of several CYP7B1-related steroids on ER activation, using an estrogen response element (ERE) reporter system. Our studies showed significant stimulation of ER by 5-androstene-3beta,17beta-diol (Aene-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-Adiol). In contrast, the CYP7B1-formed metabolites from these steroids did not activate the receptor, indicating that CYP7B1-mediated metabolism abolishes the ER-stimulating effect of these compounds. The mRNA level of HEM45, a gene known to be stimulated by estrogens, was strongly up-regulated by Aene-diol but not by its CYP7B1-formed metabolite, further supporting this concept. We did not observe stimulation by dehydroepiandrosterone (DHEA) or 7alpha-hydroxy-DHEA, previously suggested to affect ERbeta-mediated response. As part of these studies we examined metabolism of Aene-diol in pig liver which is high in CYP7B1 content. These experiments indicate that CYP7B1-mediated metabolism of Aene-diol is of a similar rate as the metabolism of the well-known CYP7B1 substrates DHEA and 3beta-Adiol. CYP7B1-mediated metabolism of 3beta-Adiol has been proposed to influence ERbeta-mediated growth suppression. Our results indicate that Aene-diol also might be important for ER-related pathways. Our data indicate that low concentrations of Aene-diol can trigger ER-mediated response equally well for both ERalpha and beta and that CYP7B1-mediated conversion of Aene-diol into a 7alpha-hydroxymetabolite will result in loss of action.

  1. Ethanol-Induced Motor Impairment Mediated by Inhibition of α7 Nicotinic Receptors

    PubMed Central

    McDaid, John; Abburi, Chandrika; Wolfman, Shannon L.; Gallagher, Keith

    2016-01-01

    Nicotine and ethanol (EtOH) are among the most widely co-abused substances, and nicotinic acetylcholine receptors (nAChRs) contribute to the behavioral effects of both drugs. Along with their role in addiction, nAChRs also contribute to motor control circuitry. The α7 nAChR subtype is highly expressed in the laterodorsal tegmental nucleus (LDTg), a brainstem cholinergic center that contributes to motor performance through its projections to thalamic motor relay centers, including the mediodorsal thalamus. We demonstrate that EtOH concentrations just above the legal limits for intoxication in humans can inhibit α7 nAChRs in LDTg neurons from rats. This EtOH-induced inhibition is mediated by a decrease in cAMP/PKA signaling. The α7 nAChR-positive allosteric modulator PNU120596 [N-(5-chloro-2,4-dimethoxyphenyl)-N′-(5-methyl-3-isoxazolyl)-urea], which interferes with receptor desensitization, completely eliminated EtOH modulation of these receptors. These data suggest that EtOH inhibits α7 responses through a PKA-dependent enhancement of receptor desensitization. EtOH also inhibited the effects of nicotine at presynaptic α7 nAChRs on glutamate terminals in the mediodorsal thalamus. In vivo administration of PNU120596 either into the cerebral ventricles or directly into the mediodorsal thalamus attenuated EtOH-induced motor impairment. Thus, α7 nAChRs are likely important mediators of the motor impairing effects of moderate EtOH consumption. SIGNIFICANCE STATEMENT The motor-impairing effects of ethanol contribute to intoxication-related injury and death. Here we explore the cellular and neural circuit mechanisms underlying ethanol-induced motor impairment. Physiologically relevant concentrations of ethanol inhibit activity of a nicotinic receptor subtype that is expressed in brain areas associated with motor control. That receptor inhibition is mediated by decreased receptor phosphorylation, suggesting an indirect modulation of cell signaling pathways to achieve

  2. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  3. GR-127935-sensitive mechanism mediating hypotension in anesthetized rats: are 5-HT5B receptors involved?

    PubMed

    Sánchez-Maldonado, Carolina; López-Sánchez, Pedro; Anguiano-Robledo, Liliana; Leopoldo, Marcello; Lacivita, Enza; Terrón, José A

    2015-04-01

    The 5-HT1B/1D receptor antagonist, GR-127935, inhibits hypotensive responses produced by the 5-HT1A, 5-HT1B/1D and 5-HT7 receptor agonist, and 5-HT5A/5B receptor ligand, 5-carboxamidotryptamine (5-CT), in rats. This work further characterized the above mechanism using more selective 5-HT1B and 5-HT1D receptor antagonists. Also, expression of 5-HT5A and 5-HT5B receptor mRNAs in blood vessels was searched by reverse transcription polymerase chain reaction. Decreases in diastolic blood pressure induced by 5-CT (0.001-10 μg/kg, intravenously) were analyzed in anesthetized rats that had received intravenous vehicle (1 mL/kg), SB-224289 (5-HT1B antagonist; 0.3 and 1.0 mg/kg), BRL15572 (5-HT1D antagonist; 0.3 and 1.0 mg/kg), SB-224289 + BRL15572 (0.3 mg/kg, each), or SB-224289 + BRL15572 (0.3 mg/kg, each) + GR-127935 (1 mg/kg). Because only the latter treatment inhibited 5-CT-induced hypotension, suggestive of a mechanism unrelated to 5-HT1B/1D receptors, the effects of antagonists/ligands at 5-HT5A (SB-699551, 1 mg/kg), 5-HT6 (SB-399885, 1 mg/kg), and 5-HT1B/1D/5A/5B/7 receptors (ergotamine, 0.1 mg/kg) on 5-CT-induced hypotension were tested. Interestingly, only ergotamine blocked 5-CT-induced responses; this effect closely paralleled that of SB-224289 + BRL-15572 + GR-127935. Neither did ergotamine nor GR-127935 inhibit hypotensive responses induced by the 5-HT7 receptor agonist, LP-44. Faint but clear bands corresponding to 5-HT5A and 5-HT5B receptor mRNAs in aorta and mesenteric arteries were detected. Results suggest that the GR-127935-sensitive mechanism mediating hypotension in rats is unrelated to 5-HT1B, 5-HT1D, 5-HT5A, 5-HT6, and 5-HT7 receptors. This mechanism, however, resembles putative 5-HT5B receptors.

  4. Different AMPA receptor subtypes mediate the distinct kinetic components of a biphasic EPSC in hippocampal interneurons

    PubMed Central

    Stincic, Todd L.; Frerking, Matthew E.

    2015-01-01

    CA1 hippocampal interneurons at the border between stratum radiatum (SR) and stratum lacunosum-moleculare (SLM) have AMPA receptor (AMPAR)-mediated excitatory postsynaptic currents (EPSCs) that consist of two distinct phases: a typical fast component (FC), and a highly unusual slow component (SC) that persists for hundreds of milliseconds. To determine whether these kinetically distinct components of the EPSC are mediated by distinct AMPAR subpopulations, we examined the relative contributions of GluA2-containing and—lacking AMPARs to the SC. GluA2-containing AMPARs mediated the majority of the FC whereas GluA2-lacking AMPARs preferentially generated the SC. When glutamate uptake through the glial glutamate transporter excitatory amino acid transporter (EAAT1) was inhibited, spill over-mediated AMPAR activation recruited an even slower third kinetic component that persisted for several seconds; however, this spillover-mediated current was mediated predominantly by GluA2-containing AMPARs and therefore was clearly distinct from the SC when uptake is intact. Thus, different AMPAR subpopulations that vary in GluA2 content mediate the distinct components of the AMPAR EPSC. The SC is developmentally downregulated in mice, declining after the second postnatal week. This downregulation affects both GluA2-containing and GluA2-lacking AMPARs mediating the SC, and is not accompanied by developmental changes in the GluA2 content of AMPARs underlying the FC. Thus, the downregulation of the SC appears to be independent of synaptic GluA2 expression, suggesting the involvement of another AMPAR subunit or an auxiliary protein. Our results therefore identify GluA2-dependent and GluA2-independent determinants of the SC: GluA2-lacking AMPARs preferentially contribute to the SC, while the developmental downregulation of the SC is independent of GluA2 content. PMID:26042027

  5. Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

    PubMed Central

    Villalón, Carlos M; Heiligers, Jan P C; Centurión, David; De Vries, Peter; Saxena, Pramod R

    1997-01-01

    It has been suggested that the tachycardic response to 5-hydroxytryptamine (5-HT) in the spinal-transected cat is mediated by ‘5-HT1-like' receptors since this effect, being mimicked by 5-carboxamidotryptamine (5-CT), is not modified by ketanserin or MDL 72222, but it is blocked by methiothepin, methysergide or mesulergine. The present study was set out to reanalyse this suggestion in terms of the IUPHAR 5-HT receptor classification schemes proposed in 1994 and 1996. Intravenous (i.v.) bolus injections of the tryptamine derivatives, 5-CT (0.01, 0.03, 0.1, 0.3, 1, 3, 10 and 30 μg kg−1), 5-HT (3, 10 and 30 μg kg−1) and 5-methoxytryptamine (3, 10 and 30 μg kg−1) as well as the atypical antipsychotic drug, clozapine (1000 and 3000 μg kg−1) resulted in dose-dependent increases in heart rate, with a rank order of agonist potency of 5-CT >> 5-HT > 5-methoxytryptamine >> clozapine. The tachycardic effects of 5-HT and 5-methoxytryptamine were dose-dependently antagonized by i.v. administration of lisuride (30 and 100 μg kg−1), ergotamine (100 and 300 μg kg−1) or mesulergine (100, 300 and 1000 μg kg−1); the highest doses of these antagonists used also blocked the tachycardic effects of 5-CT. Clozapine (1000 and 3000 μg kg−1) did not affect the 5-HT-induced tachycardia, but attenuated, with its highest dose, the responses to 5-methoxytryptamine and 5-CT. However, these doses of clozapine as well as the high doses of ergotamine (300 μg kg−1) and mesulergine (300 and 1000 μg kg−1) also attenuated the tachycardic effects of isoprenaline. In contrast, 5-HT-, 5-methoxytryptamine- and 5-CT-induced tachycardia were not significantly modified after i.v. administration of physiological saline (0.1 and 0.3 ml kg−1), the 5-HT1B/1D receptor antagonist, GR127935 (500 μg kg−1) or the 5-HT3/4 receptor antagonist, tropisetron (3000 μg kg−1). Intravenous injections of the 5-HT1 receptor agonists

  6. Modulation of NMDA and AMPA-mediated synaptic transmission by CB1 receptors in frontal cortical pyramidal cells.

    PubMed

    Li, Qiang; Yan, Haidun; Wilson, Wilkie A; Swartzwelder, H Scott

    2010-06-25

    Although the endogenous cannabinoid system modulates a variety of physiological and pharmacological processes, the specific role of cannabinoid CB1 receptors in the modulation of glutamatergic neurotransmission and neural plasticity is not well understood. Using whole-cell patch clamp recording techniques, evoked or spontaneous excitatory postsynaptic currents (eEPSCs or sEPSCs) were recorded from visualized, layer II/III pyramidal cells in frontal cortical slices from rat brain. Bath application of the CB1 receptor agonist, WIN 55212-2 (WIN), reduced the amplitude of NMDA receptor-mediated EPSCs in a concentration-dependent manner. When co-applied with the specific CB1 antagonists, AM251 or AM281, WIN did not suppress NMDA receptor-mediated EPSCs. WIN also reduced the amplitude of evoked AMPA receptor-mediated EPSCs, an effect that was also reversed by AM251. Both the frequency and amplitude of spontaneous AMPA receptor-mediated EPSCs were significantly reduced by WIN. In contrast, WIN reduced the frequency, but not the amplitude of miniature EPSCs, suggesting that the suppression of glutamatergic activity by CB1 receptors in the frontal neocortex is mediated by a presynaptic mechanism. Taken together, these data indicate a critical role for endocannabinoid signaling in the regulation of excitatory synaptic transmission in frontal neocortex, and suggest a possible neuronal mechanism whereby THC regulates cortical function.

  7. DDR2 receptor promotes MMP-2-mediated proliferation and invasion by hepatic stellate cells.

    PubMed

    Olaso, E; Ikeda, K; Eng, F J; Xu, L; Wang, L H; Lin, H C; Friedman, S L

    2001-11-01

    Type I collagen provokes activation of hepatic stellate cells during liver injury through mechanisms that have been unclear. Here, we tested the role of the discoidin domain tyrosine kinase receptor 2 (DDR2), which signals in response to type I collagen, in this pathway. DDR2 mRNA and protein are induced in stellate cells activated by primary culture or in vivo during liver injury. The receptor becomes tyrosine phosphorylated in response to either endogenous or exogenous type I collagen, whereas its expression is downregulated during cellular quiescence induced by growth on Matrigel. We developed stellate cell lines stably overexpressing either wild-type DDR2, a constitutively active chimeric DDR2 receptor (Fc-DDR2), a truncated receptor expressing the extracellular domain, or a kinase-dead DDR2 Cells overexpressing DDR2 showed enhanced proliferation and invasion through Matrigel, activities that were directly related to increased expression of active matrix metalloproteinase 2 (MMP-2). These data show that DDR2 is induced during stellate cell activation and implicate the phosphorylated receptor as a mediator of MMP-2 release and growth stimulation in response to type I collagen. Moreover, type I collagen-dependent upregulation of DDR2 expression establishes a positive feedback loop in activated stellate cells, leading to further proliferation and enhanced invasive activity.

  8. Nociceptin/orphanin FQ-NOP receptor system in inflammatory and immune-mediated diseases.

    PubMed

    Gavioli, Elaine C; de Medeiros, Iris Ucella; Monteiro, Marta C; Calo, Girolamo; Romão, Pedro R T

    2015-01-01

    The neuropeptide nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand of the G-protein-coupled receptor NOP. Cells from the immune system express the precursor preproN/OFQ and the NOP receptor, as well as secrete N/OFQ. The activation of the N/OFQ-NOP pathway can regulate inflammatory and immune responses. Several immune activities, including leukocyte migration, cytokine and chemokine production, and lymphocytes proliferation are influenced by NOP activation. It was demonstrated that cytokines and other stimuli such as Toll-like receptor agonist (e.g., lipopolysaccharide) induce N/OFQ production by cells from innate and adaptive immune response. In this context, N/OFQ could modulate the outcome of inflammatory diseases, such as sepsis and immune-mediated pathologies by mechanisms not clearly elucidated. In fact, clinical studies revealed increased levels of N/OFQ under sepsis, arthritis, and Parkinson's disease. Preclinical and clinical studies pointed to the blockade of NOP receptor signaling as successful strategy for the treatment of inflammatory diseases. This review is focused on experimental and clinical data that suggest the participation of N/OFQ-NOP receptor activation in the modulation of the immune response, highlighting the immunomodulatory potential of NOP antagonists in the inflammatory and immunological disturbances.

  9. Ganglioside mediate the interaction between Nogo receptor 1 and LINGO-1.

    PubMed

    Saha, Nayanendu; Kolev, Momchil V; Semavina, Mariya; Himanen, Juha; Nikolov, Dimitar B

    2011-09-16

    Upon spinal cord injury, the myelin inhibitors, including the myelin-associated glycoprotein (MAG), Nogo-A and the oligodendrocyte myelin glycoprotein (OMgp), bind to and signal via a single neuronal receptor/co-receptor complex comprising of Nogo receptor 1(NgR1)/LINGO-1 and p75 or TROY, impeding regeneration of injured axons. We employed a cell-free system to study the binding of NgR1 to its co-receptors and the myelin inhibitor Nogo-A, and show that gangliosides mediate the interaction of NgR1 with LINGO-1. Solid phase binding assays demonstrate that the sialic acid moieties of gangliosides and the stalk of NgR1 are the principal determinants of these molecular interactions. Moreover, the tripartite complex comprising of NgR1, LINGO-1 and ganglioside exhibits stronger binding to Nogo-A (Nogo-54) in the presence of p75, suggesting the gangliosides modulate the myelin inhibitor-receptor signaling.

  10. Activation by SLAM Family Receptors Contributes to NK Cell Mediated "Missing-Self" Recognition.

    PubMed

    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.

  11. Delta- and kappa-opioid receptors in the caudal midline medulla mediate haemorrhage-evoked hypotension.

    PubMed

    Henderson, Luke A; Keay, Kevin A; Bandler, Richard

    2002-04-16

    In mammals blood loss can trigger, shock, an abrupt, life-threatening hypotension and bradycardia. In the halothane-anaesthetised rat this response is blocked by inactivation of a discrete, vasodepressor area in the caudal midline medulla (CMM). Haemorrhagic shock is blocked also by systemic or ventricular injections of the opioid antagonist, naloxone. This study investigated, in the halothane anaesthetised rat, the contribution of delta-, kappa- and mu-opioid receptors in the CMM vasodepressor region to haemorrhage-evoked shock (i.e. hypotension and bradycardia) and its recovery. It was found that microinjections into the CMM of the delta-opioid receptor antagonist, naltrindole delayed and attenuated the hypotension and bradycardia evoked by haemorrhage, but did not promote recompensation. In contrast, CMM microinjections of the kappa-opioid receptor antagonist, nor-binaltorphamine, although it did not alter haemorrhage-evoked hypotension and bradycardia, did lead to a rapid restoration of AP, but not HR. CMM microinjections of the mu-opioid receptor antagonist, CTAP had no effect on haemorrhage-evoked shock or recompensation. These data indicate that delta- and kappa- (but not mu-) opioid receptor-mediated events within the CMM contribute to the hypotension and bradycardia evoked by haemorrhage and the effectiveness of naloxone in reversing shock.

  12. Nicotinic receptor-mediated biphasic effect on neuronal excitability in chick lateral spiriform neurons.

    PubMed

    Liu, Y-B; Guo, J-Z; Chiappinelli, V A

    2007-09-21

    Local neuronal circuits integrate synaptic information with different excitatory or inhibitory time windows. Here we report that activation of nicotinic acetylcholine receptors (nAChRs) leads to biphasic effects on excitability in chick lateral spiriform (SPL) neurons during whole cell recordings in brain slices. Carbachol (100 microM in the presence of 1 microM atropine) produced an initial short-term increase in the firing rates of SPL neurons (125+/-14% of control) that was mediated by postsynaptic nAChRs. However, after 3 min exposure to nicotinic agonists, the firing rate measured during an 800 ms depolarizing pulse declined to 19+/-7% (100 microM carbachol) or 26+/-8% (10 microM nicotine) of the control rate and remained decreased for 10-20 min after washout of the agonists. Similarly, after 60 s of electrically-stimulated release of endogenous acetylcholine (ACh) from cholinergic afferent fibers, there was a marked reduction (45+/-5% of control) in firing rates in SPL neurons. All of these effects were blocked by the nAChR antagonist dihydro-beta-erythroidine (30 microM). The inhibitory effect was not observed in Ca(2+)-free buffer. The nAChR-mediated inhibition depended on active G-proteins in SPL neurons and was prevented by the GABA(B) receptor antagonist phaclofen (200 microM), while the GABA(B) receptor agonist baclofen (10 microM) decreased firing rate in SPL neurons to 13+/-1% of control. The inhibitory response thus appears to be due to a nAChR-mediated enhancement of presynaptic GABA release, which then activates postsynaptic GABA(B) receptors. In conclusion, activation of nAChRs in the SPL initiates a limited time window for an excitatory period, after which a prolonged inhibitory effect turns off this window. The prolonged inhibitory effect may serve to protect SPL neurons from excessive excitation.

  13. Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish*

    PubMed Central

    Bai, Yan; Tan, Xungang; Zhang, Haifeng; Liu, Chengdong; Zhao, Beibei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng

    2014-01-01

    The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish. PMID:24928507

  14. Glucocorticoid receptor-interacting protein 1 mediates ligand-independent nuclear translocation and activation of constitutive androstane receptor in vivo.

    PubMed

    Min, Gyesik; Kemper, J Kim; Kemper, Byron

    2002-07-19

    Phenobarbital (PB) induction of CYP2B genes is mediated by translocation of the constitutively active androstane receptor (CAR) to the nucleus. Interaction of CAR with p160 coactivators and enhancement of CAR transactivation by the coactivators have been shown in cultured cells. In the present studies, the interaction of CAR with the p160 coactivator glucocorticoid receptor-interacting protein 1 (GRIP1) was examined in vitro and in vivo. Binding of GRIP1 to CAR was shown by glutathione S-transferase (GST) pull-down and affinity DNA binding. N- or C-terminal fragments of GRIP1 that contained the central receptor-interacting domain bound to GST-CAR, but the presence of ligand increased the binding to GST-CAR of only the fragments containing the C-terminal region. In gel shift analysis, binding to CAR was observed only with GRIP1 fragments containing the C-terminal region, and the binding was increased by a CAR agonist and decreased by a CAR antagonist. Expression of GRIP1 enhanced CAR-mediated transactivation in cultured hepatic-derived cells 2-3-fold. In hepatocytes transfected in vivo, expression of exogenous GRIP1 alone induced transactivation of the CYP2B1 PB-dependent enhancer 15-fold, whereas CAR expression alone resulted in only a 3-fold enhancement in untreated mice. Remarkably, CAR and GRIP1 together synergistically transactivated the enhancer about 150-fold, which is approximately equal to activation by PB treatment. In PB-treated mice, expression of exogenous CAR alone had little effect, expression of GRIP1 increased transactivation about 2-fold, and with CAR and GRIP, a 4-fold activation was observed. In untreated mice, expression of GRIP resulted in nuclear translocation of green fluorescent protein-CAR. These results strongly suggest that a p160 coactivator functions in CAR-mediated transactivation in vivo in response to PB treatment and that the synergistic activation of CAR by GRIP in untreated animals results from both nuclear translocation and

  15. Prosurvival NMDA 2A receptor signaling mediates postconditioning neuroprotection in the hippocampus.

    PubMed

    Zhang, Xi; Zhang, Quanguang; Tu, Jingyi; Zhu, Ying; Yang, Fang; Liu, Bin; Brann, Darrell; Wang, Ruimin

    2015-03-01

    Ischemic postconditioning (Post C), which involves administration of a brief ischemia after the initial ischemic event, has been demonstrated to be strongly neuroprotective against global cerebral ischemia (GCI) and to improve cognitive outcome. To enhance understanding of the underlying mechanisms, the current study examined the role of NMDA receptors in mediating the beneficial effects of Post C (3 min ischemia) administered 2 days after GCI in adult male rats. The results revealed that Post C was strongly neuroprotective against GCI, and that this effect was blocked by administration of the NMDA receptor antagonist MK-801. Further work revealed that the NR2A-type NMDA receptors mediate the Post C beneficial effects as administration of a NR2A-preferring antagonist (NVP-A) blocked Post C neuroprotection and cognitive enhancement, while administration of a NR2B-preferring antagonist (Ro25) was without effect. Post C significantly up-regulated NR2A levels and phosphorylation of NR2A in the hippocampal CA1 region after Post C. Post C also increased Ca(2+) influx and activation/phosphorylation of CamKIIα at Thr(286), effects that were NR2A mediated as they were blocked by NVP-A. Phosphorylation of ERK and CREB was also increased by Post C, as were two downstream CREB-dependent prosurvival factors, brain derived neurotropic factor (BDNF) and Bcl2, effects that were blocked by the NR2A antagonist, NVP-A. Taken as a whole, the current study provides evidence that NR2A-activation and downstream prosurvival signaling is a critical mediator of Post C-induced neuroprotection and cognitive enhancement following GCI.

  16. Differential Regulation of Progesterone Receptor-Mediated Transcription by CDK2 and DNA-PK.

    PubMed

    Treviño, Lindsey S; Bolt, Michael J; Grimm, Sandra L; Edwards, Dean P; Mancini, Michael A; Weigel, Nancy L

    2016-02-01

    Progesterone receptor (PR) function is altered by cell signaling, but the mechanisms of kinase-specific regulation are not well defined. To examine the role of cell signaling in the regulation of PR transcriptional activity, we have utilized a previously developed mammalian-based estrogen-response element promoter array cell model and automated cell imaging and analysis platform to visualize and quantify effects of specific kinases on different mechanistic steps of PR-mediated target gene activation. For these studies, we generated stable estrogen-response element array cell lines expressing inducible chimeric PR that contains a swap of the estrogen receptor-α DNA-binding domain for the DNA-binding domain of PR. We have focused on 2 kinases important for steroid receptor activity: cyclin-dependent kinase 2 and DNA-dependent protein kinase. Treatment with either a Cdk1/2 inhibitor (NU6102) or a DNA-dependent protein kinase inhibitor (NU7441) decreased hormone-mediated chromatin decondensation and transcriptional activity. Further, we observed a quantitative reduction in the hormone-mediated recruitment of select coregulator proteins with NU6102 that is not observed with NU7441. In parallel, we determined the effect of kinase inhibition on hormone-mediated induction of primary and mature transcripts of endogenous genes in T47D breast cancer cells. Treatment with NU6102 was much more effective than NU7441, in inhibiting induction of PR target genes that exhibit a rapid increase in primary transcript expression in response to hormone. Taken together, these results indicate that the 2 kinases regulate PR transcriptional activity by distinct mechanisms.

  17. Adolescent nicotine administration alters serotonin receptors and cell signaling mediated through adenylyl cyclase.

    PubMed

    Xu, Z; Seidler, F J; Cousins, M M; Slikker, W; Slotkin, T A

    2002-10-04

    Nicotine is a neuroteratogen that targets synaptic function during critical developmental stages and recent studies indicate that CNS vulnerability extends into adolescence, the age at which smoking typically commences. We administered nicotine to adolescent rats via continuous minipump infusions from PN30 to PN47.5, using 6 mg/kg/day, a dose rate that replicates the plasma nicotine levels found in smokers, and examined 5HT receptors and related cell signaling during nicotine administration (PN45) and in the post-treatment period (PN50, 60, 75). Adolescent nicotine decreased 5HT(2) receptor binding in brain regions containing 5HT projections (hippocampus and cerebral cortex), with selectivity for females in the cerebral cortex; regions containing 5HT cell bodies showed either an increase (midbrain in males) or no change (brainstem). In contrast, there were no significant changes in 5HT(1A) receptors; however, the ability of the receptors to signal through adenylyl cyclase (AC) showed a switch from stimulatory to inhibitory effects in females during the post-treatment period. There were also transient alterations in AC responses to beta-adrenergic receptor stimulation, as well as pronounced induction of the AC response to the non-receptor-mediated stimulant, forskolin. Our results indicate that adolescent nicotine exposure alters the concentrations and functions of postsynaptic 5HT receptors in a manner commensurate with impaired 5HT synaptic function. The direction of change, emergence of defects after the cessation of nicotine administration, and sex-preference for effects in females, all support a relationship of impaired 5HT function to the higher incidence of depression seen in adolescent smokers.

  18. Endocytosis and ligand dissociation and degradation mediated by the hepatic galactosyl receptor occur via two different pathways

    SciTech Connect

    Weigel, P.H.; Clarke, B.L.; Oka, J.A.

    1986-05-01

    Isolated rat hepatocytes express two distinct populations of surface Galactosyl receptor activity, measured by the binding of /sup 125/I-asialo-orosomucoid (ASOR), which they designate State 1 and State 2. Freshly isolated cells express only state 1 receptors. Cells equilibrated at 37/sup 0/C also express State 2 receptors, which represent 50-80% of the total surface activity. In the absence of ligand, State 2 receptor activity is reversibly decreased by metabolic energy poisons, low temperature and microtubule drugs, whereas State 1 receptor activity is unaffected. Endocytosis of /sup 125/I-ASOR by State 1 receptors is followed by a slow dissociation of /sup 125/I-ASOR from receptor but the immediate release of acid soluble degradation products. In contrast, State 2 receptors mediate endocytosis which involves a rapid dissociation step but a 20 min lag, prior to the release of degradation products. Both pathways follow first order kinetics and are functional under steady state conditions indicating coordinated receptor recycling. Degradation mediated by both pathways is inhibited by leupeptin and chloroquine. The State 1 and 2 pathways can be further differentiated by the greater sensitivity of the latter to microtubule drugs. These results suggest that there are either structurally different native receptors or that identical receptors are directed into two different functional pathways, for example by interaction with different types of coated pits.

  19. Ligand Bias at Metabotropic Glutamate 1a Receptors: Molecular Determinants That Distinguish β-Arrestin-Mediated from G Protein-Mediated Signaling

    PubMed Central

    Emery, Andrew C.; DiRaddo, John O.; Miller, Eric; Hathaway, Hannah A.; Pshenichkin, Sergey; Takoudjou, Guy Rodrigue; Grajkowska, Ewa; Yasuda, Robert P.; Wolfe, Barry B.

    2012-01-01

    The metabotropic glutamate 1a (mGlu1a) receptor is a G protein-coupled receptor linked with phosphoinositide (PI) hydrolysis and with β-arrestin-1-mediated sustained extracellular signal-regulated kinase (ERK) phosphorylation and cytoprotective signaling. Previously, we reported the existence of ligand bias at this receptor, inasmuch as glutamate induced both effects, whereas quisqualate induced only PI hydrolysis. In the current study, we showed that mGlu1 receptor agonists such as glutamate, aspartate, and l-cysteate were unbiased and activated both signaling pathways, whereas quisqualate and (S)-3,5-dihydroxyphenylglycine stimulated only PI hydrolysis. Competitive antagonists inhibited only PI hydrolysis and not the β-arrestin-dependent pathway, whereas a noncompetitive mGlu1 receptor antagonist blocked both pathways. Mutational analysis of the ligand binding domain of the mGlu1a receptor revealed that Thr188 residues were essential for PI hydrolysis but not for protective signaling, whereas Arg323 and Lys409 residues were required for β-arrestin-1-mediated sustained ERK phosphorylation and cytoprotective signaling but not for PI hydrolysis. Therefore, the mechanism of ligand bias appears to involve different modes of agonist interactions with the receptor ligand binding domain. Although some mGlu1a receptor agonists are biased toward PI hydrolysis, we identified two endogenous compounds, glutaric acid and succinic acid, as new mGlu1 receptor agonists that are fully biased toward β-arrestin-mediated protective signaling. Pharmacological studies indicated that, in producing the two effects, glutamate interacted in two distinct ways with mGlu1 receptors, inasmuch as competitive mGlu1 receptor antagonists that blocked PI hydrolysis did not inhibit cytoprotective signaling. Quisqualate, which is biased toward PI hydrolysis, failed to inhibit glutamate-induced protection, and glutaric acid, which is biased toward protection, did not interfere with glutamate

  20. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  1. Neuregulin-1-beta1 enters brain and spinal cord by receptor-mediated transport.

    PubMed

    Kastin, Abba J; Akerstrom, Victoria; Pan, Weihong

    2004-02-01

    Proteins of the neuregulin (NRG) family play important regulatory roles in neuronal survival and synaptic activity. NRG-1-beta1 has particular potential as a therapeutic agent because it enhances myelination of neurites in spinal cord explants. In this study, we determined the permeation of NRG-1-beta1 across the blood-brain and blood-spinal cord barriers (BBB and BSCB respectively). Intact radioactively labeled NRG-1-beta1 had a saturable and relatively rapid influx rate from blood to the CNS in mice. Capillary depletion studies showed that NRG-1-beta1 entered the parenchyma of the brain and spinal cord rather than being trapped in the capillaries that compose the BBB. The possible mechanism of receptor-mediated transport was shown by the ability of antibodies to erbB3 and erbB4 receptors to inhibit the influx. Lipophilicity, less important for such saturable transport mechanisms, was measured by the octanol : buffer partition coefficient and found to be low. The results indicate that NRG-1-beta1 enters spinal cord and brain by a saturable receptor-mediated mechanism, which provides the opportunity for possible therapeutic manipulation at the BBB level.

  2. Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody.

    PubMed

    Kasturirangan, Srinath; Rainey, G Jonah; Xu, Linda; Wang, Xinwei; Portnoff, Alyse; Chen, Tracy; Fazenbaker, Christine; Zhong, Helen; Bee, Jared; Zeng, Zhutian; Jenne, Craig; Wu, Herren; Gao, Changshou

    2017-03-10

    Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens.

  3. Spinal GABA receptors mediate the suppressive effect of electroacupuncture on cold allodynia in rats.

    PubMed

    Park, Jung-Hyun; Han, Jae-Bok; Kim, Sun-Kwang; Park, Jung-Hyuk; Go, Dong-Hyun; Sun, Boram; Min, Byung-Il

    2010-03-31

    This study was performed to determine whether spinal GABAergic systems mediate the relieving effects of low frequency electroacupuncture (EA) on cold allodynia in a rat tail model of neuropathic pain. For neuropathic surgery, the right superior caudal trunk was resected at the level between the S1 and S2 spinal nerves innervating the tail. Two weeks after the nerve injury, the intrathecal catheter was implanted. Five days after the catheterization, rats were intrathecally injected with gabazine (GABA(A) receptor antagonist, 0.0003, 0.001 or 0.003mug), or saclofen (GABA(B) receptor antagonist, 3, 10 or 30mug). Ten minutes after the injection, EA (2Hz) was applied to the ST36 acupoint for 30min. The cold allodynia was assessed by the tail immersion test (i.e. immersing the tail in cold (4 degrees C) water and measuring the latency of an abrupt tail movement) before and after the EA treatment. EA stimulation at ST36 significantly inhibited the cold allodynia sign, whereas EA at non-acupoint and plain acupuncture at ST36 (without electrical stimulation) did not show antiallodynic effects. Intrathecal administration of gabazine or saclofen blocked the relieving effects of ST36 EA stimulation on cold allodynia. These results suggest that spinal GABA(A) and GABA(B) receptors mediate the suppressive effect of low frequency EA on cold allodynia in the tail neuropathic rats.

  4. The syndecan family of proteoglycans. Novel receptors mediating internalization of atherogenic lipoproteins in vitro.

    PubMed Central

    Fuki, I V; Kuhn, K M; Lomazov, I R; Rothman, V L; Tuszynski, G P; Iozzo, R V; Swenson, T L; Fisher, E A; Williams, K J

    1997-01-01

    Cell-surface heparan sulfate proteoglycans have been shown to participate in lipoprotein catabolism, but the roles of specific proteoglycan classes have not been examined previously. Here, we studied the involvement of the syndecan proteoglycan family. First, transfection of CHO cells with expression vectors for several syndecan core proteins produced parallel increases in the cell association and degradation of lipoproteins enriched in lipoprotein lipase, a heparan-binding protein. Second, a chimeric construct, FcR-Synd1, that consists of the ectodomain of the IgG Fc receptor Ia linked to the highly conserved transmembrane and cytoplasmic domains of syndecan-1 directly mediated efficient internalization, in a process triggered by ligand clustering. Third, internalization of lipase-enriched lipoproteins via syndecan-1 and of clustered IgGs via the chimera showed identical kinetics (t1/2 = 1 h) and identical dose-response sensitivities to cytochalasin B, which disrupts microfilaments, and to genistein, which inhibits tyrosine kinases. In contrast, internalization of the receptor-associated protein, which proceeds via coated pits, showed a t1/2 < 15 min, limited sensitivity to cytochalasin B, and complete insensitivity to genistein. Thus, syndecan proteoglycans can directly mediate ligand catabolism through a pathway with characteristics distinct from coated pits, and might act as receptors for atherogenic lipoproteins and other ligands in vivo. PMID:9294130

  5. Fenugreek induced apoptosis in breast cancer MCF-7 cells mediated independently by fas receptor change.

    PubMed

    Alshatwi, Ali Abdullah; Shafi, Gowhar; Hasan, Tarique Noorul; Syed, Naveed Ahmed; Khoja, Kholoud Khalid

    2013-01-01

    Trigonella foenum in graecum (Fenugreek) is a traditional herbal plant used to treat disorders like diabetes, high cholesterol, wounds, inflammation, gastrointestinal ailments, and it is believed to have anti-tumor properties, although the mechanisms for the activity remain to be elucidated. In this study, we prepared a methanol extract from Fenugreek whole plants and investigated the mechanism involved in its growth-inhibitory effect on MCF- 7 human breast cancer cells. Apoptosis of MCF-7 cells was evidenced by investigating trypan blue exclusion, TUNEL and Caspase 3, 8, 9, p53, FADD, Bax and Bak by real-time PCR assays inducing activities, in the presence of FME at 65 μg/mL for 24 and 48 hours. FME induced apoptosis was mediated by the death receptor pathway as demonstrated by the increased level of Fas receptor expression after FME treatment. However, such change was found to be absent in Caspase 3, 8, 9, p53, FADD, Bax and Bak, which was confirmed by a time-dependent and dose-dependent manner. In summary, these data demonstrate that at least 90% of FME induced apoptosis in breast cell is mediated by Fas receptor-independently of either FADD, Caspase 8 or 3, as well as p53 interdependently.

  6. Targeted Fcγ Receptor (FcγR)-mediated Clearance by a Biparatopic Bispecific Antibody*

    PubMed Central

    Kasturirangan, Srinath; Rainey, G. Jonah; Xu, Linda; Wang, Xinwei; Portnoff, Alyse; Chen, Tracy; Fazenbaker, Christine; Zhong, Helen; Bee, Jared; Zeng, Zhutian; Jenne, Craig; Wu, Herren; Gao, Changshou

    2017-01-01

    Soluble ligands have commonly been targeted by antibody therapeutics for cancers and other diseases. Although monoclonal antibodies targeting such ligands can block their interactions with their cognate receptors, they can also significantly increase the half-life of their ligands by FcRn-mediated antibody recycling, thereby evading ligand renal clearance and requiring increasingly high antibody doses to neutralize the increasing pool of target. To overcome this issue, we generated a bispecific/biparatopic antibody (BiSAb) that targets two different epitopes on IL-6 to block IL-6-mediated signaling. The BiSAb formed large immune complexes with IL-6 that can bind Fcγ receptors on phagocytic cells and are rapidly internalized. In addition, rapid clearance of the BiSAb·IL-6 complex was observed in mice while the parental antibodies prolonged the serum half-life of IL-6. Intravital imaging of the liver in mice confirmed that the rapid clearance of these large immune complexes was associated with Fcγ receptor-dependent binding to Kupffer cells in the liver. The approach described here provides a general strategy for therapeutic antibodies with the ability to not only neutralize but also actively drive clearance of their soluble antigens. PMID:28100773

  7. Effects of positive AMPA receptor modulators on calpain-mediated spectrin degradation in cultured hippocampal slices.

    PubMed

    Jourdi, Hussam; Yanagihara, Ted; Martinez, Ulises; Bi, Xiaoning; Lynch, Gary; Baudry, Michel

    2005-01-01

    Positive modulators of AMPA receptors (AMPAr), also known as ampakines, are allosteric effectors of the receptors and have been extensively studied in past years due to their potential use as treatment for various diseases and ailments of the central nervous system such as mild cognitive impairment, schizophrenia, and Alzheimer's disease. Ampakines have been shown to improve performance on memory tasks in animals and in human subjects, an effect linked to their ability to increase agonist-mediated ion influx through AMPAr, thus leading to enhanced synaptic responses and facilitation of long-term potentiation (LTP) induction at glutamatergic synapses. As LTP is associated with calpain activation and spectrin degradation, we determined the effects of ampakine treatment of cultured hippocampal slices on spectrin degradation. Calpain activation was evaluated by determining the levels of the 145-150kDa degradation products of spectrin. Our data indicated that incubation of hippocampal slices with some, but not all positive modulators of AMPA receptors resulted in enhanced spectrin degradation, an effect that was blocked by a calpain inhibitor. In addition, an antagonist of AMPAr but not of NMDAr blocked ampakine-induced spectrin degradation. These results indicate that prolonged treatment with selected ampakines leads to spectrin degradation mediated by activation of the calcium-dependent protease calpain.

  8. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    PubMed

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils.

  9. Evidence for multiple receptors mediating fluid secretion in salivary glands of ticks.

    PubMed

    Kaufman, W R; Wong, D L

    1983-01-28

    Using isolated salivary glands of the ixodid tick Amblyomma hebraeum Koch, we tested the effectiveness of butaclamol and sulpiride in blocking fluid secretion stimulated by a number of agonists. (+)-Butaclamol was a potent inhibitor of dopamine, N-methyldopamine and noradrenaline (Ki congruent to 30-60 nM), but was less effective on ergometrine (Ki congruent to 310 nM). Tranylcypromine-stimulated fluid secretion in the absence and presence of (+)-butaclamol and (+/-)-sulpiride suggested that tranylcypromine's action is mediated through two receptors. (+/-)-Sulpiride, though a rather weak antagonist of ergometrine (Ki congruent to 6150 nM), was ineffectual as a dopamine blocker, indicating distinct receptor sites on this epithelium for dopamine and ergometrine. Both (+)-butaclamol and sulpiride reversed the autoinhibition associated with supramaximal levels of dopamine. Sulpiride also abolished spiperone's potentiation of dopamine. Butaclamol, on the other hand, had no such effect on spiperone's potentiation of dopamine. Finally, although the CNS of ticks contains both dopamine and noradrenaline in quantity (congruent to 650 and congruent to 370 ng . g-1 res respectively), the salivary glands contain far more dopamine than noradrenaline (congruent to 85 and congruent to 6 ng . g-1 respectively). The data support the hypothesis that dopamine is a natural transmitter substance in the tick salivary gland, and that there are distinct receptor sites in the epithelium mediating the actions of catecholamines, ergot alkaloids and butyrophenones. The physiological significance of the ergot alkaloid and butyrophenone sites is not clear.

  10. Substance P selectively modulates GABA(A) receptor-mediated synaptic transmission in striatal cholinergic interneurons.

    PubMed

    Govindaiah, G; Wang, Yanyan; Cox, Charles L

    2010-02-01

    Substance P (SP) is co-localized and co-released with gamma-amino butyric acid (GABA) from approximately 50% of GABAergic medium spiny neurons (MSNs) in the striatum. MSNs innervate several cellular targets including neighboring MSNs and cholinergic interneurons via collaterals. However, the functional role of SP release onto striatal interneurons is unknown. Here we examined SP-mediated actions on inhibitory synaptic transmission in cholinergic interneurons using whole-cell recordings in mouse corticostriatal slices. We found that SP selectively suppressed GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), but not excitatory post-synaptic currents (EPSCs) in cholinergic interneurons. In contrast, SP did not alter IPSCs in fast-spiking interneurons and MSNs. SP suppressed IPSC amplitude in a concentration-dependent and reversible manner, and the NK1 receptor antagonist RP67580 attenuated the SP-mediated suppression. In addition, RP67580 alone enhanced the evoked IPSC amplitude in cholinergic interneurons, suggesting an endogenous action of SP on regulation of inhibitory synaptic transmission. SP did not alter the paired-pulse ratio, but reduced the amplitudes of GABA(A) agonist muscimol-induced outward currents and miniature IPSCs in cholinergic interneurons, suggesting SP exerts its effects primarily at the post-synaptic site. Our results indicate that the physiological effects of SP are to enhance the activity of striatal cholinergic interneurons and provide a rationale for designing potential new antiparkinsonian agents.

  11. Uncoupling scavenger receptor A-mediated phagocytosis of bacteria from endotoxic shock resistance.

    PubMed

    Amiel, Eyal; Acker, Julie L; Collins, Ryan M; Berwin, Brent

    2009-10-01

    Unresolved infection by gram-negative bacteria can result in the potentially lethal condition known as endotoxic shock, whereby uncontrolled inflammation can lead to multiple organ failure and death of the infected host. Previous results have demonstrated that animals deficient in class A scavenger receptor (SRA), a trafficking receptor for bacteria and bacterium-derived molecules, are more susceptible to endotoxic shock. This has been proposed to be a result of impaired SRA-dependent phagocytic clearance of bacteria resulting in stronger proinflammatory stimuli. In this report, we test the hypothesis that there is an obligate reciprocal relationship between SRA-mediated phagocytosis of bacteria and susceptibility to endotoxic shock. Here, we demonstrate that both SRA-dependent and -independent gram-negative bacterial strains elicit SRA-dependent increased cytokine production in vitro and in vivo and increased susceptibility to endotoxic shock in SRA-deficient mice. This is the first evidence showing that SRA-mediated clearance of LPS is functionally distinct from the role of SRA in bacterial phagocytosis and is a formal demonstration that the SRA-dependent cytokine responses and the resultant endotoxic shock are not coupled to SRA-mediated clearance of bacteria.

  12. LRP6 protein regulates low density lipoprotein (LDL) receptor-mediated LDL uptake.

    PubMed

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-06

    Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6(R611C) mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6(WT)) and LRP6(R611C) in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6(WT) was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6(WT) forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6(R611C). These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels.

  13. Calmodulin physically interacts with the erythropoietin receptor and enhances Jak2-mediated signaling

    SciTech Connect

    Kakihana, Kazuhiko; Yamamoto, Masahide; Iiyama, Mitsuko; Miura, Osamu . E-mail: miura.hema@tmd.ac.jp

    2005-09-23

    Stimulation of the erythropoietin receptor (EpoR) induces a transient increase in intracellular Ca{sup 2+} level as well as activation of the Jak2 tyrosine kinase to stimulate various downstream signaling pathways. Here, we demonstrate that the universal Ca{sup 2+} receptor calmodulin (CaM) binds EpoR in a Ca{sup 2+}-dependent manner in vitro. Binding studies using various EpoR mutants in hematopoietic cells showed that CaM binds the membrane-proximal 65-amino-acid cytoplasmic region (amino acids 258-312) of EpoR that is critical for activation of Jak2-mediated EpoR signaling. Structurally unrelated CaM antagonists, W-13 and CMZ, inhibited activation of Jak2-mediated EpoR signaling pathways, whereas W-12, a W-13 analog, did not show any significant inhibitory effect. Moreover, overexpression of CaM augmented Epo-induced tyrosine phosphorylation of the EpoR. W-13, but not W-12, also inhibited Epo-induced proliferation and survival. Together, these results indicate that CaM binds to the membrane-proximal EpoR cytoplasmic region and plays an essential role in activation of Jak2-mediated EpoR signaling.

  14. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    SciTech Connect

    Majumdar, S.; Basu, S.K. )

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections.

  15. Activation of histamine H3-receptors inhibits carrier-mediated norepinephrine release during protracted myocardial ischemia. Comparison with adenosine A1-receptors and alpha2-adrenoceptors.

    PubMed

    Imamura, M; Lander, H M; Levi, R

    1996-03-01

    We previously showed that prejunctional histamine H3-receptors downregulate norepinephrine exocytosis, which is markedly enhanced in early myocardial ischemia. In the present study, we investigated whether H3-receptors modulate nonexocytotic norepinephrine release during protracted myocardial ischemia. In this setting, decreased pH(i) in sympathetic nerve endings sequentially leads to a compensatory activation of the Na+-H+ antiporter (NHE), accumulation of intracellular Na+, reversal of the neuronal uptake of norepinephrine, and thus carrier-mediated release of norepinephrine. Accordingly, norepinephrine overflow from isolated guinea pig hearts undergoing 20-minute global ischemia and 45-minute reperfusion was attenuated approximately 80% by desipramine (10 nmol/L) and 70% by 5-(N-ethyl-N-isopropyl)-amiloride (EIPA, 10 micromol/L), inhibitors of norepinephrine uptake and NHE, respectively. The H3-receptor agonist imetit (0.1 micromol/L) decreased carrier-mediated norepinephrine release by approximately 50%. This effect was blocked by the H3-receptor antagonist thioperamide (0.3 micromol/L), indicating that H-receptor activation inhibits carrier-mediated norepinephrine release. At lower concentrations, imetit (10 nmol/L) or EIPA (3 micromol/L) did not inhibit carrier-mediated norepinephrine release. However, a 25% inhibition occurred with imetit (10 nmol/L) and EIPA (3 micromol/L) combined. This synergism suggests an association between H-receptors and NHE. Conceivably, activation of H-receptors may lead to inhibition of NHE. In fact, alpha2-adrenoceptor activation, which is known to stimulate NHE, enhanced norepinephrine release, whereas alpha2-adrenoceptor blockade attenuated it. Furthermore, activation of adenosine A1-receptors markedly attenuated norepinephrine release, whereas their inhibition potentiated it. Because norepinephrine directly correlated with the severity of reperfusion arrhythmia and imetit reduced the incidence of ventricular fibrillation by 50

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

    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.

  18. Ligation of human Fc receptor like-2 by monoclonal antibodies down-regulates B-cell receptor-mediated signalling

    PubMed Central

    Shabani, Mahdi; Bayat, Ali Ahmad; Jeddi-Tehrani, Mahmood; Rabbani, Hodjatallah; Hojjat-Farsangi, Mohammad; Ulivieri, Cristina; Amirghofran, Zahra; Baldari, Cosima Tatiana; Shokri, Fazel

    2014-01-01

    B-cell antigen receptor (BCR) signalling and its regulation through negative and positive regulators are critical for balancing B-cell response and function. Human Fc receptor like-2 (FCRL2), a member of the newly identified FCRL family, could influence B-cell signalling due to possession of both immunoreceptor tyrosine-based activation and inhibitory motifs (ITAM and ITIM). Since the natural ligand of FCRL2 has not been identified, we generated FCRL2-specific monoclonal antibodies (mAbs) and employed them to investigate the influence of FCRL2 stimulation on BCR signalling in an FCRL2-expressing B-cell line. Two anti-FCRL2 mAb-producing hybridoma clones (5A7-E7 and 3D8-G8) were selected. None of the mAbs displayed any cross-reactivity with the other members of the FCRL family including recombinant FCRL1, -3, -4 and -5, as tested by FACS and ELISA techniques. Engagement of the FCRL2 by these mAbs resulted in significant inhibition of BCR signalling mediators such as calcium mobilization and phosphorylation of the mitogen-activated protein kinases Erk, p38 and Jnk. These findings indicate that the FCRL2 ITIM motifs are functional and the anti-FCRL2 mAbs may mimic the natural ligand of FCRL2 by induction of inhibitory signals in B cells. PMID:24797767

  19. PCSK9-mediated degradation of the LDL receptor generates a 17 kDa C-terminal LDL receptor fragment.

    PubMed

    Tveten, Kristian; Strøm, Thea Bismo; Berge, Knut Erik; Leren, Trond P

    2013-06-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the LDL receptor (LDLR) at the cell surface and reroutes the internalized LDLR to intracellular degradation. In this study, we have shown that PCSK9-mediated degradation of the full-length 160 kDa LDLR generates a 17 kDa C-terminal LDLR fragment. This fragment was not generated from mutant LDLRs resistant to PCSK9-mediated degradation or when degradation was prevented by chemicals such as ammonium chloride or the cysteine cathepsin inhibitor E64d. The observation that the 17 kDa fragment was only detected when the cells were cultured in the presence of the γ-secretase inhibitor DAPT indicates that this 17 kDa fragment undergoes γ-secretase cleavage within the transmembrane domain. The failure to detect the complementary 143 kDa ectodomain fragment is likely to be due to its rapid degradation in the endosomal lumen. The 17 kDa C-terminal LDLR fragment was also generated from a Class 5 mutant LDLR undergoing intracellular degradation. Thus, one may speculate that an LDLR with bound PCSK9 and a Class 5 LDLR with bound LDL are degraded by a similar mechanism that could involve ectodomain cleavage in the endosome.

  20. Glycine receptors mediate excitation of subplate neurons in neonatal rat cerebral cortex.

    PubMed

    Kilb, W; Hanganu, I L; Okabe, A; Sava, B A; Shimizu-Okabe, C; Fukuda, A; Luhmann, H J

    2008-08-01

    The development of the cerebral cortex depends on genetic factors and early electrical activity patterns that form immature neuronal networks. Subplate neurons (SPn) are involved in the construction of thalamocortical innervation, generation of oscillatory network activity, and in the proper formation of the cortical columnar architecture. Because glycine receptors play an important role during early corticogenesis, we analyzed the functional consequences of glycine receptor activation in visually identified SPn in neocortical slices from postnatal day 0 (P0) to P4 rats using whole cell and perforated patch-clamp recordings. In all SPn the glycinergic agonists glycine, beta-alanine, and taurine induced dose-dependent inward currents with the affinity for glycine being higher than that for beta-alanine and taurine. Glycine-induced responses were blocked by the glycinergic antagonist strychnine, but were unaffected by either the GABAergic antagonist gabazine, the N-methyl-d-aspartate-receptor antagonist d-2-amino-5-phosphonopentanoic acid, or picrotoxin and cyanotriphenylborate, antagonists of alpha-homomeric and alpha1-subunit-containing glycine receptors, respectively. Under perforated-patch conditions, glycine induced membrane depolarizations that were sufficient to trigger action potentials (APs) in most cells. Furthermore, glycine and taurine decreased the injection currents as well as the synaptic stimulation strength required to elicit APs, indicating that glycine receptors have a consistent excitatory effect on SPn. Inhibition of taurine transport and application of hypoosmolar solutions induced strychnine-sensitive inward currents, suggesting that taurine can act as a possible endogenous agonist on SPn. In summary, these results demonstrate that SPn express glycine receptors that mediate robust excitatory membrane responses during early postnatal development.

  1. The chemokine receptor CXCR7 is highly expressed in human glioma cells and mediates antiapoptotic effects.

    PubMed

    Hattermann, Kirsten; Held-Feindt, Janka; Lucius, Ralph; Müerköster, Susanne Sebens; Penfold, Mark E T; Schall, Thomas J; Mentlein, Rolf

    2010-04-15

    The chemokine CXCL12/stromal cell-derived factor-1 and its receptor CXCR4 play a major role in tumor invasion, proliferation, and metastasis. Recently, CXCR7 was identified as a novel, alternate receptor for CXCL12 and CXCL11/I-TAC. Because both chemokines are expressed abundantly in human astrocytomas and glioblastomas, we investigated the occurrence and function of both receptors in astroglial tumors. In situ, CXCR7 is highly expressed on tumor endothelial, microglial, and glioma cells whereas CXCR4 has a much more restricted localization; CXCL12 is often colocalized with CXCR7. CXCR7 transcription in tumor homogenates increased with malignancy. In vitro, CXCR7 was highly expressed in all glioma cell lines investigated whereas CXCR4 was only scarcely transcribed on one of eight lines. In contrast, a tumor stem-like cell line preferentially expressed CXCR4 which diminished upon differentiation, whereas CXCR7 increased drastically. Stimulation of CXCR7-positive glioma cells (CXCR4- and CXCR3-negative) by CXCL12 induced transient phosphorylation of extracellular signal-regulated kinases Erk1/2, indicating that the receptor is functionally active. The phosphoinositide-specific phospholipase C inhibitor U73122 effectively inhibited Erk activation and suggests that the mitogen-activated protein kinase pathway is activated indirectly. Whereas proliferation and migration were little influenced, chemokine stimulation prevented camptothecin- and temozolomide-induced apoptosis. The selective CXCR7 antagonist CCX733 reduced the antiapoptotic effects of CXCL12 as shown by nuclear (Nicoletti) staining, caspase-3/7 activity assays, and cleavage of poly(ADP-ribose) polymerase-1. Thus, CXCR7 is a functional receptor for CXCL12 in astrocytomas/glioblastomas and mediates resistance to drug-induced apoptosis. Whereas CXCR7 is found on "differentiated" glioma cells, the alternate receptor CXCR4 is also localized on glioma stem-like cells.

  2. PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP in isolated mouse atria.

    PubMed

    Hoover, Donald B; Girard, Beatrice M; Hoover, Jeffrey L; Parsons, Rodney L

    2013-08-05

    PACAP and VIP have prominent effects on cardiac function in several species, but little is known about their influence on the murine heart. Accordingly, we evaluated the expression of PACAP/VIP receptors in mouse heart and the response of isolated atria to peptide agonists. Quantitative PCR demonstrated that PAC₁, VPAC₁, and VPAC₂ receptor mRNAs are present throughout the mouse heart. Expression of all three receptor transcripts was low, PAC₁ being the lowest. No regional differences in expression were detected for individual receptor mRNAs after normalization to L32. Pharmacological effects of PACAP-27, VIP, and the selective PAC₁ agonist maxadilan were evaluated in isolated, spontaneously beating atria from C57BL/6 mice of either sex. Incremental additions of PACAP-27 at 1 min intervals caused a concentration-dependent tachycardia with a logEC₅₀=-9.08 ± 0.15 M (n=7) and a maximum of 96.3 ± 5.9% above baseline heart rate. VIP and maxadilan also caused tachycardia but their potencies were about two orders of magnitude less. Increasing the dosing interval to 5 min caused a leftward shift of the concentration-response curve to maxadilan but no changes in the curves for PACAP-27 or VIP. Under this condition, neither the potency nor the efficacy of maxadilan differed from those of PACAP-27. Neither PACAP-27 nor maxadilan caused tachyphylaxis, and maximal responses to maxadilan were maintained for at least 2 h. We conclude that all three VIP/PACAP family receptors are expressed by mouse cardiac tissue, but only PAC₁ receptors mediate positive chronotropic responses to PACAP-27 and VIP.

  3. Serotonin receptor antagonists discriminate between PKA- and PKC-mediated plasticity in aplysia sensory neurons.

    PubMed

    Dumitriu, Bogdan; Cohen, Jonathan E; Wan, Qin; Negroiu, Andreea M; Abrams, Thomas W

    2006-04-01

    Highly selective serotonin (5-hydroxytryptamine, 5-HT) receptor antagonists developed for mammals are ineffective in Aplysia due to the evolutionary divergence of neurotransmitter receptors and because the higher ionic strength of physiological saline for marine invertebrates reduces antagonist affinity. It has therefore been difficult to identify antagonists that specifically block individual signaling cascades initiated by 5-HT. We studied two broad-spectrum 5-HT receptor antagonists that have been characterized biochemically in Aplysia CNS: methiothepin and spiperone. Methiothepin is highly effective in inhibiting adenylyl cyclase (AC)-coupled 5-HT receptors in Aplysia. Spiperone, which blocks phospholipase C (PLC)-coupled 5-HT receptors in mammals, does not block AC-coupled 5-HT receptors in Aplysia. In electrophysiological studies, we explored whether methiothepin and spiperone can be used in parallel to distinguish between the AC-cAMP and PLC-protein kinase C (PKC) modulatory cascades that are initiated by 5-HT. 5-HT-induced broadening of the sensory neuron action potential in the presence of tetraethylammonium/nifedipine, which is mediated by modulation of the S-K+ currents, was used an assay for the AC-cAMP cascade. Spike broadening initiated by 5 microM 5-HT was unaffected by 100 microM spiperone, whereas it was effectively blocked by 100 microM methiothepin. Facilitation of highly depressed sensory neuron-to-motor neuron synapses by 5-HT was used as an assay for the PLC-PKC cascade. Spiperone completely blocked facilitation of highly depressed synapses by 5 microM 5-HT. In contrast, methiothepin produced a modest, nonsignificant, reduction in the facilitation of depressed synapses. Interestingly, these experiments revealed that the PLC-PKC cascade undergoes desensitization during exposure to 5-HT.

  4. PACAP causes PAC1/VPAC2 receptor mediated hypertension and sympathoexcitation in normal and hypertensive rats.

    PubMed

    Farnham, M M J; Lung, M S Y; Tallapragada, V J; Pilowsky, P M

    2012-10-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is an excitatory neuropeptide that plays an important role in hypertension and stress responses. PACAP acts at three G protein-coupled receptors [PACAP type 1 receptor (PAC(1)) and vasoactive intestinal peptide receptor types 1 and 2 (VPAC(1) and VPAC(2))] and is localized to sites involved in cardiovascular control, most significantly the rostral ventrolateral medulla (RVLM). The RVLM is crucial for the tonic and reflex control of efferent sympathetic activity. Increases in sympathetic activity are observed in most types of hypertension and heart failure. PACAP delivered intrathecally also causes massive sympathoexcitation. We aimed to determine the presence and abundance of the three PACAP receptors in the RVLM, the role, in vivo, of PACAP in the RVLM on tonic and reflex cardiovascular control, and the contribution of PACAP to hypertension in the spontaneously hypertensive rat (SHR). Data were obtained using quantitative PCR and microinjection of PACAP and its antagonist, PACAP(6-38), into the RVLM of anesthetized artificially ventilated normotensive rats or SHRs. All three receptors were present in the RVLM. PACAP microinjection into the RVLM caused sustained sympathoexcitation and tachycardia with a transient hypertension but did not affect homeostatic reflexes. The responses were partially mediated through PAC(1)/VPAC(2) receptors since the effect of PACAP was attenuated (∼50%) by PACAP(6-38). PACAP was not tonically active in the RVLM in this preparation because PACAP(6-38) on its own had no inhibitory effect. PACAP has long-lasting cardiovascular effects, but altered PACAP signaling within the RVLM is not a cause of hypertension in the SHR.

  5. Presynaptic long-term depression mediated by Gi/o-coupled receptors

    PubMed Central

    Atwood, Brady K.; Lovinger, David M.; Mathur, Brian N.

    2014-01-01

    Long-term depression (LTD) of the efficacy of synaptic transmission is now recognized as an important mechanism for regulation of information storage and control of actions, as well as synapse, neuron, and circuit development. Studies of LTD mechanisms have focused mainly on postsynaptic AMPA receptor trafficking. However, the focus has now expanded to include presynaptically expressed plasticity; the predominant form being initiated by presynaptically expressed Gi/o-coupled metabotropic receptor (Gi/o-GPCR) activation. Several forms of LTD involving activation of different presynaptic Gi/o-GPCRs as a “common pathway” are described. Here, we review the literature on presynaptic Gi/o-GPCR-mediated LTD, discuss known mechanisms, gaps in our knowledge, and evaluate if all Gi/o-GPCR are capable of inducing presynaptic LTD. PMID:25160683

  6. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-02-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk.

  7. Highly chlorinated PCBs inhibit the human xenobiotic response mediated by the steroid and xenobiotic receptor (SXR).

    PubMed Central

    Tabb, Michelle M; Kholodovych, Vladyslav; Grün, Felix; Zhou, Changcheng; Welsh, William J; Blumberg, Bruce

    2004-01-01

    Polychlorinated biphenyls (PCBs) are a family of persistent organic contaminants suspected to cause adverse effects in wildlife and humans. In rodents, PCBs bind to the aryl hydrocarbon (AhR) and pregnane X receptors (PXR) inducing the expression of catabolic cytochrome p450 enzymes of the CYP1A and 3A families. We found that certain highly chlorinated PCBs are potent activators of rodent PXR but antagonize its human ortholog, the steroid and xenobiotic receptor (SXR), inhibiting target gene induction. Thus, exposure to PCBs may blunt the human xenobiotic response, inhibiting the detoxification of steroids, bioactive dietary compounds, and xenobiotics normally mediated by SXR. The antagonistic PCBs are among the most stable and abundant in human tissues. These findings have important implications for understanding the biologic effects of PCB exposure and the use of animal models to predict the attendant risk. PMID:14754570

  8. The anorectic effect of neurotensin is mediated via a histamine H1 receptor in mice.

    PubMed

    Ohinata, Kousaku; Shimano, Tomoko; Yamauchi, Rena; Sakurada, Shinobu; Yanai, Kazuhiko; Yoshikawa, Masaaki

    2004-12-01

    Neurotensin (NT), a tridecapeptide found in the mammalian brain and peripheral tissues, induces a decrease in food intake after central administration. In this investigation, we examine whether the histaminergic system is involved in NT-induced suppression of feeding. Intracerebroventricular injection of NT (0.1-1 nmol/mouse) led to dose-dependent inhibition of food intake in fasted ddY mice. The anorectic effect induced by NT (0.1 nmol/mouse) was ameliorated upon co-administration of pyrilamine (3 nmol/mouse), an antagonist for histomine H1 receptor. The NT-induced anorectic effect was partially ameliorated in H1 knockout mice. The findings suggest that the H1 receptor in part mediates the NT-induced suppression of food intake.

  9. Using GFP--ligand fusions to measure receptor-mediated endocytosis in living cells.

    PubMed

    Medina-Kauwe, Lali K; Chen, Xinhua

    2002-01-01

    Recombinant DNA technology has enabled the production of many types of chimeric proteins containing heterologous functional domains that have served a variety of useful capacities for cell biology research. Among proteins gaining wide use as a fusion partner is Aequorea victoria green fluorescent protein (GFP). GFP has been employed by numerous groups as a reporter gene for cell transfection and as an autofluorescent tag by recombinant fusion to foreign sequences. Here we describe the use of GFP as a tag for ligands, and provide examples of how purified recombinant GFP-ligand fusion proteins may be used to detect ligand-receptor interactions, including receptor-mediated endocytosis. Both its utility and limitations are discussed.

  10. Presynaptic long-term depression mediated by Gi/o-coupled receptors.

    PubMed

    Atwood, Brady K; Lovinger, David M; Mathur, Brian N

    2014-11-01

    Long-term depression (LTD) of the efficacy of synaptic transmission is now recognized as an important mechanism for the regulation of information storage and the control of actions, as well as for synapse, neuron, and circuit development. Studies of LTD mechanisms have focused mainly on postsynaptic AMPA-type glutamate receptor trafficking. However, the focus has now expanded to include presynaptically expressed plasticity, the predominant form being initiated by presynaptically expressed Gi/o-coupled metabotropic receptor (Gi/o-GPCR) activation. Several forms of LTD involving activation of different presynaptic Gi/o-GPCRs as a 'common pathway' are described. We review here the literature on presynaptic Gi/o-GPCR-mediated LTD, discuss known mechanisms, gaps in our knowledge, and evaluate whether all Gi/o-GPCRs are capable of inducing presynaptic LTD.

  11. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells

    PubMed Central

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-01

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  12. The aryl hydrocarbon receptor mediates raloxifene-induced apoptosis in estrogen receptor-negative hepatoma and breast cancer cells.

    PubMed

    O'Donnell, E F; Koch, D C; Bisson, W H; Jang, H S; Kolluri, S K

    2014-01-30

    Identification of new molecular targets for the treatment of breast cancer is an important clinical goal, especially for triple-negative breast cancer, which is refractory to existing targeted treatments. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known primarily as the mediator of dioxin toxicity. However, the AhR can also inhibit cellular proliferation in a ligand-dependent manner and act as a tumor suppressor in mice, and thus may be a potential anticancer target. To investigate the AhR as an anticancer target, we conducted a small molecule screen to discover novel AhR ligands with anticancer properties. We identified raloxifene, a selective estrogen receptor (ER) modulator currently used in the clinic for prevention of ER-positive breast cancer and osteoporosis in post-menopausal women, as an AhR activator. Raloxifene directly bound the AhR and induced apoptosis in ER-negative mouse and human hepatoma cells in an AhR-dependent manner, indicating that the AhR is a molecular target of raloxifene and mediates raloxifene-induced apoptosis in the absence of ER. Raloxifene selectively induced apoptosis of triple-negative MDA-MB-231 breast cancer cells compared with non-transformed mammary epithelial cells via the AhR. Combined with recent data showing that raloxifene inhibits triple-negative breast cancer xenografts in vivo (Int J Oncol. 43(3):785-92, 2013), our results support the possibility of repurposing of raloxifene as an AhR-targeted therapeutic for triple-negative breast cancer patients. To this end, we also evaluated the role of AhR expression on survival of patients diagnosed with breast cancer. We found that higher expression of the AhR is significantly associated with increased overall survival and distant metastasis-free survival in both hormone-dependent (ER-positive) and hormone-independent (ER and progesterone receptor (PR)-negative) breast cancers. Together, our data strongly support the possibility of using the Ah

  13. 5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

    PubMed Central

    Verheggen, R; Hundeshagen, A G; Brown, A M; Schindler, M; Kaumann, A J

    1998-01-01

    In the human temporal artery both 5-HT1-like and 5-HT2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT1-like receptors resemble more closely recombinant 5-HT1B than 5-HT1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT1B, 5-HT1D and other 5-HT receptors.The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent KB=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).Sumatriptan evoked contractions (EC50, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).The potency of 5-HT (EC50) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.We detected arterial receptor mRNA for the following receptors (incidence): 5-HT1B (8/8), 5-HT1D (2/8), 5-HT1F (0/4), 5-HT2A (0/8), 5-HT2B (0/8), 5-HT2C (0/8), 5-HT4 (4/8) and 5-HT7 (4/8).We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT1D receptors as mediators of the contractile effects of 5-HT and sumatriptan. PMID:9723944

  14. Insulin and IGF-1 receptors regulate FoxO-mediated signaling in muscle proteostasis

    PubMed Central

    O’Neill, Brian T.; Lee, Kevin Y.; Klaus, Katherine; Softic, Samir; Krumpoch, Megan T.; Stanford, Kristin I.; Robinson, Matthew M.; Cai, Weikang; Kleinridders, Andre; Pereira, Renata O.; Hirshman, Michael F.; Accili, Domenico; Goodyear, Laurie J.; Nair, K. Sreekumaran

    2016-01-01

    Diabetes strongly impacts protein metabolism, particularly in skeletal muscle. Insulin and IGF-1 enhance muscle protein synthesis through their receptors, but the relative roles of each in muscle proteostasis have not been fully elucidated. Using mice with muscle-specific deletion of the insulin receptor (M-IR–/– mice), the IGF-1 receptor (M-IGF1R–/– mice), or both (MIGIRKO mice), we assessed the relative contributions of IR and IGF1R signaling to muscle proteostasis. In differentiated muscle, IR expression predominated over IGF1R expression, and correspondingly, M-IR–/– mice displayed a moderate reduction in muscle mass whereas M-IGF1R–/– mice did not. However, these receptors serve complementary roles, such that double-knockout MIGIRKO mice displayed a marked reduction in muscle mass that was linked to increases in proteasomal and autophagy-lysosomal degradation, accompanied by a high-protein-turnover state. Combined muscle-specific deletion of FoxO1, FoxO3, and FoxO4 in MIGIRKO mice reversed increased autophagy and completely rescued muscle mass without changing proteasomal activity. These data indicate that signaling via IR is more important than IGF1R in controlling proteostasis in differentiated muscle. Nonetheless, the overlap of IR and IGF1R signaling is critical to the regulation of muscle protein turnover, and this regulation depends on suppression of FoxO-regulated, autophagy-mediated protein degradation. PMID:27525440

  15. Lactate modulates the activity of primary cortical neurons through a receptor-mediated pathway.

    PubMed

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism.

  16. Lactate Modulates the Activity of Primary Cortical Neurons through a Receptor-Mediated Pathway

    PubMed Central

    Bozzo, Luigi; Puyal, Julien; Chatton, Jean-Yves

    2013-01-01

    Lactate is increasingly described as an energy substrate of the brain. Beside this still debated metabolic role, lactate may have other effects on brain cells. Here, we describe lactate as a neuromodulator, able to influence the activity of cortical neurons. Neuronal excitability of mouse primary neurons was monitored by calcium imaging. When applied in conjunction with glucose, lactate induced a decrease in the spontaneous calcium spiking frequency of neurons. The effect was reversible and concentration dependent (IC50 ∼4.2 mM). To test whether lactate effects are dependent on energy metabolism, we applied the closely related substrate pyruvate (5 mM) or switched to different glucose concentrations (0.5 or 10 mM). None of these conditions reproduced the effect of lactate. Recently, a Gi protein-coupled receptor for lactate called HCA1 has been introduced. To test if this receptor is implicated in the observed lactate sensitivity, we incubated cells with pertussis toxin (PTX) an inhibitor of Gi-protein. PTX prevented the decrease of neuronal activity by L-lactate. Moreover 3,5-dyhydroxybenzoic acid, a specific agonist of the HCA1 receptor, mimicked the action of lactate. This study indicates that lactate operates a negative feedback on neuronal activity by a receptor-mediated mechanism, independent from its intracellular metabolism. PMID:23951229

  17. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor

    PubMed Central

    Hennen, Stephanie; Kodra, János T.; Soroka, Vladyslav; Krogh, Berit O.; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G.; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S.; Reedtz-Runge, Steffen

    2016-01-01

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors. PMID:27196125

  18. Ultraslow Water-Mediated Transmembrane Interactions Regulate the Activation of A2A Adenosine Receptor

    NASA Astrophysics Data System (ADS)

    Lee, Yoonji; Kim, Songmi; Choi, Sun; Hyeon, Changbong

    2016-09-01

    Water molecules inside G-protein coupled receptor have recently been spotlighted in a series of crystal structures. To decipher the dynamics and functional roles of internal waters in GPCR activity, we studied A$_{\\text{2A}}$ adenosine receptor using $\\mu$sec-molecular dynamics simulations. Our study finds that the amount of water flux across the transmembrane (TM) domain varies depending on the receptor state, and that the water molecules of the TM channel in the active state flow three times slower than those in the inactive state. Depending on the location in solvent-protein interface as well as the receptor state, the average residence time of water in each residue varies from $\\sim\\mathcal{O}(10^2)$ psec to $\\sim\\mathcal{O}(10^2)$ nsec. Especially, water molecules, exhibiting ultraslow relaxation ($\\sim\\mathcal{O}(10^2)$ nsec) in the active state, are found around the microswitch residues that are considered activity hotspots for GPCR function. A continuous allosteric network spanning the TM domain, arising from water-mediated contacts, is unique in the active state, underscoring the importance of slow waters in the GPCR activation.

  19. M2-like macrophages are responsible for collagen degradation through a mannose receptor-mediated pathway.

    PubMed

    Madsen, Daniel H; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S; Brenner, David A; Burgdorf, Sven; Engelholm, Lars H; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto; Bugge, Thomas H

    2013-09-16

    Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase-dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor-associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process.

  20. The Fc and not CD4 Receptor Mediates Antibody Enhancement of HIV Infection in Human Cells

    NASA Astrophysics Data System (ADS)

    Homsy, Jacques; Meyer, Mia; Tateno, Masatoshi; Clarkson, Sarah; Levy, Jay A.

    1989-06-01

    Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.

  1. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed Central

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-01-01

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism. PMID:6896821

  2. A new Kupffer cell receptor mediating plasma clearance of carcinoembryonic antigen by the rat.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1982-05-15

    Native human carcinoembryonic antigen is rapidly removed from the circulation by the rat liver Kupffer cell after intravenous injection. The molecule is subsequently transferred to the hepatocyte in an immunologically identifiable form. Carcinoembryonic antigen has a circulatory half-life of 3.7 (+/- 0.8) min, and cellular entry is by receptor-mediated endocytosis. Non-specific fluid pinocytosis and phagocytosis can be excluded as possible mechanisms by the kinetics of clearance and failure of colloidal carbon to inhibit uptake. Substances with known affinity for the hepatic receptors for mannose, N-acetylglucosamine, fucose and galactose all fail to inhibit carcinoembryonic antigen clearance. After two cycles of the Smith degradation, carcinoembryonic antigen is still able to inhibit clearance of the native molecule. Receptor specificity is apparently not dependent on those non-reducing terminal sugars of the native molecule. Performic acid-oxidized carcinoembryonic antigen also inhibits clearance of carcinoembryonic antigen in vivo. Receptor binding is not dependent on tertiary protein conformation. Non-specific cross-reacting antigen, a glycoprotein structurally similar to carcinoembryonic antigen, is cleared by the same mechanism.

  3. Structural insight into antibody-mediated antagonism of the Glucagon-like peptide-1 Receptor.

    PubMed

    Hennen, Stephanie; Kodra, János T; Soroka, Vladyslav; Krogh, Berit O; Wu, Xiaoai; Kaastrup, Peter; Ørskov, Cathrine; Rønn, Sif G; Schluckebier, Gerd; Barbateskovic, Silvia; Gandhi, Prafull S; Reedtz-Runge, Steffen

    2016-05-19

    The Glucagon-like peptide-1 receptor (GLP-1R) is a member of the class B G protein-coupled receptor (GPCR) family and a well-established target for the treatment of type 2 diabetes. The N-terminal extracellular domain (ECD) of GLP-1R is important for GLP-1 binding and the crystal structure of the GLP-1/ECD complex was reported previously. The first structure of a class B GPCR transmembrane (TM) domain was solved recently, but the full length receptor structure is still not well understood. Here we describe the molecular details of antibody-mediated antagonism of the GLP-1R using both in vitro pharmacology and x-ray crystallography. We showed that the antibody Fab fragment (Fab 3F52) blocked the GLP-1 binding site of the ECD directly and thereby acts as a competitive antagonist of native GLP-1. Interestingly, Fab 3F52 also blocked a short peptide agonist believed to engage primarily the transmembrane and extracellular loop region of GLP-1R, whereas functionality of an allosteric small-molecule agonist was not inhibited. This study has implications for the structural understanding of the GLP-1R and related class B GPCRs, which is important for the development of new and improved therapeutics targeting these receptors.

  4. Transient receptor potential channels function as a coincidence signal detector mediating phosphatidylserine exposure.

    PubMed

    Harper, Matthew T; Londoño, Juan E Camacho; Quick, Kathryn; Londoño, Julia Camacho; Flockerzi, Veit; Philipp, Stephan E; Birnbaumer, Lutz; Freichel, Marc; Poole, Alastair W

    2013-06-25

    Blood platelet aggregation must be tightly controlled to promote clotting at injury sites but avoid inappropriate occlusion of blood vessels. Thrombin, which cleaves and activates Gq-coupled protease-activated receptors, and collagen-related peptide, which activates the receptor glycoprotein VI, stimulate platelets to aggregate and form thrombi. Coincident activation by these two agonists synergizes, causing the exposure of phosphatidylserine on the cell surface, which is a marker of cell death in many cell types. Phosphatidylserine exposure is also essential to produce additional thrombin on platelet surfaces, which contributes to thrombosis. We found that activation of either thrombin receptors or glycoprotein VI alone produced a calcium signal that was largely dependent only on store-operated Ca(2+) entry. In contrast, experiments with platelets from knockout mice showed that the presence of both ligands activated nonselective cation channels of the transient receptor potential C (TRPC) family, TRPC3 and TRPC6. These channels principally allowed entry of Na(+), which coupled to reverse-mode Na(+)/Ca(2+) exchange to allow calcium influx and thereby contribute to Ca(2+) signaling and phosphatidylserine exposure. Thus, TRPC channels act as coincidence detectors to coordinate responses to multiple signals in cells, thereby indirectly mediating in platelets an increase in intracellular calcium concentrations and exposure of prothrombotic phosphatidylserine.

  5. β-arrestins: regulatory role and therapeutic potential in opioid and cannabinoid receptor-mediated analgesia.

    PubMed

    Raehal, Kirsten M; Bohn, Laura M

    2014-01-01

    Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of "pharmacological" interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects.

  6. Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression

    PubMed Central

    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

  7. β-Arrestins: Regulatory Role and Therapeutic Potential in Opioid and Cannabinoid Receptor-Mediated Analgesia

    PubMed Central

    Bohn, Laura M.

    2016-01-01

    Pain is a complex disorder with neurochemical and psychological components contributing to the severity, the persistence, and the difficulty in adequately treating the condition. Opioid and cannabinoids are two classes of analgesics that have been used to treat pain for centuries and are arguably the oldest of “pharmacological” interventions used by man. Unfortunately, they also produce several adverse side effects that can complicate pain management. Opioids and cannabinoids act at G protein-coupled receptors (GPCRs), and much of their effects are mediated by the mu-opioid receptor (MOR) and cannabinoid CB1 receptor (CB1R), respectively. These receptors couple to intracellular second messengers and regulatory proteins to impart their biological effects. In this chapter, we review the role of the intracellular regulatory proteins, β-arrestins, in modulating MOR and CB1R and how they influence the analgesic and side-effect profiles of opioid and cannabinoid drugs in vivo. This review of the literature suggests that the development of opioid and cannabinoid agonists that bias MOR and CB1R toward G protein signaling cascades and away from β-arrestin interactions may provide a novel mechanism by which to produce analgesia with less severe adverse effects. PMID:24292843

  8. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells.

    PubMed

    Joshi, Mahesh S; Ferguson, T Bruce; Johnson, Fruzsina K; Johnson, Robert A; Parthasarathy, Sampath; Lancaster, Jack R

    2007-06-12

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and alpha-2 adrenoceptors (alpha-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and alpha-2 adrenoceptors, partly inhibited L-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of alpha-2 AR, at very low concentrations completely inhibited NO formation. Like L-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of alpha-2 AR was very potent in activating cellular NO, thus indicating a possible role for alpha-2 AR in L-arginine-mediated NO synthesis. D-arginine also activated NO production and could be inhibited by imidazoline and alpha-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated L-arginine-mediated NO synthesis, thus indicating mediation via G proteins. L-type Ca(2+) channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the L-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of L-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development.

  9. Prolactin mediates neuroprotection against excitotoxicity in primary cell cultures of hippocampal neurons via its receptor.

    PubMed

    Vergara-Castañeda, E; Grattan, D R; Pasantes-Morales, H; Pérez-Domínguez, M; Cabrera-Reyes, E A; Morales, T; Cerbón, M

    2016-04-01

    Recently it has been reported that prolactin (PRL) exerts a neuroprotective effect against excitotoxicity in hippocampus in the rat in vivo models. However, the exact mechanism by which PRL mediates this effect is not completely understood. The aim of our study was to assess whether prolactin exerts neuroprotection against excitotoxicity in an in vitro model using primary cell cultures of hippocampal neurons, and to determine whether this effect is mediated via the prolactin receptor (PRLR). Primary cell cultures of rat hippocampal neurons were used in all experiments, gene expression was evaluated by RT-qPCR, and protein expression was assessed by Western blot analysis and immunocytochemistry. Cell viability was assessed by using the MTT method. The results demonstrated that PRL treatment of neurons from primary cultures did not modify cell viability, but that it exerted a neuroprotective effect, with cells treated with PRL showing a significant increase of viability after glutamate (Glu)--induced excitotoxicity as compared with neurons treated with Glu alone. Cultured neurons expressed mRNA for both PRL and its receptor (PRLR), and both PRL and PRLR expression levels changed after the excitotoxic insult. Interestingly, the PRLR protein was detected as two main isoforms of 100 and 40 kDa as compared with that expressed in hypothalamic cells, which was present only as a 30 kDa variant. On the other hand, PRL was not detected in neuron cultures, either by western blot or by immunohistochemistry. Neuroprotection induced by PRL was significantly blocked by specific oligonucleotides against PRLR, thus suggesting that the PRL role is mediated by its receptor expressed in these neurons. The overall results indicated that PRL induces neuroprotection in neurons from primary cell cultures.

  10. Receptor-mediated activation of nitric oxide synthesis by arginine in endothelial cells

    PubMed Central

    Joshi, Mahesh S.; Ferguson, T. Bruce; Johnson, Fruzsina K.; Johnson, Robert A.; Parthasarathy, Sampath; Lancaster, Jack R.

    2007-01-01

    Arginine contains the guanidinium group and thus has structural similarity to ligands of imidazoline and α-2 adrenoceptors (α-2 AR). Therefore, we investigated the possibility that exogenous arginine may act as a ligand for these receptors in human umbilical vein endothelial cells and activate intracellular nitric oxide (NO) synthesis. Idazoxan, a mixed antagonist of imidazoline and α-2 adrenoceptors, partly inhibited l-arginine-initiated NO formation as measured by a Griess reaction. Rauwolscine, a highly specific antagonist of α-2 AR, at very low concentrations completely inhibited NO formation. Like l-arginine, agmatine (decarboxylated arginine) also activated NO synthesis, however, at much lower concentrations. We found that dexmedetomidine, a specific agonist of α-2 AR was very potent in activating cellular NO, thus indicating a possible role for α-2 AR in l-arginine-mediated NO synthesis. d-arginine also activated NO production and could be inhibited by imidazoline and α-2 AR antagonists, thus indicating nonsubstrate actions of arginine. Pertussis toxin, an inhibitor of G proteins, attenuated l-arginine-mediated NO synthesis, thus indicating mediation via G proteins. l-type Ca2+ channel blocker nifedipine and phospholipase C inhibitor U73122 inhibited NO formation and thus implicated participation of a second messenger pathway. Finally, in isolated rat gracilis vessels, rauwolscine completely inhibited the l-arginine-initiated vessel relaxation. Taken together, these data provide evidence for binding of arginine to membrane receptor(s), leading to the activation of endothelial NO synthase (eNOS) NO production through a second messenger pathway. These findings provide a previously unrecognized mechanistic explanation for the beneficial effects of l-arginine in the cardiovascular system and thus provide new potential avenues for therapeutic development. PMID:17535904

  11. Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

    PubMed Central

    Limberg, Jacqueline K.; Peltonen, Garrett L.; Johansson, Rebecca E.; Harrell, John W.; Kellawan, Jeremy M.; Eldridge, Marlowe W.; Sebranek, Joshua J.; Walker, Benjamin J.; Schrage, William G.

    2016-01-01

    Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control. PMID:27375493

  12. Communication: a simple analytical formula for the free energy of ligand-receptor-mediated interactions.

    PubMed

    Angioletti-Uberti, Stefano; Varilly, Patrick; Mognetti, Bortolo M; Tkachenko, Alexei V; Frenkel, Daan

    2013-01-14

    Recently [P. Varilly, S. Angioletti-Uberti, B. M. Mognetti, and D. Frenkel, "A general theory of DNA-mediated and other valence-limited colloidal interactions," J. Chem. Phys. 137, 094108 (2012)], we presented a general theory for calculating the strength and properties of colloidal interactions mediated by ligand-receptor bonds (such as those that bind DNA-coated colloids). In this Communication, we derive a surprisingly simple analytical form for the interaction free energy, which was previously obtainable only via a costly numerical thermodynamic integration. As a result, the computational effort to obtain potentials of interaction is significantly reduced. Moreover, we can gain insight from this analytic expression for the free energy in limiting cases. In particular, the connection of our general theory to other previous specialised approaches is now made transparent. This important simplification will significantly broaden the scope of our theory.

  13. Constitutive aryl hydrocarbon receptor signaling constrains type I interferon-mediated antiviral innate defense.

    PubMed

    Yamada, Taisho; Horimoto, Hiromasa; Kameyama, Takeshi; Hayakawa, Sumio; Yamato, Hiroaki; Dazai, Masayoshi; Takada, Ayato; Kida, Hiroshi; Bott, Debbie; Zhou, Angela C; Hutin, David; Watts, Tania H; Asaka, Masahiro; Matthews, Jason; Takaoka, Akinori

    2016-06-01

    Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxic activity of many environmental xenobiotics. However, its role in innate immune responses during viral infection is not fully understood. Here we demonstrate that constitutive AHR signaling negatively regulates the type I interferon (IFN-I) response during infection with various types of virus. Virus-induced IFN-β production was enhanced in AHR-deficient cells and mice and resulted in restricted viral replication. We found that AHR upregulates expression of the ADP-ribosylase TIPARP, which in turn causes downregulation of the IFN-I response. Mechanistically, TIPARP interacted with the kinase TBK1 and suppressed its activity by ADP-ribosylation. Thus, this study reveals the physiological importance of endogenous activation of AHR signaling in shaping the IFN-I-mediated innate response and, further, suggests that the AHR-TIPARP axis is a potential therapeutic target for enhancing antiviral responses.

  14. On the calcium receptor that mediates depolarization-secretion coupling at cholinergic motor nerve terminals

    PubMed Central

    Silinsky, E.M.

    1981-01-01

    1 The behaviour of the divalent cations Ca and Sr as agonists for receptors that mediate the synchronous evoked secretion of acetylcholine (ACh) was studied in the hope of determining whether the relationship between Ca binding and ACh secretion is determined only by the law of mass action or by the mathematical framework of receptor theory. Experiments were designed to evaluate the assumption that maximum effect requires occupation of all receptors by testing for the presence of spare Ca receptors on presynaptic terminals. Frog cutaneous nerve-muscle preparations were employed in conjunction with conventional electrophysiological methods. 2 Curves of log [Ca] or log [Sr] against the mean number of ACh quanta released (m̄) were constructed to saturation. The log [Sr]-m̄ relationship was shifted to the right and had a smaller maximum than the log [Ca]-m̄ curve. This suggests that Ca has a higher efficacy than Sr and raises the possibility that spare binding sites are present for Ca. 3 As a qualitative test for spare Ca receptors, La3+ (≥0.5 μm) or 2-chloroadenosine (25 μm) was employed as an irreversible antagonist of the effects of extracellular Ca on evoked ACh release. Despite the irreversible blockade of a proportion of receptors, increases in the [Ca] overcame this antagonism and produced a parallel shift in the log [Ca]-m̄ relation to the right. This suggests an apparent receptor reserve for Ca. Antagonism of Sr-mediated ACh release by either La3+ or 2-chloroadenosine could not be overcome by increasing the [Sr]. 4 As a quantitative test for spare Ca binding sites, the equilibrium affinity constant for Sr(KSr) as a competitive inhibitor of Ca was determined and compared with values for KSr calculated by two other methods which invoke the spare receptor assumption. All three methods produced comparable results. (KSr = 0.24-0.27 mm-1). 5 The equilibrium affinity constant for Ca (KCa) was calculated by comparing reciprocal plots of the concentrations of

  15. Synaptic NMDA receptor-mediated currents in anterior piriform cortex are reduced in the adult fragile X mouse.

    PubMed

    Gocel, James; Larson, John

    2012-09-27

    Fragile X syndrome is a neurodevelopmental condition caused by the transcriptional silencing of the fragile X mental retardation 1 (FMR1) gene. The Fmr1 knockout (KO) mouse exhibits age-dependent deficits in long term potentiation (LTP) at association (ASSN) synapses in anterior piriform cortex (APC). To investigate the mechanisms for this, whole-cell voltage-clamp recordings of ASSN stimulation-evoked synaptic currents were made in APC of slices from adult Fmr1-KO and wild-type (WT) mice, using the competitive N-methyl-D-aspartate (NMDA) receptor antagonist, CPP, to distinguish currents mediated by NMDA and AMPA receptors. NMDA/AMPA current ratios were lower in Fmr1-KO mice than in WT mice, at ages ranging from 3-18months. Since amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) mediated by AMPA receptors were no different in Fmr1-KO and WT mice at these ages, the results suggest that NMDA receptor-mediated currents are selectively reduced in Fmr1-KO mice. Analyses of voltage-dependence and decay kinetics of NMDA receptor-mediated currents did not reveal differences between Fmr1-KO and WT mice, suggesting that reduced NMDA currents in Fmr1-KO mice are due to fewer synaptic receptors rather than differences in receptor subunit composition. Reduced NMDA receptor signaling may help to explain the LTP deficit seen at APC ASSN synapses in Fmr1-KO mice at 6-18months of age, but does not explain normal LTP at these synapses in mice 3-6months old. Evoked currents and mEPSCs were also examined in senescent Fmr1-KO and WT mice at 24-28months of age. NMDA/AMPA ratios were similar in senescent WT and Fmr1-KO mice, due to a decrease in the ratio in the WT mice, without significant change in AMPA receptor-mediated mEPSCs.

  16. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    PubMed

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  17. Med1 Subunit of the Mediator Complex in Nuclear Receptor-Regulated Energy Metabolism, Liver Regeneration, and Hepatocarcinogenesis

    PubMed Central

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K.

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  18. Pharmacological profile of the receptors that mediate external carotid vasoconstriction by 5-HT in vagosympathectomized dogs.

    PubMed

    Villalón, C M; Ramírez-San Juan, E; Castillo, C; Castillo, E; López-Muñoz, F J; Terrón, J A

    1995-11-01

    1. 5-Hydroxytryptamine (5-HT) can produce vasodilatation or vasoconstriction of the canine external carotid bed depending upon the degree of carotid sympathetic tone. Hence, external carotid vasodilatation to 5-HT in dogs with intact sympathetic tone is primarily mediated by prejunctional 5-HT1-like receptors similar to the 5-HT1D subtype, which inhibit the carotid sympathetic outflow. The present investigation is devoted to the pharmacological analysis of the receptors mediating external carotid vasoconstriction by 5-HT in vagosympathectomized dogs. 2. Intracarotid (i.c.) infusions for 1 min of 5-HT (0.3, 1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent decreases in both external carotid blood flow and the corresponding conductance; both mean arterial blood pressure and heart rate remained unchanged during the infusions of 5-HT. These responses to 5-HT were resistant to blockade by antagonists at 5-HT2 (ritanserin) and 5-HT3/5-HT4 (tropisetron) receptors, but were partly blocked by the 5-HT1-like and 5-HT2 receptor antagonist, methiothepin (0.3 mg kg-1); higher doses of methiothepin (1 and 3 mg kg-1) caused little, if any, further blockade. These methiothepin (3 mg kg-1)-resistant responses to 5-HT were not significantly antagonized by MDL 72222 (0.3 mg kg-1) or tropisetron (3 mg kg-1). 3. The external carotid vasoconstrictor effects of 5-HT were mimicked by the selective 5-HT1-like receptor agonist, sumatriptan (3, 10, 30 and 100 micrograms during 1 min, i.c.), which produced dose-dependent decreases in external carotid blood flow and the corresponding conductance; these effects of sumatriptan were dose-dependently antagonized by methiothepin (0.3, 1 and 3 mg kg-1), but not by 5-HT1D-like receptor blocking doses of metergoline (0.1 mg kg-1). 4. The above vasoconstrictor effects of 5-HT remained unaltered after administration of phentolamine, propranolol, atropine, hexamethonium, brompheniramine, cimetidine and haloperidol, thus excluding the

  19. Pharmacological profile of the receptors that mediate external carotid vasoconstriction by 5-HT in vagosympathectomized dogs.

    PubMed Central

    Villalón, C. M.; Ramírez-San Juan, E.; Castillo, C.; Castillo, E.; López-Muñoz, F. J.; Terrón, J. A.

    1995-01-01

    1. 5-Hydroxytryptamine (5-HT) can produce vasodilatation or vasoconstriction of the canine external carotid bed depending upon the degree of carotid sympathetic tone. Hence, external carotid vasodilatation to 5-HT in dogs with intact sympathetic tone is primarily mediated by prejunctional 5-HT1-like receptors similar to the 5-HT1D subtype, which inhibit the carotid sympathetic outflow. The present investigation is devoted to the pharmacological analysis of the receptors mediating external carotid vasoconstriction by 5-HT in vagosympathectomized dogs. 2. Intracarotid (i.c.) infusions for 1 min of 5-HT (0.3, 1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent decreases in both external carotid blood flow and the corresponding conductance; both mean arterial blood pressure and heart rate remained unchanged during the infusions of 5-HT. These responses to 5-HT were resistant to blockade by antagonists at 5-HT2 (ritanserin) and 5-HT3/5-HT4 (tropisetron) receptors, but were partly blocked by the 5-HT1-like and 5-HT2 receptor antagonist, methiothepin (0.3 mg kg-1); higher doses of methiothepin (1 and 3 mg kg-1) caused little, if any, further blockade. These methiothepin (3 mg kg-1)-resistant responses to 5-HT were not significantly antagonized by MDL 72222 (0.3 mg kg-1) or tropisetron (3 mg kg-1). 3. The external carotid vasoconstrictor effects of 5-HT were mimicked by the selective 5-HT1-like receptor agonist, sumatriptan (3, 10, 30 and 100 micrograms during 1 min, i.c.), which produced dose-dependent decreases in external carotid blood flow and the corresponding conductance; these effects of sumatriptan were dose-dependently antagonized by methiothepin (0.3, 1 and 3 mg kg-1), but not by 5-HT1D-like receptor blocking doses of metergoline (0.1 mg kg-1). 4. The above vasoconstrictor effects of 5-HT remained unaltered after administration of phentolamine, propranolol, atropine, hexamethonium, brompheniramine, cimetidine and haloperidol, thus excluding the

  20. Targeting Toll-like receptor 4 prevents cobalt-mediated inflammation.

    PubMed

    Lawrence, Helen; Mawdesley, Amy Elizabeth; Holland, James Patrick; Kirby, John Andrew; Deehan, David John; Tyson-Capper, Alison Jane

    2016-02-16

    Cobalt-chrome alloy is a widely used biomaterial in joint replacements, dental implants and spinal rods. Although it is an effective and biocompatible material, adverse reactions to metal debris (ARMD) have arisen in a minority of patients, particularly in those with metal-on-metal bearing hip replacements. There is currently no treatment for ARMD and once progressive, early revision surgery of the implant is necessary. Therapeutic agents to prevent, halt or reverse ARMD would therefore be advantageous. Cobalt ions activate Toll-like receptor 4 (TLR4), an innate immune receptor responsible for inflammatory responses to bacterial lipopolysaccharide (LPS) resulting in the production of pro-inflammatory cytokines and chemokines. We hypothesised that anti-TLR4 neutralising antibodies, reported to inhibit TLR4-mediated inflammation, could prevent the inflammatory response to cobalt ions in an in vitro macrophage cell culture model. This study shows that a monoclonal anti-TLR4 antibody inhibited cobalt-mediated increases in pro-inflammatory IL8, CCL20 and IL1A expression, as well as IL-8 secretion. In contrast, a polyclonal antibody did not prevent the effect of cobalt ions on either IL-8 or IL1A expression, although it did have a small effect on the CCL20 response. Interestingly, both antibodies inhibited cobalt-mediated neutrophil migration although the greater effect was observed with the monoclonal antibody. In summary our data shows that a monoclonal anti-TLR4 antibody can inhibit cobalt-mediated inflammatory responses while a polyclonal antibody only inhibits the effect of specific cytokines. Anti-TLR4 antibodies have therapeutic potential in ARMD although careful antibody design is required to ensure that the LPS response is preserved.

  1. NR2D-containing NMDA receptors mediate tissue plasminogen activator-promoted neuronal excitotoxicity.

    PubMed

    Baron, A; Montagne, A; Cassé, F; Launay, S; Maubert, E; Ali, C; Vivien, D

    2010-05-01

    Although the molecular bases of its actions remain debated, tissue-type plasminogen activator (tPA) is a paradoxical brain protease, as it favours some learning/memory processes, but increases excitotoxic neuronal death. Here, we show that, in cultured cortical neurons, tPA selectively promotes NR2D-containing N-methyl-D-aspartate receptor (NMDAR)-dependent activation. We show that tPA-mediated signalling and neurotoxicity through the NMDAR are blocked by co-application of an NR2D antagonist (phenanthrene derivative (2S(*), 3R(*))-1-(phenanthrene-2-carbonyl)piperazine-2,3-dicarboxylic acid, PPDA) or knockdown of neuronal NR2D expression. In sharp contrast with cortical neurons, hippocampal neurons do not exhibit NR2D both in vitro and in vivo and are consequently resistant to tPA-promoted NMDAR-mediated neurotoxicity. Moreover, we have shown that activation of synaptic NMDAR prevents further tPA-dependent NMDAR-mediated neurotoxicity and sensitivity to PPDA. This study shows that the earlier described pro-neurotoxic effect of tPA is mediated by NR2D-containing NMDAR-dependent extracellular signal-regulated kinase activation, a deleterious effect prevented by synaptic pre-activation.

  2. IgE Receptor-Mediated Mast-Cell Renin Release

    PubMed Central

    Aldi, Silvia; Robador, Pablo A.; Tomita, Kengo; Di Lorenzo, Annarita; Levi, Roberto

    2015-01-01

    Renin is a newly discovered constituent of mast cells. Given that mast cells play a major role in IgE-mediated allergic hypersensitivity, we investigated whether activation of the high-affinity IgE receptor FcεRI elicits release of mast-cell renin. Cross-linking of FcεRI on the surface of mature bone marrow–derived mast cells elicited release of enzymatically active renin protein. The angiotensin I–forming activity of the renin protein was completely blocked by the selective renin inhibitor BILA 2157, which excludes formation of angiotensin I by proteases other than renin. FcεRI-mediated mast-cell renin release was inhibited by dexamethasone and potentiated by the proinflammatory mediator PGE2. Furthermore, cross-linking of mast-cell FcεRI in ex vivo murine hearts passively sensitized with monoclonal anti-DNP IgE also resulted in mast-cell degranulation and overflow of renin. Our findings indicate that IgE-mediated allergic hypersensitivity provokes release of renin from both cultured and resident cardiac mast cells, a process likely to be exacerbated in a chronic inflammatory background. Given the widespread distribution of mast cells, and the presence of angiotensinogen and angiotensin-converting enzyme in many tissues, renin release in immediate hypersensitivity reactions could result in local angiotensin II generation and multiorgan dysfunctions. PMID:24262755

  3. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed

    Villalón, C M; Contreras, J; Ramírez-San Juan, E; Castillo, C; Perusquía, M; Terrón, J A

    1995-12-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  4. Characterization of prejunctional 5-HT receptors mediating inhibition of sympathetic vasopressor responses in the pithed rat.

    PubMed Central

    Villalón, C. M.; Contreras, J.; Ramírez-San Juan, E.; Castillo, C.; Perusquía, M.; Terrón, J. A.

    1995-01-01

    1. It has recently been shown that continuous infusions of 5-hydroxytryptamine (5-HT) are able to inhibit, in a dose-dependent manner, the pressor responses induced by preganglionic (T7-T9) sympathetic stimulation in pithed rats pretreated with desipramine (50 micrograms kg-1, i.v.). This inhibitory effect, besides being significantly more pronounced at lower frequencies of stimulation (0.03-I Hz) and devoid of tachyphylaxis, is reversible after interrupting the infusions of 5-HT (up to 5.6 micrograms kg-1 min-1). In the present study we have characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-HT. 2. The inhibition induced by 5.6 micrograms kg-1 min-1 of 5-HT on sympathetically-induced pressor responses was not blocked after i.v. treatment with physiological saline (1 ml kg-1), ritanserin (0.1 mg kg-1), MDL 72222 (0.15 mg kg-1) or tropisetron (3 mg kg-1), which did not modify the sympathetically-induced pressor responses per se, but was significantly antagonized by the 5-HT1-like and 5-HT2 receptor antagonist, methysergide (0.3 mg kg-1), which also produced a slight attenuation of the pressor responses to 0.03 and 0.1 Hz per se. 3. Unexpectedly and contrasting with methysergide, the 5-HT1-like and 5-HT2 receptor antagonists, methiothepin (0.01, 0.03 and 0.1 mg kg-1) and metergoline (1 and 3 mg kg-1), apparently failed to block the above 5-HT-induced inhibition. Nevertheless, it is noteworthy that these antagonists also blocked the electrically-induced pressor responses per se, presumably by blockade of vascular alpha 1-adrenoceptors and, indeed, this property might have masked their potential antagonism at the inhibitory 5-HT1-like receptors. 4. Consistent with the above findings, 5-carboxamidotryptamine (5-CT, a potent 5-HT1-like receptor agonist), metergoline and methysergide mimicked the inhibitory action of 5-HT with the following rank order of agonist potency: 5CT > > 5-HT > metergoline > or = methysergide. 5

  5. Heterogeneity of human lymphocyte Fc receptors. II. Relationship to antibody-dependent, cell-mediated cytotoxicity

    PubMed Central

    Gormus, B. J.; Woodson, Mildred; Kaplan, M. E.

    1978-01-01

    Human peripheral blood lymphocytes (PBL) were incubated (stripped) with pronase or papain and compared with unstripped lymphocytes for their ability to mediate antibody-dependent, cell-mediated cytotoxicity (ADCC). Despite marked removal or inactivation of receptors for heat-aggregated IgG (aggG) by proteolytic digestion, and pronounced changes in the percentages of cells rosetting with IgG-sensitized erythrocytes (EA) (decreased by papain, increased by pronase), stripped PBL functioned normally in ADCC. Stripped and unstripped lymphocytes were pre-treated with aggG to determine the role of aggG receptors in ADCC. AggG almost totally abolished ADCC by unstripped PBL, but inhibited ADCC by enzyme-stripped lymphocytes relatively poorly. Neither untreated nor stripped PBL were able to induce cytotoxicity of chicken erythrocyte (CRBC) target cells sensitized with the Fab'2 fragment of anti-CRBC IgG antibody (CRBC-A). Exposure of PBL to EA monolayers composed of CRBC-A or of sheep erythrocytes (SRBC) sensitized with rabbit anti-SRBC IgG antibody (SRBC-A) depleted PBL of cells that rosetted with CRBC-A and with human Rh-positive, type O erythrocytes sensitized with the human anti-Rh serum Ripley (HRBC-A Ripley). Non-adherent cells were incapable of binding aggG and had markedly diminished cytotoxicity in ADCC. Similarly, exposure of PBL to HRBC-A Ripley monolayers resulted in non-adherent cells that were incapable of rosette formation with HRBC-A or CRBC-A, failed to bind aggG, and exhibited significantly diminished ADCC activity. These studies indicated that: (1) cytotoxic effector PBL active in ADCC (K cells) have receptors for aggG and for EA; (2) PBL deficient in functional aggG receptors (enzymatically inactivated or removed) are capable of inducing normal levels of ADCC; (3) aggG and EA receptors appear to be closely associated on native K-cell membranes; (4) there is no clear-cut relationship in a given lymphocyte population between the presence of either aggG or

  6. The endogenous cannabinoid anandamide inhibits alpha7 nicotinic acetylcholine receptor-mediated responses in Xenopus oocytes.

    PubMed

    Oz, Murat; Ravindran, Arippa; Diaz-Ruiz, Oscar; Zhang, Li; Morales, Marisela

    2003-09-01

    The effect of the endogenous cannabinoid ligand anandamide on the function of the cloned alpha7 subunit of the nicotinic acetylcholine (ACh) receptor expressed in Xenopus oocytes was investigated by using the two-electrode voltage-clamp technique. Anandamide reversibly inhibited nicotine (10 microM) induced-currents in a concentration-dependent manner (10 nM to 30 microM), with an IC50 value of 229.7 +/- 20.4 nM. The effect of anandamide was neither dependent on the membrane potential nor meditated by endogenous Ca2+ dependent Cl- channels since it was unaffected by intracellularly injected BAPTA and perfusion with Ca2+-free bathing solution containing 2 mM Ba2+. Anandamide decreased the maximal nicotine-induced responses without significantly affecting its potency, indicating that it acts as a noncompetitive antagonist on nicotinic acetylcholine (nACh) alpha7 receptors. This effect was not mediated by CB1 or CB2 receptors, as neither the selective CB1 receptor antagonist N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR 141716A) nor CB2 receptor antagonist N-((1S)-endo-1,3,3-trimethyl-bicyclo-heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide (SR 144528) reduced the inhibition by anandamide. In addition, inhibition of nicotinic responses by anandamide was not sensitive to either pertussis toxin treatment or to the membrane permeable cAMP analog 8-Br-cAMP (0.2 mM). Inhibitors of enzymes involved in anandamide metabolism including phenylmethylsulfonyl fluoride, superoxide dismutase, and indomethacin, or the anandamide transport inhibitor AM404 did not prevent anandamide inhibition of nicotinic responses, suggesting that anandamide itself acted on nicotinic receptors. In conclusion, these results demonstrate that the endogenous cannabinoid anandamide inhibits the function of nACh alpha7 receptors expressed in Xenopus oocytes in a cannabinoid receptor-independent and

  7. Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events.

    PubMed

    Gouwy, Mieke; Struyf, Sofie; Noppen, Samuel; Schutyser, Evemie; Springael, Jean-Yves; Parmentier, Marc; Proost, Paul; Van Damme, Jo

    2008-08-01

    CC and CXC chemokines coinduced in fibroblasts and leukocytes by cytokines and microbial agents determine the number of phagocytes infiltrating into inflamed tissues. Interleukin-8/CXCL8 and stromal cell-derived factor-1/CXCL12 significantly and dose-dependently increased the migration of monocytes, expressing the corresponding CXC chemokine receptors CXCR2 and CXCR4, toward suboptimal concentrations of the monocyte chemotactic proteins CCL2 or CCL7. These findings were confirmed using different chemotaxis assays and monocytic THP-1 cells. In contrast, the combination of two CC chemokines (CCL2 plus CCL7) or two CXC chemokines (CXCL8 plus CXCL12) did not provide synergy in monocyte chemotaxis. These data show that chemokines competing for related receptors and using similar signaling pathways do not synergize. Receptor heterodimerization is probably not essential for chemokine synergy as shown in CXCR4/CCR2 cotransfectants. It is noteworthy that CCL2 mediated extracellular signal-regulated kinase 1/2 phosphorylation and calcium mobilization was significantly enhanced by CXCL8 in monocytes, indicating cooperative downstream signaling pathways during enhanced chemotaxis. Moreover, in contrast to intact CXCL12, truncated CXCL12(3-68), which has impaired receptor signaling capacity but can still desensitize CXCR4, was unable to synergize with CCL2 in monocytic cell migration. Furthermore, AMD3100 and RS102895, specific CXCR4 and CCR2 inhibitors, respectively, reduced the synergistic effect between CCL2 and CXCL12 significantly. These data indicate that for synergistic interaction between chemokines binding and signaling of the two chemokines via their proper receptors is necessary.

  8. Potentiating effect of eszopiclone on GABA(A) receptor-mediated responses in pedunculopontine neurons.

    PubMed

    Ye, Meijun; Garcia-Rill, Edgar

    2009-07-01

    The pedunculopontine nucleus (PPN) is part of the cholinergic arm of the reticular activating system, which is mostly active during waking and REM sleep. GABAergic modulation of this area appears to regulate sleep-wake cycles. Eszopiclone (ESZ), a nonbenzodiazepine hypnotic agent, appears to modulate GABAergic receptors. However, the action site of ESZ in the brain is still unresolved. We tested the hypothesis that ESZ acts by potentiating GABA(A) receptors on PPN neurons. Wholecell voltage clamp recordings were performed on PPN neurons in 7-15 day rat brainstem slices, and the potentiating effects of ESZ on the responses to the GABA(A) receptor agonist isoguvacine (IGV), and on GABA(A) receptor-mediated inhibitory post-synaptic currents (IPSCs), were determined. In the presence of tetrodotoxin, ESZ (1) increased the amplitude of the outward current induced by IGV, (2) increased its duration, and (3) enhanced the IGV-induced decrease in input resistance (Rin). The GABA(A) receptor antagonist gabazine (GBZ) blocked these effects. ESZ alone did not induce detectable currents or change Rin at a holding potential of -60 mV, but when held at 0 mV, ESZ induced an outward current in 13/21 PPN cells, an effect blocked by GBZ. ESZ also increased the amplitude (n = 18/21), duration (n = 17/21), and frequency (n = 13/15) of IPSCs. ESZ may potentiate GABA(A) inhibition in the PPN via pre- and post-synaptic modulation, which may underlie the hypnotic effects of ESZ. The differential effects of ESZ on both pre- and post-synaptic sites may partially explain why it has less significant side effects compared to other hypnotic agents.

  9. Renal uptake of myoglobin is mediated by the endocytic receptors megalin and cubilin.

    PubMed

    Gburek, Jakub; Birn, Henrik; Verroust, Pierre J; Goj, Bogusława; Jacobsen, Christian; Moestrup, Søren K; Willnow, Thomas E; Christensen, Erik I

    2003-09-01

    Nephrotoxicity of myoglobin is well recognized as playing a part in the development of acute renal failure in settings of myoglobinuria. However, the molecular mechanism of myoglobin uptake in renal proximal tubules has not been clarified. Here, we report that the endocytic receptors megalin and cubilin are involved in renal reabsorption of myoglobin. Both receptors were captured from solubilized renal brush-border membranes by affinity chromatography using myoglobin-Sepharose. Myoglobin bound to purified megalin and cubilin with Kd values of 2.0 and 3 microM, respectively, as evaluated by surface plasmon resonance analysis. Apomyoglobin bound to megalin with the same affinity, and the affinity of apomyoglobin to cubilin was reduced (Kd = 5 microM). Radioiodinated myoglobin could be displaced by apomyoglobin in inhibition studies using isolated renal brush-border membranes (Ki approximately 2 microM). Receptor-associated protein as well as antibodies directed against megalin and cubilin markedly inhibited the uptake of fluorescent-labeled myoglobin by cultured yolk sac BN-16 cells. The significance of megalin- and cubilin-mediated endocytosis for myoglobin uptake in vivo was demonstrated by use of kidney-specific megalin knockout mice. Injected myoglobin was extensively reabsorbed by megalin-expressing proximal tubular cells, whereas there was very little uptake in the megalin-deficient cells. In conclusion, this study establishes the molecular mechanism of myoglobin uptake in the renal proximal tubule involving the endocytic receptors megalin and cubilin. Identification of the receptors for tubular uptake of myoglobin may be essential for development of new therapeutic strategies for myoglobinuric acute renal failure.

  10. Lipid-Mediated Regulation of Embedded Receptor Kinases via Parallel Allosteric Relays.

    PubMed

    Ghosh, Madhubrata; Wang, Loo Chien; Ramesh, Ranita; Morgan, Leslie K; Kenney, Linda J; Anand, Ganesh S

    2017-02-28

    Membrane-anchored receptors are essential cellular signaling elements for stimulus sensing, propagation, and transmission inside cells. However, the contributions of lipid interactions to the function and dynamics of embedded receptor kinases have not been described in detail. In this study, we used amide hydrogen/deuterium exchange mass spectrometry, a sensitive biophysical approach, to probe the dynamics of a membrane-embedded receptor kinase, EnvZ, together with functional assays to describe the role of lipids in receptor kinase function. Our results reveal that lipids play an important role in regulating receptor function through interactions with transmembrane segments, as well as through peripheral interactions with nonembedded domains. Specifically, the lipid membrane allosterically modulates the activity of the embedded kinase by altering the dynamics of a glycine-rich motif that is critical for phosphotransfer from ATP. This allostery in EnvZ is independent of membrane composition and involves direct interactions with transmembrane and periplasmic segments, as well as peripheral interactions with nonembedded domains of the protein. In the absence of the membrane-spanning regions, lipid allostery is propagated entirely through peripheral interactions. Whereas lipid allostery impacts the phosphotransferase function of the kinase, extracellular stimulus recognition is mediated via a four-helix bundle subdomain located in the cytoplasm, which functions as the osmosensing core through osmolality-dependent helical stabilization. Our findings emphasize the functional modularity in a membrane-embedded kinase, separated into membrane association, phosphotransferase function, and stimulus recognition. These components are integrated through long-range communication relays, with lipids playing an essential role in regulation.

  11. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis.

    PubMed

    Richards, David M; Endres, Robert G

    2016-05-31

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery.

  12. Molecular mediators for raft-dependent endocytosis of syndecan-1, a highly conserved, multifunctional receptor.

    PubMed

    Chen, Keyang; Williams, Kevin Jon

    2013-05-17

    Endocytosis via rafts has attracted considerable recent interest, but the molecular mediators remain incompletely characterized. Here, we focused on the syndecan-1 heparan sulfate proteoglycan, a highly conserved, multifunctional receptor that we previously showed to undergo raft-dependent endocytosis upon clustering. Alanine scanning mutagenesis of three to five consecutive cytoplasmic residues at a time revealed that a conserved juxtamembrane motif, MKKK, was the only region required for efficient endocytosis after clustering. Endocytosis of clustered syndecan-1 occurs in two phases, each requiring a kinase and a corresponding cytoskeletal partner. In the initial phase, ligands trigger rapid MKKK-dependent activation of ERK and the localization of syndecan-1 into rafts. Activation of ERK drives the dissociation of syndecan-1 from α-tubulin, a molecule that may act as an anchor for syndecan-1 at the plasma membrane in the basal state. In the second phase, Src family kinases phosphorylate tyrosyl residues within the transmembrane and cytoplasmic regions of syndecan-1, a process that also requires MKKK. Tyrosine phosphorylation of syndecan-1 triggers the robust recruitment of cortactin, which we found to be an essential mediator of efficient actin-dependent endocytosis. These findings represent the first detailed characterization of the molecular events that drive endocytosis of a raft-dependent receptor and identify a novel endocytic motif, MKKK. Moreover, the results provide new tools to study syndecan function and regulation during uptake of its biologically and medically important ligands, such as HIV-1, atherogenic postprandial remnant lipoproteins, and molecules implicated in Alzheimer disease.

  13. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  14. G-protein coupled receptor 15 mediates angiogenesis and cytoprotective function of thrombomodulin.

    PubMed

    Pan, Bin; Wang, Xiangmin; Nishioka, Chie; Honda, Goichi; Yokoyama, Akihito; Zeng, Lingyu; Xu, Kailin; Ikezoe, Takayuki

    2017-04-06

    Thrombomodulin (TM) stimulates angiogenesis and protects vascular endothelial cells (ECs) via its fifth epidermal growth factor-like region (TME5); however, the cell surface receptor that mediates the pro-survival signaling activated by TM has remained unknown. We applied pull-down assay followed by MALDI-TOF MS and western blot analysis, and identified G-protein coupled receptor 15 (GPR15) as a binding partner of TME5. TME5 rescued growth inhibition and apoptosis caused by calcineurin inhibitor FK506 in vascular ECs isolated from wild type (WT) C57BL/6 mice. On the other hand, TME5 failed to protect ECs isolated from GPR15 knockout (GPR15 KO) mice from FK506-caused vascular injury. TME5 induced activation of extracellular signal-regulated kinase (ERK) and increased level of anti-apoptotic proteins in a GPR15 dependent manner. In addition, in vivo Matrigel plug angiogenesis assay found that TME5 stimulated angiogenesis in mice. TME5 promoted endothelial migration in vitro. Furthermore, TME5 increased production of NO in association with activated endothelial NO synthase (eNOS) in ECs. All these pro-angiogenesis functions of TME5 were abolished by knockout of GPR15. Our findings suggest that GPR15 plays an important role in mediating cytoprotective function as well as angiogenesis of TM.

  15. Internalization of the receptor for advanced glycation end products (RAGE) is required to mediate intracellular responses.

    PubMed

    Sevillano, Natalia; Girón, María D; Salido, Mercedes; Vargas, Alberto M; Vilches, José; Salto, Rafael

    2009-01-01

    To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.

  16. Toll-like receptor 4-mediated lymphocyte influx induces neonatal necrotizing enterocolitis.

    PubMed

    Egan, Charlotte E; Sodhi, Chhinder P; Good, Misty; Lin, Joyce; Jia, Hongpeng; Yamaguchi, Yukihiro; Lu, Peng; Ma, Congrong; Branca, Maria F; Weyandt, Samantha; Fulton, William B; Niño, Diego F; Prindle, Thomas; Ozolek, John A; Hackam, David J

    2016-02-01

    The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

  17. Activation of oncogenic tyrosine kinase signaling promotes insulin receptor-mediated cone photoreceptor survival

    PubMed Central

    Rajala, Ammaji; Wang, Yuhong; Rajala, Raju V.S.

    2016-01-01

    In humans, daylight vision is primarily mediated by cone photoreceptors. These cells die in age-related retinal degenerations. Prolonging the life of cones for even one decade would have an enormous beneficial effect on usable vision in an aging population. Photoreceptors are postmitotic, but shed 10% of their outer segments daily, and must synthesize the membrane and protein equivalent of a proliferating cell each day. Although activation of oncogenic tyrosine kinase and inhibition of tyrosine phosphatase signaling is known to be essential for tumor progression, the cellular regulation of this signaling in postmitotic photoreceptor cells has not been studied. In the present study, we report that a novel G-protein coupled receptor–mediated insulin receptor (IR) signaling pathway is regulated by non-receptor tyrosine kinase Src through the inhibition of protein tyrosine phosphatase IB (PTP1B). We demonstrated the functional significance of this pathway through conditional deletion of IR and PTP1B in cones, in addition to delaying the death of cones in a mouse model of cone degeneration by activating the Src. This is the first study demonstrating the molecular mechanism of a novel signaling pathway in photoreceptor cells, which provides a window of opportunity to save the dying cones in retinal degenerative diseases. PMID:27391439

  18. Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

    PubMed Central

    Egan, Charlotte E.; Sodhi, Chhinder P.; Good, Misty; Lin, Joyce; Jia, Hongpeng; Yamaguchi, Yukihiro; Lu, Peng; Ma, Congrong; Branca, Maria F.; Weyandt, Samantha; Fulton, William B.; Niño, Diego F.; Prindle, Thomas; Ozolek, John A.; Hackam, David J.

    2015-01-01

    The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification. PMID:26690704

  19. Inflammatory and neuropathic cold allodynia are selectively mediated by the neurotrophic factor receptor GFRα3

    PubMed Central

    Lippoldt, Erika K.; Ongun, Serra; Kusaka, Geoffrey K.; McKemy, David D.

    2016-01-01

    Tissue injury prompts the release of a number of proalgesic molecules that induce acute and chronic pain by sensitizing pain-sensing neurons (nociceptors) to heat and mechanical stimuli. In contrast, many proalgesics have no effect on cold sensitivity or can inhibit cold-sensitive neurons and diminish cooling-mediated pain relief (analgesia). Nonetheless, cold pain (allodynia) is prevalent in many inflammatory and neuropathic pain settings, with little known of the mechanisms promoting pain vs. those dampening analgesia. Here, we show that cold allodynia induced by inflammation, nerve injury, and chemotherapeutics is abolished in mice lacking the neurotrophic factor receptor glial cell line-derived neurotrophic factor family of receptors-α3 (GFRα3). Furthermore, established cold allodynia is blocked in animals treated with neutralizing antibodies against the GFRα3 ligand, artemin. In contrast, heat and mechanical pain are unchanged, and results show that, in striking contrast to the redundant mechanisms sensitizing other modalities after an insult, cold allodynia is mediated exclusively by a single molecular pathway, suggesting that artemin–GFRα3 signaling can be targeted to selectively treat cold pain. PMID:27051069

  20. Kinetics of membrane adhesion mediated by ligand-receptor interaction studied with a biomimetic system.

    PubMed Central

    Boulbitch, A; Guttenberg, Z; Sackmann, E

    2001-01-01

    We report the first measurement of the kinetics of adhesion of a single giant vesicle controlled by the competition between membrane-substrate interaction mediated by ligand-receptor interaction, gravitation, and Helfrich repulsion. To model the cell-tissue interaction, we doped the vesicles with lipid-coupled polymers (mimicking the glycocalix) and the reconstituted ligands selectively recognized by alpha(IIb)beta(3) integrin-mediating specific attraction forces. The integrin was grafted on glass substrates to act as a target cell. The adhesion of the vesicle membrane to the integrin-covered surface starts with the spontaneous formation of a small (approximately 200 nm) domain of tight adhesion, which then gradually grows until the whole adhesion area is in the state of tight adhesion. The time of adhesion varies from few tens of seconds to about one hour depending on the ligand and lipopolymer concentration. At small ligand concentrations, we observed the displacement xi of the front of tight adhesion following the square root law xi approximately t(1/2), whereas, at high concentrations, we found a linear law xi approximately t. We show both experimentally and theoretically that the t(1/2)-regime is dominated by diffusion of ligands, and the xi approximately t-regime by the kinetics of ligands-receptors association. PMID:11606287

  1. Mutational analysis of the Ve1 immune receptor that mediates Verticillium resistance in tomato.

    PubMed

    Zhang, Zhao; Song, Yin; Liu, Chun-Ming; Thomma, Bart P H J

    2014-01-01

    Pathogenic Verticillium species are economically important plant pathogens that cause vascular wilt diseases in hundreds of plant species. The Ve1 gene of tomato confers resistance against race 1 strains of Verticillium dahliae and V. albo-atrum. Ve1 encodes an extracellular leucine-rich repeat (eLRR) receptor-like protein (RLP) that serves as a cell surface receptor for recognition of the recently identified secreted Verticillium effector Ave1. To investigate recognition of Ave1 by Ve1, alanine scanning was performed on the solvent exposed β-strand/β-turn residues across the eLRR domain of Ve1. In addition, alanine scanning was also employed to functionally characterize motifs that putatively mediate protein-protein interactions and endocytosis in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. Functionality of the mutant proteins was assessed by screening for the occurrence of a hypersensitive response upon co-expression with Ave1 upon Agrobacterium tumefaciens-mediated transient expression (agroinfiltration). In order to confirm the agroinfiltration results, constructs encoding Ve1 mutants were transformed into Arabidopsis and the transgenes were challenged with race 1 Verticillium. Our analyses identified several regions of the Ve1 protein that are required for functionality.

  2. Liver X receptor (LXR) mediates negative regulation of mouse and human Th17 differentiation

    PubMed Central

    Cui, Guoliang; Qin, Xia; Wu, Lili; Zhang, Yuebo; Sheng, Xiaoyan; Yu, Qiwen; Sheng, Hongguang; Xi, Beili; Zhang, Jingwu Z.; Zang, Ying Qin

    2011-01-01

    Th17 cells are a subset of CD4+ T cells with an important role in clearing certain bacterial and fungal pathogens. However, they have also been implicated in autoimmune diseases such as multiple sclerosis. Exposure of naive CD4+ T cells to IL-6 and TGF-β leads to Th17 cell differentiation through a process in which many proteins have been implicated. We report here that ectopic expression of liver X receptor (LXR) inhibits Th17 polarization of mouse CD4+ T cells, while LXR deficiency promotes Th17 differentiation in vitro. LXR activation in mice ameliorated disease in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis, whereas LXR deficiency exacerbated disease. Further analysis revealed that Srebp-1, which is encoded by an LXR target gene, mediated the suppression of Th17 differentiation by binding to the E-box element on the Il17 promoter, physically interacting with aryl hydrocarbon receptor (Ahr) and inhibiting Ahr-controlled Il17 transcription. The putative active site (PAS) domain of Ahr and the N-terminal acidic region of Srebp-1 were essential for this interaction. Additional analyses suggested that similar LXR-dependent mechanisms were operational during human Th17 differentiation in vitro. This study reports what we believe to be a novel signaling pathway underlying LXR-mediated regulation of Th17 cell differentiation and autoimmunity. PMID:21266776

  3. Receptor mediated disruption of retinal pigment epithelium function in acute glycated-albumin exposure

    PubMed Central

    Dahrouj, Mohammad; Desjardins, Danielle M.; Liu, Yueying; Crosson, Craig E.; Ablonczy, Zsolt

    2015-01-01

    Diabetic macular edema (DME) is a major cause of visual impairment. Although DME is generally believed to be a microvascular disease, dysfunction of the retinal pigment epithelium (RPE) can also contribute to its development. Advanced glycation end-products (AGE) are thought to be one of the key factors involved in the pathogenesis of diabetes in the eye, and we have previously demonstrated a rapid breakdown of RPE function following glycated-albumin (Glyc-alb, a common AGE mimetic) administration in monolayer cultures of fetal human RPE cells. Here we present new evidence that this response is attributed to apically oriented AGE receptors (RAGE). Moreover, time-lapse optical coherence tomography in Dutch-belted rabbits 48 hours post intravitreal Glyc-alb injections demonstrated a significant decrease in RPE-mediated fluid resorption in vivo. In both the animal and tissue culture models, the response to Glyc-alb was blocked by the relatively selective RAGE antagonist, FPS-ZM1 and was also inhibited by ZM323881, a relatively selective vascular endothelial growth factor receptor 2 (VEGF-R2) antagonist. Our data establish that the Glyc-alb-induced breakdown of RPE function is mediated via specific RAGE and VEGF-R2 signaling both in vitro and in vivo. These results are consistent with the notion that the RPE is a key player in the pathogenesis of DME. PMID:26070987

  4. The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis

    PubMed Central

    Gimenez-Ibanez, Selena; Ntoukakis, Vardis

    2009-01-01

    Plants use pattern recognition receptors (PRRs) to perceive pathogen-associated molecular pattern (PAMPs) and initiate defence responses. PAMP-triggered immunity (PTI) plays an important role in general resistance, and constrains the growth of most microbes on plants. Despite the importance of PRRs in plant immunity, the vast majority of them remain to be identified. We recently showed that the Arabidopsis LysM receptor kinase CERK1 is required not only for chitin signalling and fungal resistance, but plays an essential role in restricting bacterial growth on plants. We proposed that CERK1 may mediate the perception of a bacterial PAMP, or an endogenous plant cell wall component released during infection, through its extracellular carbohydrate-binding LysM-motifs. Here we report reduced activation of a PAMP-induced defence response on plants lacking the CERK1 gene after treatment with crude bacterial extracts. This demonstrates that CERK1 mediates perception of an unknown bacterial PAMP in Arabidopsis. PMID:19816132

  5. Pharmacologically Distinct Nicotinic Acetylcholine Receptors Drive Efferent-Mediated Excitation in Calyx-Bearing Vestibular Afferents

    PubMed Central

    Kewin, Kevin; Jordan, Paivi M.; Cameron, Peter; Klapczynski, Marcin; McIntosh, J. Michael; Crooks, Peter A.; Dwoskin, Linda P.; Lysakowski, Anna

    2015-01-01

    Electrical stimulation of vestibular efferent neurons rapidly excites the resting discharge of calyx/dimorphic (CD) afferents. In turtle, this excitation arises when acetylcholine (ACh), released from efferent terminals, directly depolarizes calyceal endings by activating nicotinic ACh receptors (nAChRs). Although molecular biological data from the peripheral vestibular system implicate most of the known nAChR subunits, specific information about those contributing to efferent-mediated excitation of CD afferents is lacking. We sought to identify the nAChR subunits that underlie the rapid excitation of CD afferents and whether they differ from α9α10 nAChRs on type II hair cells that drive efferent-mediated inhibition in adjacent bouton afferents. We recorded from CD and bouton afferents innervating the turtle posterior crista during electrical stimulation of vestibular efferents while applying several subtype-selective nAChR agonists and antagonists. The α9α10 nAChR antagonists, α-bungarotoxin and α-conotoxin RgIA, blocked efferent-mediated inhibition in bouton afferents while leaving efferent-mediated excitation in CD units largely intact. Conversely, 5-iodo-A-85380, sazetidine-A, varenicline, α-conotoxin MII, and bPiDDB (N,N-dodecane-1,12-diyl-bis-3-picolinium dibromide) blocked efferent-mediated excitation in CD afferents without affecting efferent-mediated inhibition in bouton afferents. This pharmacological profile suggested that calyceal nAChRs contain α6 and β2, but not α9, nAChR subunits. Selective blockade of efferent-mediated excitation in CD afferents distinguished dimorphic from calyx afferents by revealing type II hair cell input. Dimorphic afferents differed in having higher mean discharge rates and a mean efferent-mediated excitation that was smaller in amplitude yet longer in duration. Molecular biological data demonstrated the expression of α9 in turtle hair cells and α4 and β2 in associated vestibular ganglia. PMID:25716861

  6. Activity-dependent bidirectional regulation of GABAA receptor channels by the 5-HT4 receptor-mediated signalling in rat prefrontal cortical pyramidal neurons

    PubMed Central

    Cai, Xiang; Flores-Hernandez, Jorge; Feng, Jian; Yan, Zhen

    2002-01-01

    Emerging evidence has implicated a potential role for 5-HT4 receptors in cognition and anxiolysis. One of the main target structures of 5-HT4 receptors on ‘cognitive and emotional’ pathways is the prefrontal cortex (PFC). As GABAergic signalling plays a key role in regulating PFC functions, we examined the effect of 5-HT4 receptors on GABAA receptor channels in PFC pyramidal neurons. Application of 5-HT4 receptor agonists produced either an enhancement or a reduction of GABA-evoked currents in PFC neurons, which are both mediated by anchored protein kinase A (PKA). Although PKA phosphorylation of GABAA receptor β3 or β1 subunits leads to current enhancement or reduction respectively in heterologous expression systems, we found that β3 and β1 subunits are co-expressed in PFC pyramidal neurons. Interestingly, altering PKA activation levels can change the direction of the dual effect, switching enhancement to reduction and vice versa. In addition, increased neuronal activity in PFC slices elevated the PKA activation level, changing the enhancing effect of 5-HT4 receptors on the amplitude of GABAergic inhibitory postsynaptic currents (IPSCs) to a reduction. These results suggest that 5-HT4 receptors can modulate GABAergic signalling bidirectionally, depending on the basal PKA activation levels that are determined by neuronal activity. This modulation provides a unique and flexible mechanism for 5-HT4 receptors to dynamically regulate synaptic transmission and neuronal excitability in the PFC network. PMID:11986365

  7. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  8. Pharmacological characterization of muscarinic receptor subtypes mediating vasoconstriction of human umbilical vein

    PubMed Central

    Pujol Lereis, Virginia Andrea; Hita, Francisco Javier; Gobbi, Mauro Darío; Verdi, Marcela Gomez; Rodriguez, María Cecilia; Rothlin, Rodolfo Pedro

    2006-01-01

    The present study attempted to pharmacologically characterize the muscarinic receptor subtypes mediating contraction of human umbilical vein (HUV). HUV rings were mounted in organ baths and concentration–response curves were constructed for acetylcholine (ACh) (pEC50: 6.16±0.04; maximum response 80.00±1.98% of the responses induced by serotonin 10 μM). The absence of endothelium did not modify the contractile responses of ACh in this tissue. The role of cholinesterases was evaluated: neither neostigmine (acetylcholinesterase inhibitor) nor iso-OMPA (butyrylcholinesterase inhibitor) modified ACh responses. When both enzymes were simultaneously inhibited, a significantly but little potentiation was observed (control: pEC50 6.33±0.03; double inhibition: pEC50 6.57±0.05). Atropine, nonselective muscarinic receptors antagonist, inhibited ACh-induced contraction (pKB 9.67). The muscarinic receptors antagonists pirenzepine (M1), methoctramine (M2) and pFHHSiD (M3) also antagonized responses to ACh. The affinity values estimated for these antagonists against responses evoked by ACh were 7.58, 6.78 and 7.94, respectively. On the other hand, PD 102807 (M4 selective muscarinic receptors antagonist) was ineffective against ACh-induced contraction. In presence of a blocking concentration of pirenzepine, pFHHSiFD produced an additional antagonism activity on ACh-induced responses. The M1 muscarinic receptors agonist McN-A-343 produced similar maximum but less potent responses than ACh in HUV. The calculated pA2 for pirenzepine against McN-A-343 induced responses was 8.54. In conclusion, the data obtained in this study demonstrate the role of M1 muscarinic receptor subtypes and suggest the involvement of M3 muscarinic receptor subtypes in ACh-induced vasoconstriction in HUV rings. In addition, the vasomotor activity evoked by ACh does not seem to be modulated by endothelial factors, and their enzymatic degradation appears to have little functional relevance in this

  9. Agonist ligands mediate the transcriptional response of nuclear receptor heterodimers through distinct stoichiometric assemblies with coactivators.

    PubMed

    Pavlin, Mark Remec; Brunzelle, Joseph S; Fernandez, Elias J

    2014-09-05

    The constitutive androstane (CAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors of the nuclear receptor protein superfamily. Functional CAR:RXR heterodimers recruit coactivator proteins, such as the steroid receptor coactivator-1 (SRC1). Here, we show that agonist ligands can potentiate transactivation through both coactivator binding sites on CAR:RXR, which distinctly bind two SRC1 molecules. We also observe that SRC1 transitions from a structurally plastic to a compact form upon binding CAR:RXR. Using small angle x-ray scattering (SAXS) we show that the CAR(tcp):RXR(9c)·SRC1 complex can encompass two SRC1 molecules compared with the CAR(tcp):RXR·SRC1, which binds only a single SRC1. Moreover, sedimentation coefficients and molecular weights determined by analytical ultracentrifugation confirm the SAXS model. Cell-based transcription assays show that disrupting the SRC1 binding site on RXR alters the transactivation by CAR:RXR. These data suggest a broader role for RXR within heterodimers, whereas offering multiple strategies for the assembly of the transcription complex.

  10. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  11. Muscarinic receptor subtypes mediating the mucosal response to neural stimulation of guinea pig ileum

    SciTech Connect

    Carey, H.V.; Tien, X.Y.; Wallace, L.J.; Cooke, H.J.

    1987-09-01

    Muscarinic receptors involved in the secretory response evoked by electrical stimulation of submucosal neutrons were investigated in muscle-stripped flat sheets of guinea pig ileum set up in flux chambers. Neural stimulation produced a biphasic increase in short-circuit current due to active chloride secretion. Atropine and 4-diphenylacetoxy-N-methylpiperadine methiodide (4-DAMP) (10/sup -7/ M) were more potent inhibitors of the cholinergic phase of the response than was pirenzepine. Dose-dependent increases in base-line short-circuit current were evoked by carbachol and bethanechol; 4-hydroxy-2-butynyl trimethylammonium chloride (McN A343) produced a much smaller effect. Tetrodotoxin abolished the effects of McN A343 but did not alter the responses of carbachol and bethanechol. McN A343 significantly reduced the cholinergic phase of the neurally evoked response and caused a rightward shift of the carbachol dose-response curve. All muscarinic compounds inhibited (/sup 3/H)quinuclidinyl benzilate binding to membranes from muscosal scrapings, with a rank order of potency of 4-DAMP > pirenzepine > McN A343 > carbachol > bethanechol. These results suggest that acetylcholine released from submucosal neurons mediates chloride secretion by interacting with muscarinic cholinergic receptors that display a high binding affinity for 4-DAMP. Activation of neural muscarinic receptors makes a relatively small contribution to the overall secretory response.

  12. Extrasynaptic NMDA receptor-induced tau overexpression mediates neuronal death through suppressing survival signaling ERK phosphorylation

    PubMed Central

    Sun, Xu-Ying; Tuo, Qing-Zhang; Liuyang, Zhen-Yu; Xie, Ao-Ji; Feng, Xiao-Long; Yan, Xiong; Qiu, Mei; Li, Shen; Wang, Xiu-Lian; Cao, Fu-Yuan; Wang, Xiao-Chuan; Wang, Jian-Zhi; Liu, Rong

    2016-01-01

    Intracellular accumulation of the hyperphosphorylated tau is a pathological hallmark in the brain of Alzheimer disease. Activation of extrasynaptic NMDA receptors (E-NMDARs) induces excitatory toxicity that is involved in Alzheimer's neurodegeneration. However, the intrinsic link between E-NMDARs and the tau-induced neuronal damage remains elusive. In the present study, we showed in cultured primary cortical neurons that activation of E-NMDA receptors but not synaptic NMDA receptors dramatically increased tau mRNA and protein levels, with a simultaneous neuronal degeneration and decreased neuronal survival. Memantine, a selective antagonist of E-NMDARs, reversed E-NMDARs-induced tau overexpression. Activation of E-NMDARs in wild-type mouse brains resulted in neuron loss in hippocampus, whereas tau deletion in neuronal cultures and in the mouse brains rescued the E-NMDARs-induced neuronal death and degeneration. The E-NMDARs-induced tau overexpression was correlated with a reduced ERK phosphorylation, whereas the increased MEK activity, decreased binding and activity of ERK phosphatase to ERK, and increased ERK phosphorylation were observed in tau knockout mice. On the contrary, addition of tau proteins promoted ERK dephosphorylation in vitro. Taking together, these results indicate that tau overexpression mediates the excitatory toxicity induced by E-NMDAR activation through inhibiting ERK phosphorylation. PMID:27809304

  13. Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

    PubMed

    Toth, C A; Thomas, P; Broitman, S A; Zamcheck, N

    1985-01-01

    In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

  14. Aryl hydrocarbon receptor-dependent liver development and hepatotoxicity are mediated by different cell types.

    PubMed

    Walisser, Jacqueline A; Glover, Edward; Pande, Kalyan; Liss, Adam L; Bradfield, Christopher A

    2005-12-06

    The aryl hydrocarbon receptor (AHR) plays a role in three areas of biology that include the adaptive metabolism of xenobiotics, the toxic responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), and vascular remodeling of the developing embryo. To test the hypothesis that receptor signaling in different cell types is responsible for these aspects of AHR biology, we generated a conditional Ahr allele where exon 2 is flanked by loxP sites. Through the use of Cre-lox technology, we then investigated the role of AHR signaling in hepatocytes or endothelial cells in mediating prototypical endpoints of adaptive, toxic, or developmental signaling. Using this model, we provide evidence that AHR signaling in endothelial/hematopoietic cells is necessary for developmental closure of the ductus venosus, whereas AHR signaling in hepatocytes is necessary to generate adaptive and toxic responses of the liver in response to dioxin exposure. Taken together, these data illustrate the importance of cell-specific receptor signaling for the generation of distinct AHR-dependent physiological outcomes.

  15. Peripheral NMDA Receptors Mediate Antidromic Nerve Stimulation-Induced Tactile Hypersensitivity in the Rat.

    PubMed

    Jang, Jun Ho; Nam, Taick Sang; Jun, Jaebeom; Jung, Se Jung; Kim, Dong-Wook; Leem, Joong Woo

    2015-01-01

    We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.pl.) injection was administered into the L4 dermatome before ES, NMDAR and group-I metabotropic Glu receptor (mGluR) antagonists and group-II mGluR agonist but not AMPA/kainate receptor antagonist prevented ES-induced hypersensitivity. I.pl. injection of PKA or PKC inhibitors also prevented ES-induced hypersensitivity. When the same injections were administered after establishment of ES-induced hypersensitivity, hypersensitivity was partially reduced by NMDAR antagonist only. In naïve animals, i.pl. Glu injection into the L4 dermatome induced tactile hypersensitivity, which was blocked by NMDAR antagonist and PKA and PKC inhibitors. These results suggest that the peripheral release of Glu, induced by antidromic nerve stimulation, leads to the expansion of tactile hypersensitive skin probably via nociceptor sensitization spread due to the diffusion of Glu into the skin near the release site. In addition, intracellular PKA- and PKC-dependent mechanisms mediated mainly by NMDAR activation are involved in Glu-induced nociceptor sensitization and subsequent hypersensitivity.

  16. Involvement of neuropeptide Y Y1 receptor in the regulation of amphetamine-mediated appetite suppression.

    PubMed

    Kuo, Dong-Yih; Chen, Pei-Ni; Yu, Ching-Han; Kuo, Meng-Hsien; Hsieh, Yih-Shou; Chu, Shu-Chen

    2012-10-01

    Recently, we reported that an initial decrease followed by recovery of food intake was observed during four days of amphetamine (AMPH) treatment and suggested that these changes in response were mediated by changes in neuropeptide Y (NPY) and proopiomelanocortin (POMC). Here we investigated if Y1 receptor (Y1R) and/or Y5 receptor (Y5R) might be involved in this regulation. Rats were treated daily with AMPH for four days. Changes in the expression levels of Y1R, Y5R, melanocortin receptor 3 (MC3R), and NPY were assessed and compared. Results showed that Y1R and MC3R increased, with a maximal increase of about 210% on Day 2 but with a restoration to the normal level on Day 4. In contrast, NPY decreased with a biggest reduction of about 45% on Day 2 and the pattern of expression during AMPH treatment was opposite to those of Y1R and MC3R, while the expression of Y5R was not changed. Central inhibitions of NPY formation or Y1R activity modulated the anorectic response of AMPH and the reciprocal regulation of NPY and MC3R, revealing a crucial role of Y1R in this action. It is suggested that Y1R participates in the reciprocal regulation of NPY- and MC3R-containing neurons in the hypothalamus during the anorectic effect of AMPH. These results may further the understanding of Y1R in the control of eating.

  17. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    SciTech Connect

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro; Imagawa, Masayoshi

    2009-12-18

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  18. Differential GABAB-receptor-mediated effects in perisomatic- and dendrite-targeting parvalbumin interneurons.

    PubMed

    Booker, Sam A; Gross, Anna; Althof, Daniel; Shigemoto, Ryuichi; Bettler, Bernhard; Frotscher, Michael; Hearing, Matthew; Wickman, Kevin; Watanabe, Masahiko; Kulik, Ákos; Vida, Imre

    2013-05-01

    Inhibitory parvalbumin-containing interneurons (PVIs) control neuronal discharge and support the generation of theta- and gamma-frequency oscillations in cortical networks. Fast GABAergic input onto PVIs is crucial for their synchronization and oscillatory entrainment, but the role of metabotropic GABA(B) receptors (GABA(B)Rs) in mediating slow presynaptic and postsynaptic inhibition remains unknown. In this study, we have combined high-resolution immunoelectron microscopy, whole-cell patch-clamp recording, and computational modeling to investigate the subcellular distribution and effects of GABA(B)Rs and their postsynaptic effector Kir3 channels in rat hippocampal PVIs. Pre-embedding immunogold labeling revealed that the receptors and channels localize at high levels to the extrasynaptic membrane of parvalbumin-immunoreactive dendrites. Immunoreactivity for GABA(B)Rs was also present at lower levels on PVI axon terminals. Whole-cell recordings further showed that synaptically released GABA in response to extracellular stimulation evokes large GABA(B)R-mediated slow IPSCs in perisomatic-targeting (PT) PVIs, but only small or no currents in dendrite-targeting (DT) PVIs. In contrast, paired recordings demonstrated that GABA(B)R activation results in presynaptic inhibition at the output synapses of both PT and DT PVIs, but more strongly in the latter. Finally, computational analysis indicated that GABA(B) IPSCs can phasically modulate the discharge of PT interneurons at theta frequencies. In summary, our results show that GABA(B)Rs differentially mediate slow presynaptic and postsynaptic inhibition in PVIs and can contribute to the dynamic modulation of their activity during oscillations. Furthermore, these data provide evidence for a compartment-specific molecular divergence of hippocampal PVI subtypes, suggesting that activation of GABA(B)Rs may shift the balance between perisomatic and dendritic inhibition.

  19. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells.

    PubMed

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ERalpha crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1alpha or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFkappaB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  20. COBALTOUS CHLORIDE AND HYPOXIA INHIBIT ARYL HYDROCARBON RECEPTOR-MEDIATED RESPONSES IN BREAST CANCER CELLS

    PubMed Central

    Khan, Shaheen; Liu, Shengxi; Stoner, Matthew; Safe, Stephen

    2007-01-01

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-Erα crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1α or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NFκB which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 hr, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 hr, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 hr enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide. PMID:17599377

  1. Cobaltous chloride and hypoxia inhibit aryl hydrocarbon receptor-mediated responses in breast cancer cells

    SciTech Connect

    Khan, Shaheen; Liu Shengxi; Stoner, Matthew; Safe, Stephen

    2007-08-15

    The aryl hydrocarbon receptor (AhR) is expressed in estrogen receptor (ER)-positive ZR-75 breast cancer cells. Treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces CYP1A1 protein and mRNA levels and also activates inhibitory AhR-ER{alpha} crosstalk associated with hormone-induced reporter gene expression. In ZR-75 cells grown under hypoxia, induction of these AhR-mediated responses by TCDD was significantly inhibited. This was not accompanied by decreased nuclear AhR levels or decreased interaction of the AhR complex with the CYP1A1 gene promoter as determined in a chromatin immunoprecipitation assay. Hypoxia-induced loss of Ah-responsiveness was not associated with induction of hypoxia-inducible factor-1{alpha} or other factors that sequester the AhR nuclear translocation (Arnt) protein, and overexpression of Arnt under hypoxia did not restore Ah-responsiveness. The p65 subunit of NF{kappa}B which inhibits AhR-mediated transactivation was not induced by hypoxia and was primarily cytosolic in ZR-75 cells grown under hypoxic and normoxic conditions. In ZR-75 cells maintained under hypoxic conditions for 24 h, BRCA1 (an enhancer of AhR-mediated transactivation in breast cancer cells) was significantly decreased and this contributed to loss of Ah-responsiveness. In cells grown under hypoxia for 6 h, BRCA1 was not decreased, but induction of CYP1A1 by TCDD was significantly decreased. Cotreatment of ZR-75 cells with TCDD plus the protein synthesis inhibitor cycloheximide for 6 h enhanced CYP1A1 expression in cells grown under hypoxia and normoxia. These results suggest that hypoxia rapidly induces protein(s) that inhibit Ah-responsiveness and these may be similar to constitutively expressed inhibitors of Ah-responsiveness (under normoxia) that are also inhibited by cycloheximide.

  2. Regulation of AMPA and NMDA receptor-mediated EPSPs in dendritic trees of thalamocortical cells

    PubMed Central

    Lajeunesse, Francis; Kröger, Helmut

    2013-01-01

    Two main excitatory synapses are formed at the dendritic arbor of first-order nuclei thalamocortical (TC) neurons. Ascending sensory axons primarily establish contacts at large proximal dendrites, whereas descending corticothalamic fibers form synapses on thin distal dendrites. With the use of a multicomparment computational model based on fully reconstructed TC neurons from the ventroposterolateral nucleus of the cat, we compared local responses at the site of stimulation as well as somatic responses induced by both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)- and N-methyl-d-aspartate receptor (NMDAR)-mediated currents. We found that AMPAR-mediated responses, when synapses were located at proximal dendrites, induced a larger depolarization at the level of soma, whereas NMDAR-mediated responses were more efficient for synapses located at distal dendrites. The voltage transfer and transfer impedance were higher for NMDAR than for AMPAR activation at any location. For both types of synaptic current and for both input locations at the dendritic arbor, somatic responses were characterized by a low variability despite the large variability found in local responses in dendrites. The large neurons had overall smaller somatic responses than small neurons, but this relation was not found in local dendritic responses. We conclude that in TC cells, the dendritic location of small synaptic inputs does not play a major role in the amplitude of a somatic response, but the size of the neuron does. The variability of response amplitude between cells was much larger than the variability within cells. This suggests possible functional segregation of TC neurons of different size. PMID:23100131

  3. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase

    PubMed Central

    Ruisanchez, Éva; Dancs, Péter; Kerék, Margit; Németh, Tamás; Faragó, Bernadett; Balogh, Andrea; Patil, Renukadevi; Jennings, Brett L.; Liliom, Károly; Malik, Kafait U.; Smrcka, Alan V.; Tigyi, Gabor; Benyó, Zoltán

    2014-01-01

    Lysophosphatidic acid (LPA) has been implicated as a mediator of several cardiovascular functions, but its potential involvement in the control of vascular tone is obscure. Here, we show that both LPA (18:1) and VPC31143 (a synthetic agonist of LPA1–3 receptors) relax intact mouse thoracic aorta with similar Emax values (53.9 and 51.9% of phenylephrine-induced precontraction), although the EC50 of LPA- and VPC31143-induced vasorelaxations were different (400 vs. 15 nM, respectively). Mechanical removal of the endothelium or genetic deletion of endothelial nitric oxide synthase (eNOS) not only diminished vasorelaxation by LPA or VPC31143 but converted it to vasoconstriction. Freshly isolated mouse aortic endothelial cells expressed LPA1, LPA2, LPA4 and LPA5 transcripts. The LPA1,3 antagonist Ki16425, the LPA1 antagonist AM095, and the genetic deletion of LPA1, but not that of LPA2, abolished LPA-induced vasorelaxation. Inhibition of the phosphoinositide 3 kinase–protein kinase B/Akt pathway by wortmannin or MK-2206 failed to influence the effect of LPA. However, pharmacological inhibition of phospholipase C (PLC) by U73122 or edelfosine, but not genetic deletion of PLCε, abolished LPA-induced vasorelaxation and indicated that a PLC enzyme, other than PLCε, mediates the response. In summary, the present study identifies LPA as an endothelium-dependent vasodilator substance acting via LPA1, PLC, and eNOS.—Ruisanchez, É., Dancs, P., Kerék, M., Németh, T., Faragó, B., Balogh, A., Patil, R., Jennings, B. L., Liliom, K., Malik, K. U., Smrcka, A. V., Tigyi, G., Benyó, Z. Lysophosphatidic acid induces vasodilation mediated by LPA1 receptors, phospholipase C, and endothelial nitric oxide synthase. PMID:24249637

  4. Fc receptor-mediated immune responses: new tools but increased complexity in HIV prevention.

    PubMed

    Vargas-Inchaustegui, Diego A; Robert-Guroff, Marjorie

    2013-07-01

    The modest success of the RV144 HIV vaccine trial in Thailand and the ensuing suggestion that a Fc-receptormediated antibody activity might have played a role in the protection observed have intensified investigations on Fcrelated immune responses. HIV neutralizing antibodies have been and continue to be the focal point of research into humoral immune protection. However, recent knowledge that their protective efficacy can be augmented by Fc-FcR interactions has increased the complexity of identifying immune correlates of protection. If anything, continued studies of both humoral and cellular immune mechanisms point to the lack of a single protective anti-HIV immune response. Here we focus on humoral immunity, analyzing the role played by Fc receptor-related responses and discussing how new knowledge of their interactions requires further investigation, but may also spur novel vaccination approaches. We initially address classical Fc-receptor mediated anti-viral mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell mediated viral inhibition (ADCVI), and antibody-dependent cellular phagocytosis (ADCP), as well as the effector cells that mediate these functions. Next, we summarize key aspects of FcR-Fc interactions that are important for potential control of HIV/SIV such as FcR polymorphisms and post-transcriptional modifications. Finally we discuss less commonly studied non-mechanistic anti-HIV immune functions: antibody avidity and envelopespecific B cell memory. Overall, a spectrum of immune responses, reflecting the immune system's redundancy, will likely be needed to prevent HIV infection and/or disease progression. Aside from elicitation of critical immune mechanisms, a successful vaccine will need to induce mature B cell responses and long-lasting immune memory.

  5. Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

    PubMed

    Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

    2015-07-01

    In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

  6. α4-Containing GABAA Receptors in the Nucleus Accumbens Mediate Moderate Intake of Alcohol

    PubMed Central

    Rewal, Mridula; Jurd, Rachel; Gill, T. Michael; He, Dao-Yao; Ron, Dorit; Janak, Patricia H.

    2009-01-01

    Alcohol has subjective and behavioral effects at the pharmacological levels typically reached during the consumption of one or two alcoholic drinks. Here we provide evidence that an α4-subunit-containing gamma-amino-butyric acid A (GABAA) receptor contributes to the consumption of low-to-moderate levels of alcohol. Using viral-mediated RNA-interference (RNAi), we found that reduced expression of the α4 subunit in the nucleus accumbens (NAc) shell of rats decreased their free consumption of and preference for alcohol. The time course for the reduced alcohol intake paralleled the time course of α4 mRNA reductions achieved after viral-mediated RNAi for α4. Further, the reduction in drinking was region- and alcohol-specific: there was no effect of reductions in α4 expression in the NAc core on alcohol intake, and reductions in α4 expression in the NAc shell did not alter sucrose or water intake. These results indicate that the GABAAR α4 subunit in the NAc shell mediates alcohol intake. PMID:19144854

  7. A novel posttranscriptional mechanism for dietary cholesterol-mediated suppression of liver LDL receptor expression.

    PubMed

    Singh, Amar Bahadur; Kan, Chin Fung Kelvin; Shende, Vikram; Dong, Bin; Liu, Jingwen

    2014-07-01

    It is well-established that over-accumulation of dietary cholesterol in the liver inhibits sterol-regulatory element binding protein (SREBP)-mediated LDL receptor (LDLR) gene transcription leading to a reduced hepatic LDLR mRNA level in hypercholesterolemic animals. However, it is unknown whether elevated cholesterol levels can elicit a cellular response to increase LDLR mRNA turnover to further repress LDLR expression in liver tissue. In the current study, we examined the effect of a high cholesterol diet on the hepatic expression of LDLR mRNA binding proteins in three different animal models and in cultured hepatic cells. Our results demonstrate that high cholesterol feeding specifically elevates the hepatic expression of LDLR mRNA decay promoting factor heterogeneous nuclear ribonucleoprotein (HNRNP)D without affecting expressions of other LDLR mRNA binding proteins in vivo and in vitro. Employing the approach of adenovirus-mediated gene knockdown, we further show that depletion of HNRNPD in the liver results in a marked reduction of serum LDL-cholesterol and a substantial increase in liver LDLR expression in hyperlipidemic mice. Additional studies of gene knockdown in albumin-luciferase-untranslated region (UTR) transgenic mice provide strong evidence supporting the essential role of 3'UTR in HNRNPD-mediated LDLR mRNA degradation in liver tissue. Altogether, this work identifies a novel posttranscriptional regulatory mechanism by which dietary cholesterol inhibits liver LDLR expression via inducing HNRNPD to accelerate LDLR mRNA degradation.

  8. Vacuolar Sorting Receptor-Mediated Trafficking of Soluble Vacuolar Proteins in Plant Cells.

    PubMed

    Kang, Hyangju; Hwang, Inhwan

    2014-08-25

    Vacuoles are one of the most prominent organelles in plant cells, and they play various important roles, such as degradation of waste materials, storage of ions and metabolites, and maintaining turgor. During the past two decades, numerous advances have been made in understanding how proteins are specifically delivered to the vacuole. One of the most crucial steps in this process is specific sorting of soluble vacuolar proteins. Vacuolar sorting receptors (VSRs), which are type I membrane proteins, are involved in the sorting and packaging of soluble vacuolar proteins into transport vesicles with the help of various accessory proteins. To date, large amounts of data have led to the development of two different models describing VSR-mediated vacuolar trafficking that are radically different in multiple ways, particularly regarding the location of cargo binding to, and release from, the VSR and the types of carriers utilized. In this review, we summarize current literature aimed at elucidating VSR-mediated vacuolar trafficking and compare the two models with respect to the sorting signals of vacuolar proteins, as well as the molecular machinery involved in VSR-mediated vacuolar trafficking and its action mechanisms.

  9. Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

    PubMed

    Cheng, Wei-Erh; Ying Chang, Miao; Wei, Jyun-Yan; Chen, Yen-Jen; Maa, Ming-Chei; Leu, Tzeng-Horng

    2015-06-15

    Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

  10. Ran GTPase promotes cancer progression via Met receptor-mediated downstream signaling

    PubMed Central

    Yuen, Hiu-Fung; Chan, Ka-Kui; Platt-Higgins, Angela; Dakir, El-Habib; Matchett, Kyle B.; Haggag, Yusuf Ahmed; Jithesh, Puthen V.; Habib, Tanwir; Faheem, Ahmed; Dean, Fennell A.; Morgan, Richard; Rudland, Philip S.; El-Tanani, Mohamed

    2016-01-01

    It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival. Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator. PMID:27716616

  11. Inhibition of peroxisome proliferator-activated receptor (PPAR)-mediated keratinocyte differentiation by lipoxygenase inhibitors.

    PubMed Central

    Thuillier, Philippe; Brash, Alan R; Kehrer, James P; Stimmel, Julie B; Leesnitzer, Lisa M; Yang, Peiying; Newman, Robert A; Fischer, Susan M

    2002-01-01

    Lipoxygenase (LOX) metabolites from arachidonic acid and linoleic acid have been implicated in atherosclerosis, inflammation, keratinocyte differentiation and tumour progression. We previously showed that peroxisome proliferator-activated receptors (PPARs) play a role in keratinocyte differentiation and that the PPARalpha ligand 8S-hydroxyeicosatetraenoic acid is important in this process. We hypothesized that blocking LOX activity would block PPAR-mediated keratinocyte differentiation. Three LOX inhibitors, nordihydroguaiaretic acid, quercetin and morin, were studied for their effects on primary keratinocyte differentiation and PPAR activity. All three LOX inhibitors blocked calcium-induced expression of the differentiation marker keratin 1. In addition, activity of a PPAR-responsive element was inhibited in the presence of all three inhibitors, and this effect was mediated primarily through PPARalpha and PPARgamma. LOX inhibitors decreased the activity of a chimaeric PPAR-Gal4-ligand-binding domain reporter system and this effect was reversed by addition of PPAR ligands. Ligand-binding studies revealed that the LOX inhibitors bind directly to PPARs and demonstrate a novel mechanism for these inhibitors in altering PPAR-mediated gene expression. PMID:12069687

  12. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  13. Oviductal estrogen receptor α signaling prevents protease-mediated embryo death

    PubMed Central

    Winuthayanon, Wipawee; Bernhardt, Miranda L; Padilla-Banks, Elizabeth; Myers, Page H; Edin, Matthew L; Lih, Fred B; Hewitt, Sylvia C; Korach, Kenneth S; Williams, Carmen J

    2015-01-01

    Development of uterine endometrial receptivity for implantation is orchestrated by cyclic steroid hormone-mediated signals. It is unknown if these signals are necessary for oviduct function in supporting fertilization and preimplantation development. Here we show that conditional knockout (cKO) mice lacking estrogen receptor α (ERα) in oviduct and uterine epithelial cells have impaired fertilization due to a dramatic reduction in sperm migration. In addition, all successfully fertilized eggs die before the 2-cell stage due to persistence of secreted innate immune mediators including proteases. Elevated protease activity in cKO oviducts causes premature degradation of the zona pellucida and embryo lysis, and wild-type embryos transferred into cKO oviducts fail to develop normally unless rescued by concomitant transfer of protease inhibitors. Thus, suppression of oviductal protease activity mediated by estrogen-epithelial ERα signaling is required for fertilization and preimplantation embryo development. These findings have implications for human infertility and post-coital contraception. DOI: http://dx.doi.org/10.7554/eLife.10453.001 PMID:26623518

  14. Insulin receptor Thr1160 phosphorylation mediates lipid-induced hepatic insulin resistance

    PubMed Central

    Petersen, Max C.; Madiraju, Anila K.; Gassaway, Brandon M.; Marcel, Michael; Nasiri, Ali R.; Butrico, Gina; Marcucci, Melissa J.; Zhang, Dongyan; Abulizi, Abudukadier; Zhang, Xian-Man; Philbrick, William; Hubbard, Stevan R.; Samuel, Varman T.; Rinehart, Jesse

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D), but whether NAFLD plays a causal role in the pathogenesis of T2D is uncertain. One proposed mechanism linking NAFLD to hepatic insulin resistance involves diacylglycerol-mediated (DAG-mediated) activation of protein kinase C-ε (PKCε) and the consequent inhibition of insulin receptor (INSR) kinase activity. However, the molecular mechanism underlying PKCε inhibition of INSR kinase activity is unknown. Here, we used mass spectrometry to identify the phosphorylation site Thr1160 as a PKCε substrate in the functionally critical INSR kinase activation loop. We hypothesized that Thr1160 phosphorylation impairs INSR kinase activity by destabilizing the active configuration of the INSR kinase, and our results confirmed this prediction by demonstrating severely impaired INSR kinase activity in phosphomimetic T1160E mutants. Conversely, the INSR T1160A mutant was not inhibited by PKCε in vitro. Furthermore, mice with a threonine-to-alanine mutation at the homologous residue Thr1150 (InsrT1150A mice) were protected from high fat diet–induced hepatic insulin resistance. InsrT1150A mice also displayed increased insulin signaling, suppression of hepatic glucose production, and increased hepatic glycogen synthesis compared with WT controls during hyperinsulinemic clamp studies. These data reveal a critical pathophysiological role for INSR Thr1160 phosphorylation and provide further mechanistic links between PKCε and INSR in mediating NAFLD-induced hepatic insulin resistance. PMID:27760050

  15. Dual actions of (-)-stepholidine on the dopamine receptor-mediated adenylate cyclase activity in rat corpus striatum.

    PubMed

    Dong, Z J; Guo, X; Chen, L J; Han, Y F; Jin, G Z

    1997-01-01

    (-)-Stepholidine (SPD) is an antagonist of normosensitive dopamine (DA) receptors, but it exhibits D1 agonistic action on rotational behaviour in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the substantia nigra pars compacta (SNC). In the present study, agonistic and antagonistic effects of SPD on the DA receptor-mediated synaptosomal adenylate cyclase (AC) activity in rat striatum were investigated. After blockade of D2 receptors, SPD augmented AC activity dose-dependently. The EC50 value was 41.1 +/- 8.6 micromol/L. At the concentration of 10 micromol/L, SPD increased cAMP formation from a basal level (50.8 +/- 10.3 pmol/mg protein/min) to 133.7 +/- 31.8 pmol/mg protein/min. The SPD-induced stimulation of AC activity was almost completely reversed by 10 micromol/L Sch23390. These results indicate that SPD possesses an agonistic action on the D1 receptor. Forskolin-stimulated adenylate cyclase (FSAC) activity was used as a model to elucidate the effect of SPD on D2 receptors. The results indicate that DA inhibited FSAC activity dose-dependently, while SPD partially restored FSAC activity. Taken together, these results support the conclusion that SPD has dual actions on DA receptors that mediate AC activity, i.e., an agonistic action on D1 receptors and an antagonistic action on D2 receptors.

  16. P2Y6 Receptor-Mediated Proinflammatory Signaling in Human Bronchial Epithelia

    PubMed Central

    Hao, Yuan; Liang, Jocelyn F.; Chow, Alison W.; Cheung, Wing-tai; Ko, Wing-hung

    2014-01-01

    P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions. Our data show damage to the human bronchial epithelial cell line, 16HBE14o-, by poly-L-arginine-induced UDP release into the extracellular medium. Activation of P2Y6 receptor by its natural ligand, UDP, or its specific agonist, MRS 2693, led to the production of two proinflammatory cytokines, interleukin (IL)-6 and IL-8. This may have resulted from increased IL-6 and IL-8 mRNA expression, and activation of p38 and ERK1/2 MAPK, and NF-κB pathways. Our previous study demonstrated that UDP stimulated transepithelial Cl− secretion via both Ca2+- and cAMP-dependent pathways in 16HBE14o- epithelia. This was further confirmed in this study by simultaneous imaging of Ca2+ and cAMP levels in single cells using the Fura-2 fluorescence technique and a FRET-based approach, respectively. Moreover, the P2Y6 receptor-mediated production of IL-6 and IL-8 was found to be dependent on Ca2+, but not the cAMP/PKA pathway. Together, these studies show that nucleotides released during the airway inflammatory processes will activate P2Y6 receptors, which will lead to further release of inflammatory cytokines. The secretion of cytokines and the formation of such “cytokine networks” play an important role in sustaining the airway inflammatory disease. PMID:25243587

  17. Distinct Glucocorticoid Receptor Transcriptional Regulatory Surfaces Mediate the Cytotoxic and Cytostatic Effects of Glucocorticoids

    PubMed Central

    Rogatsky, Inez; Hittelman, Adam B.; Pearce, David; Garabedian, Michael J.

    1999-01-01

    Glucocorticoids act through the glucocorticoid receptor (GR), which can function as a transcriptional activator or repressor, to elicit cytostatic and cytotoxic effects in a variety of cells. The molecular mechanisms regulating these events and the target genes affected by the activated receptor remain largely undefined. Using cultured human osteosarcoma cells as a model for the GR antiproliferative effect, we demonstrate that in U20S cells, GR activation leads to irreversible growth inhibition, apoptosis, and repression of Bcl2. This cytotoxic effect is mediated by GR’s transcriptional repression function, since transactivation-deficient mutants and ligands still bring about apoptosis and Bcl2 down-regulation. In contrast, the antiproliferative effect of GR in SAOS2 cells is reversible, does not result in apoptosis or repression of Bcl2, and is a function of the receptor’s ability to stimulate transcription. Thus, the cytotoxic versus cytostatic outcome of glucocorticoid treatment is cell context dependent. Interestingly, the cytostatic effect of glucocorticoids in SAOS2 cells involves multiple GR activation surfaces. GR mutants and ligands that disrupt individual transcriptional activation functions (activation function 1 [AF-1] and AF-2) or receptor dimerization fail to fully inhibit cellular proliferation and, remarkably, discriminate between the targets of GR’s cytostatic action, the cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1. Induction of p21Cip1 is agonist dependent and requires AF-2 but not AF-1 or GR dimerization. In contrast, induction of p27Kip1 is agonist independent, does not require AF-2 or AF-1, but depends on GR dimerization. Our findings indicate that multiple GR transcriptional regulatory mechanisms that employ distinct receptor surfaces are used to evoke either the cytostatic or cytotoxic response to glucocorticoids. PMID:10373553

  18. Sequential phosphorylation mediates receptor- and kinase-induced inhibition of TREK-1 background potassium channels.

    PubMed

    Murbartián, Janet; Lei, Qiubo; Sando, Julianne J; Bayliss, Douglas A

    2005-08-26

    Background potassium channels determine membrane potential and input resistance and serve as prominent effectors for modulatory regulation of cellular excitability. TREK-1 is a two-pore domain background K+ channel (KCNK2, K2P2.1) that is sensitive to a variety of physicochemical and humoral factors. In this work, we used a recombinant expression system to show that activation of G alpha(q)-coupled receptors leads to inhibition of TREK-1 channels via protein kinase C (PKC), and we identified a critical phosphorylation site in a key regulatory domain that mediates inhibition of the channel. In HEK 293 cells co-expressing TREK-1 and either the thyrotropin-releasing hormone receptor (TRHR1) or the Orexin receptor (Orx1R), agonist stimulation induced robust channel inhibition that was suppressed by a bisindolylmaleimide PKC inhibitor but not by a protein kinase A blocker ((R(p))-cAMP-S). Channel inhibition by agonists or by direct activators of PKC (phorbol dibutyrate) and PKA (forskolin) was disrupted not only by alanine or aspartate mutations at an identified PKA site (Ser-333) in the C terminus, but also at a more proximal regulatory site in the cytoplasmic C terminus (Ser-300); S333A and S300A mutations enhanced basal TREK-1 current, whereas S333D and S300D substitutions mimicked phosphorylation and strongly diminished currents. When studied in combination, TREK-1 current density was enhanced in S300A/S333D but reduced in S300D/S333A mutant channels. Channel mutants were expressed and appropriately targeted to cell membranes. Together, these data support a sequential phosphorylation model in which receptor-induced kinase activation drives modification at Ser-333 that enables subsequent phosphorylation at Ser-300 to inhibit TREK-1 channel activity.

  19. Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

    PubMed Central

    Cozens-Roberts, C.; Lauffenburger, D. A.; Quinn, J. A.

    1990-01-01

    The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities. We compare the predictions of the probabilistic model with those of a deterministic model, and show how a deterministic approach can yield some inaccurate results; e.g., it cannot account for temporally continuous cell attach mentor detachment, it can underestimate the time needed for cell attachment, it can overestimate the time required for cell detachment for a given level of force, and it can overestimate the force necessary for cell detachment. PMID:2174271

  20. Characterization of 5-HT receptors mediating constriction of porcine carotid arteriovenous anastomoses; involvement of 5-HT1B/1D and novel receptors

    PubMed Central

    De Vries, Peter; Villalón, Carlos M; Heiligers, Jan P C; Saxena, Pramod R

    1998-01-01

    It was previously shown that porcine cranial arteriovenous anastomoses (AVAs) constrict to 5-hydroxytryptamine (5-HT), ergotamine, dihydroergotamine, as well as sumatriptan and that sumatriptan acts exclusively via 5-HT1B/1D receptors. The present study was devoted to establish the contribution of 5-HT1B/1D receptors in the constriction of AVAs elicited by 5-HT (in presence of 0.5 mg kg−1 ketanserin), ergotamine and dihydroergotamine in anaesthetized pigs.Intracarotid infusion of 5-HT (2 μg kg−1 min−1) and intravenous doses of ergotamine (2.5–20 μg kg−1) and dihydroergotamine (3–100 μg kg−1) reduced AVA and increased nutrient blood flows and vascular conductances. The vasodilator response to 5-HT, observed mainly in the skin and ear, was much more prominent than that of the ergot alkaloids.Treatment with the 5-HT1B/1D receptor antagonist GR127935 (0.5 mg kg−1, i.v.) significantly attenuated both ergot-induced AVA constriction and arteriolar dilatation, whereas GR127935 only slightly affected the carotid vascular effects of 5-HT.The results suggest that 5-HT constricts carotid AVAs primarily via receptors, which seem to differ from those (5-HT1B/1D) stimulated by sumatriptan. The ergot alkaloids produce AVA constriction for a substantial part via 5-HT1B/1D receptors, but also stimulate unidentified receptors. Both these non-5-HT1B/1D receptors may be targets for the development of novel antimigraine drugs.The moderate vasodilator response to the ergot derivatives seems to be mediated, at least in part, by 5-HT1B/1D receptors, whereas the arteriolar dilatation caused by 5-HT may be mediated by other, possibly 5-HT7 receptors. PMID:9605562

  1. Rho/ROCK acts downstream of lysophosphatidic acid receptor 1 in modulating P2X3 receptor-mediated bone cancer pain in rats

    PubMed Central

    Wu, Jing-xiang; Yuan, Xiao-min; Wang, Qiong; Wei, Wang

    2016-01-01

    Background Lysophosphatidic acid receptor 1 and Rho/ROCK signaling is implicated in bone cancer pain development. However, it remains unknown whether the two signaling pathways function together in P2X3 receptor-mediated bone cancer pain. Results In this study, using a rat model of bone cancer, we examined the expression of P2X3 and lysophosphatidic acid receptor 1 in rat dorsal root ganglion neurons and further dissected whether lysophosphatidic acid receptor 1 and Rho/ROCK-mediated pathways interacted in modulating rat pain behavior. Bone cancer was established by inoculating Walker 256 cells into the left tibia of female Wistar rats. We observed a gradual and yet significant decline in mean paw withdrawal threshold in rats with bone cancer, but not in control rats. Our immunohistochemical staining revealed that the number of P2X3- and lysophosphatidic acid receptor 1-positive dorsal root ganglion neurons was significantly greater in rats with bone cancer than control rats. Lysophosphatidic acid receptor 1 blockade with VPC32183 significantly attenuated decline in mean paw withdrawal threshold. Flinching behavior test further showed that lysophosphatidic acid receptor 1 inhibition with VPC32183 transiently but significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Rho inhibition by intrathecal BoTXC3 caused a rapid reversal in decline in mean paw withdrawal threshold of rats with bone cancer. Flinching behavior test showed that BoTXC3 transiently and significantly attenuated α,β-meATP-induced increase in paw lift time per minute. Similar findings were observed with ROCK inhibition by intrathecal Y27632. Furthermore, VPC32183 and BoTXC3 effectively aborted the appearance of lysophosphatidic acid-induced calcium influx peak. Conclusions Lysophosphatidic acid and its receptor LPAR1, acting through the Rho-ROCK pathway, regulate P2X3 receptor in the development of both mechanical and spontaneous pain in bone cancer. PMID:27094551

  2. Adenosine Signaling Increases Proinflammatory and Profibrotic Mediators through Activation of a Functional Adenosine 2B Receptor in Renal Fibroblasts.

    PubMed

    Wilkinson, Patrick F; Farrell, Francis X; Morel, Diane; Law, William; Murphy, Suzanne

    2016-07-01

    Interstitial renal fibrosis is a major pathophysiological manifestation of patients diagnosed with Chronic Kidney Disease (CKD), Diabetic Nephropathy (DN) and other inflammatory diseases. Adenosine signaling is an innate autocrine and paracrine cellular signaling pathway involving several key mediators that are elevated in the blood and kidneys of patients with DN. In these studies, we hypothesized that extracellular adenosine signals through one or more functional adenosine GPCRs on renal fibroblasts which increases profibrotic and proinflammatory mediators by inducing an activated fibroblast phenotype. Utilizing the renal fibroblast cell line NRK-49F, the presence and relative abundance of adenosine receptors (AR) A1, A2A, A2B, and A3 were quantified by RT-PCR. Under normal homeostatic conditions, only AR1 and AR2B were detected. The functionality of each receptor was then assessed by receptor specific pharmacological agonism and antagonism and assessed for modulation of the GPCR associated secondary messenger molecule, cyclic adenosine monophosphate (cAMP). Agonism of the AR2B receptor resulted in increased intracellular cAMP while agonism of the AR1 receptor inhibited cAMP modulation. Upon direct agonism of the AR2B receptor, transcripts for profibrotic and inflammatory mediators including SMA-α, IL-6, TGF-β, CTGF, and fibronectin were elevated between 2-4 fold. These data indicate that renal fibroblasts express a functional AR1 receptor that inhibits cAMP upon stimulation, leading to a functional AR2B receptor that increases cAMP upon stimulation and also induces an activated fibroblast phenotype resulting in increased fibrotic and inflammatory mediators.

  3. The chemokine receptors CXCR1 and CXCR2 couple to distinct G protein-coupled receptor kinases to mediate and regulate leukocyte functions

    PubMed Central

    Raghuwanshi, Sandeep K.; Su, Yingjun; Singh, Vandana; Hayes, Katherine; Richmond, Ann; Richardson, Ricardo M.

    2012-01-01

    The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized and internalized. In this study we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, shRNA was used to inhibit GRK 2, 3, 5 and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6, respectively, increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization and internalization, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect in CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6−/−) displayed an increase in CXCR2-mediated G-protein activation, but in vitro exhibited a decrease in chemotaxis, receptor desensitization and internalization relative to wild type (GRK6+/+) cells. In contrast, neutrophil recruitment in vivo in GRK6−/− mice was increased in response to delivery of CXCL1 through the air-pouch model. In a wound closure assay, GRK6−/− mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure as compared to GRK6+/+ animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis. PMID:22869904

  4. Nicotine enhances the cyclic AMP-dependent protein kinase-mediated phosphorylation of alpha4 subunits of neuronal nicotinic receptors.

    PubMed

    Hsu, Y N; Edwards, S C; Wecker, L

    1997-12-01

    Studies determined whether alpha4beta2 or alpha3beta2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha4beta2 and 500 nM for alpha3beta2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha4beta2 receptors were incubated with [gamma-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha4 subunit was present. Phosphorylation of alpha4 subunits of alpha4beta2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing alpha3beta2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha4 and not alpha3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.

  5. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  6. Enhanced Functional Activity of the Cannabinoid Type-1 Receptor Mediates Adolescent Behavior

    PubMed Central

    Kasanetz, Fernando; Lynch, Diane L.; Friemel, Chris M.; Lassalle, Olivier; Hurst, Dow P.; Steindel, Frauke; Monory, Krisztina; Schäfer, Carola; Miederer, Isabelle; Leweke, F. Markus; Schreckenberger, Mathias; Lutz, Beat; Reggio, Patricia H.; Manzoni, Olivier J.; Spanagel, Rainer

    2015-01-01

    Adolescence is characterized by drastic behavioral adaptations and comprises a particularly vulnerable period for the emergence of various psychiatric disorders. Growing evidence reveals that the pathophysiology of these disorders might derive from aberrations of normal neurodevelopmental changes in the adolescent brain. Understanding the molecular underpinnings of adolescent behavior is therefore critical for understanding the origin of psychopathology, but the molecular mechanisms that trigger adolescent behavior are unknown. Here, we hypothesize that the cannabinoid type-1 receptor (CB1R) may play a critical role in mediating adolescent behavior because enhanced endocannabinoid (eCB) signaling has been suggested to occur transiently during adolescence. To study enhanced CB1R signaling, we introduced a missense mutation (F238L) into the rat Cnr1 gene that encodes for the CB1R. According to our hypothesis, rats with the F238L mutation (Cnr1F238L) should sustain features of adolescent behavior into adulthood. Gain of function of the mutated receptor was demonstrated by in silico modeling and was verified functionally in a series of biochemical and electrophysiological experiments. Mutant rats exhibit an adolescent-like phenotype during adulthood compared with wild-type littermates, with typical high risk/novelty seeking, increased peer interaction, enhanced impulsivity, and augmented reward sensitivity for drug and nondrug reward. Partial inhibition of CB1R activity in Cnr1F238L mutant rats normalized behavior and led to a wild-type phenotype. We conclude that the activity state and functionality of the CB1R is critical for mediating adolescent behavior. These findings implicate the eCB system as an important research target for the neuropathology of adolescent-onset mental health disorders. SIGNIFICANCE STATEMENT We present the first rodent model with a gain-of-function mutation in the cannabinoid type-1 receptor (CB1R). Adult mutant rats exhibit an adolescent

  7. Riluzole mediates anti-tumor properties in breast cancer cells independent of metabotropic glutamate receptor-1.

    PubMed

    Speyer, Cecilia L; Nassar, Mahdy A; Hachem, Ali H; Bukhsh, Miriam A; Jafry, Waris S; Khansa, Rafa M; Gorski, David H

    2016-06-01

    Riluzole, the only drug approved by the FDA for treating amyotrophic lateral sclerosis, inhibits melanoma proliferation through its inhibitory effect on glutamatergic signaling. We demonstrated that riluzole also inhibits the growth of triple-negative breast cancer (TNBC) and described a role for metabotropic glutamate receptor-1 (GRM1) in regulating TNBC cell growth and progression. However, the role of GRM1 in mediating riluzole's effects in breast cancer has not been fully elucidated. In this study, we seek to determine how much of riluzole's action in breast cancer is mediated through GRM1. We investigated anti-tumor properties of riluzole in TNBC and ER+ cells using cell growth, invasion, and soft-agar assays and compared riluzole activity with GRM1 levels. Using Lentiviral vectors expressing GRM1 or shGRM1, these studies were repeated in cells expressing high or low GRM1 levels where the gene was either silenced or overexpressed. Riluzole inhibited proliferation, invasion, and colony formation in both TNBC and ER+ cells. There was a trend between GRM1 expression in TNBC cells and their response to riluzole in both cell proliferation and invasion assays. However, silencing and overexpression studies had no effect on cell sensitivity to riluzole. Our results clearly suggest a GRM1-independent mechanism through which riluzole mediates its effects on breast cancer cells. Understanding the mechanism by which riluzole mediates breast cancer progression will be useful in identifying new therapeutic targets for treating TNBC and in facilitating stratification of patients in clinical trials using riluzole in conjunction with conventional therapy.

  8. The effects of soluble recombinant complement receptor 1 on complement-mediated experimental glomerulonephritis.

    PubMed

    Couser, W G; Johnson, R J; Young, B A; Yeh, C G; Toth, C A; Rudolph, A R

    1995-05-01

    Complement is a major mediator of tissue injury in several types of glomerulonephritis. However, no therapeutic agents that inhibit complement activation are available for human use. sCR1 (TP10, BRL 55736) is a recombinant, soluble human complement receptor 1 (CR1) molecule lacking transmembrane and cytoplasmic domains that inhibits C3 and C5 convertase activity by preferentially binding C4b and C3b. To test the efficacy of sCR1 on complement-mediated glomerulonephritis, rats were pretreated with sCR1 (60 mg/kg per day) before and during the induction of three models of complement-dependent glomerulonephritis (concanavalin A and antithymocyte serum models of proliferative glomerulonephritis, passive Heyman nephritis). Daily sCR1 and complement hemolytic activity levels were measured, and renal histology and urine protein excretion were examined. Mean serum sCR1 levels of 100 to 200 micrograms/mL were maintained with a reduction in complement hemolytic activity to less than 15% in most animals. In the antithymocyte serum model, sCR1-treated animals had significant reductions in mesangiolysis, glomerular platelet and macrophage infiltrates, and proteinuria at 48 h. In the concanavalin A model, sCR1 significantly reduced glomerular C3 and fibrin deposits, platelet infiltrates, and proteinuria at 48 h. In passive Heymann nephritis, proteinuria was also significantly reduced (199 +/- 8.5 versus 125 +/- 16 mg/day, P < 0.002) at 5 days. It was concluded that sCR1 significantly reduces both morphologic and functional consequences of several different types of complement-mediated glomerulonephritis and deserves evaluation as a potential therapeutic agent in complement-mediated immune glomerular disease in humans.

  9. Selective inhibition of beta(2)-adrenergic receptor-mediated cAMP generation by activation of the P2Y(2) receptor in mouse pineal gland tumor cells.

    PubMed

    Suh, B C; Kim, J S; Namgung, U; Han, S; Kim, K T

    2001-06-01

    Rhythmic noradrenergic signaling from the hypothalamic clock in the suprachiasmatic nucleus to the pineal gland causes an increase in intracellular cAMP which regulates the circadian fluctuation of melatonin synthesis. The activation of phospholipase C (PLC)-coupled P2Y(2) receptors upon treatment with ATP and UTP exclusively inhibited the isoproterenol-stimulated cAMP production in mouse pineal gland tumor cells. However, the activation of other PLC-coupled receptors including P2Y(1) and bombesin receptors had little or no effect on the isoproterenol-stimulated cAMP production. Also, ATP did not inhibit cAMP production caused by forskolin, prostaglandin E(2), or the adenosine analog NECA. These results suggest a selective coupling between signalings of P2Y(2) and beta(2)-adrenergic receptors. The binding of [(3)H]CGP12177 to beta(2)-adrenergic receptors was not effected by the presence of ATP or UTP. Ionomycin decreased the isoproterenol-stimulated cAMP production, whereas phorbol 12-myristate 13-acetate slightly potentiated the isoproterenol response. Chelation of intracellular Ca(2+), however, had little effect on the ATP-induced inhibition of cAMP production, while it completely reversed the ionomycin-induced inhibition. Treatment of cells with pertussis toxin almost completely blocked the inhibitory effect of nucleotides. Pertussis toxin also inhibited the nucleotide-induced increase in intracellular Ca(2+) and inositol 1,4,5-trisphosphate production by 30-40%, suggesting that the ATP-mediated inhibition of the cAMP generation and the partial activation of PLC are mediated by pertussis toxin-sensitive G(i)-protein. We conclude that one of the functions of P2Y(2) receptors on the pineal gland is the selective inhibition of beta-adrenergic receptor-mediated signaling pathways via the inhibitory G-proteins.

  10. Prevention of RhoA activation and cofilin-mediated actin polymerization mediates the antihypertrophic effect of adenosine receptor agonists in angiotensin II- and endothelin-1-treated cardiomyocytes.

    PubMed

    Zeidan, Asad; Gan, Xiaohong Tracey; Thomas, Ashley; Karmazyn, Morris

    2014-01-01

    Adenosine receptor activation has been shown to be associated with diminution of cardiac hypertrophy and it has been suggested that endogenously produced adenosine may serve to blunt pro-hypertrophic processes. In the present study, we determined the effects of two pro-hypertrophic stimuli, angiotensin II (Ang II, 100 nM) and endothelin-1 (ET-1, 10 nM) on Ras homolog gene family, member A (RhoA)/Rho-associated, coiled-coil containing protein kinase (ROCK) activation in cultured neonatal rat ventricular myocytes and whether the latter serves as a target for the anti-hypertrophic effect of adenosine receptor activation. Both hypertrophic stimuli potently increased RhoA activity with peak activation occurring 15-30 min following agonist addition. These effects were associated with significantly increased phosphorylation (inactivation) of cofilin, a downstream mediator of RhoA, an increase in actin polymerization, and increased activation and nuclear import of p38 mitogen activated protein kinase. The ability of both Ang II and ET-1 to activate the RhoA pathway was completely prevented by the adenosine A1 receptor agonist N (6)-cyclopentyladenosine, the A2a receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine, the A3 receptor agonist N (6)-(3-iodobenzyl)adenosine-5'-methyluronamide as well as the nonspecific adenosine analog 2-chloro adenosine. All effects of specific receptor agonists were prevented by their respective receptor antagonists. Moreover, all adenosine agonists prevented either Ang II- or ET-1-induced hypertrophy, a property shared by the RhoA inhibitor Clostridium botulinum C3 exoenzyme, the ROCK inhibitor Y-27632 or the actin depolymerizing agent latrunculin B. Our study therefore demonstrates that both Ang II and ET-1 can activate the RhoA pathway and that prevention of the hypertrophic response to both agonists by adenosine receptor activation is mediated by prevention of RhoA stimulation and actin polymerization.

  11. β-arrestin-1 mediates the TCR-triggered re-routing of distal receptors to the immunological synapse by a PKC-mediated mechanism

    PubMed Central

    Fernández-Arenas, Elena; Calleja, Enrique; Martínez-Martín, Nadia; Gharbi, Severine I; Navajas, Rosana; García-Medel, Noel; Penela, Petronila; Alcamí, Antonio; Mayor, Federico; Albar, Juan P; Alarcón, Balbino

    2014-01-01

    T-cell receptors (TCR) recognize their antigen ligand at the interface between T cells and antigen-presenting cells, known as the immunological synapse (IS). The IS provides a means of sustaining the TCR signal which requires the continual supply of new TCRs. These are endocytosed and redirected from distal membrane locations to the IS. In our search for novel cytoplasmic effectors, we have identified β-arrestin-1 as a ligand of non-phosphorylated resting TCRs. Using dominant-negative and knockdown approaches we demonstrate that β-arrestin-1 is required for the internalization and downregulation of non-engaged bystander TCRs. Furthermore, TCR triggering provokes the β-arrestin-1-mediated downregulation of the G-protein coupled chemokine receptor CXCR4, but not of other control receptors. We demonstrate that β-arrestin-1 recruitment to the TCR, and bystander TCR and CXCR4 downregulation, are mechanistically mediated by the TCR-triggered PKC-mediated phosphorylation of β-arrestin-1 at Ser163. This mechanism allows the first triggered TCRs to deliver a stop migration signal, and to promote the internalization of distal TCRs and CXCR4 and their translocation to the IS. This receptor crosstalk mechanism is critical to sustain the TCR signal. PMID:24502978

  12. Estrogen receptor-alpha mediates estrogen facilitation of baroreflex heart rate responses in conscious mice.

    PubMed

    Pamidimukkala, Jaya; Xue, Baojian; Newton, Leslie G; Lubahn, Dennis B; Hay, Meredith

    2005-03-01

    Estrogen facilitates baroreflex heart rate responses evoked by intravenous infusion of ANG II and phenylephrine (PE) in ovariectomized female mice. The present study aims to identify the estrogen receptor subtype involved in mediating these effects of estrogen. Baroreflex responses to PE, ANG II, and sodium nitroprusside (SNP) were tested in intact and ovariectomized estrogen receptor-alpha knockout (ERalphaKO) with (OvxE+) or without (OvxE-) estrogen replacement. Wild-type (WT) females homozygous for the ERalpha(+/+) were used as controls. Basal mean arterial pressures (MAP) and heart rates were comparable in all the groups except the ERalphaKO-OvxE+ mice. This group had significantly smaller resting MAP, suggesting an effect of estrogen on resting vascular tone possibly mediated by the ERbeta subtype. Unlike the WT females, estrogen did not facilitate baroreflex heart rate responses to either PE or ANG II in the ERalphaKO-OvxE+ mice. The slope of the line relating baroreflex heart rate decreases with increases in MAP evoked by PE was comparable in ERalphaKO-OvxE- (-6.97 +/- 1.4 beats.min(-1).mmHg(-1)) and ERalphaKO-OvxE+ (-6.18 +/- 1.3) mice. Likewise, the slope of the baroreflex bradycardic responses to ANG II was similar in ERalphaKO-OvxE- (-3.87 +/- 0.5) and ERalphaKO-OvxE+(-2.60 +/- 0.5) females. Data suggest that estrogen facilitation of baroreflex responses to PE and ANG II is predominantly mediated by ERalpha subtype. A second important observation in the present study is that the slope of ANG II-induced baroreflex bradycardia is significantly blunted compared with PE in the intact as well as the ERalphaKO-OvxE+ females. We have previously reported that this ANG II-mediated blunting of cardiac baroreflexes is observed only in WT males and not in ovariectomized WT females independent of their estrogen replacement status. The present data suggest that in females lacking ERalpha, ANG II causes blunting of cardiac baroreflexes similar to males and may be

  13. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  14. Adenosine A1 receptors mediate inhibition of cAMP formation in vitro in the pontine, REM sleep induction zone.

    PubMed

    Marks, Gerald A; Birabil, Christian G; Speciale, Samuel G

    2005-11-09

    Microinjection of adenosine A1 receptor agonist or an inhibitor of adenylyl cyclase into the caudal, oral pontine reticular formation (PnOc) of the rat induces a long-lasting increase in REM sleep. Here, we report significant inhibition of forskolin-stimulated cAMP in dissected pontine tissue slices containing the PnOc incubated with the A1 receptor agonist, cyclohexaladenosine (10(-8) M). These data are consistent with adenosine A1 receptor agonist actions on REM sleep mediated through inhibition of cAMP.

  15. Methods for recording and measuring tonic GABAA receptor-mediated inhibition.

    PubMed

    Bright, Damian P; Smart, Trevor G

    2013-12-05

    Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current.

  16. Avian orientation: the pulse effect is mediated by the magnetite receptors in the upper beak.

    PubMed

    Wiltschko, Wolfgang; Munro, Ursula; Ford, Hugh; Wiltschko, Roswitha

    2009-06-22

    Migratory silvereyes treated with a strong magnetic pulse shift their headings by approximately 90 degrees , indicating an involvement of magnetite-based receptors in the orientation process. Structures containing superparamagnetic magnetite have been described in the inner skin at the edges of the upper beak of birds, while single-domain magnetite particles are indicated in the nasal cavity. To test which of these structures mediate the pulse effect, we subjected migratory silvereyes, Zosterops l. lateralis, to a strong pulse, and then tested their orientation, while the skin of their upper beak was anaesthetized with a local anaesthetic to temporarily deactivate the magnetite-containing structures there. After the pulse, birds without anaesthesia showed the typical shift, whereas when their beak was anaesthetized, they maintained their original headings. This indicates that the superparamagnetic magnetite-containing structures in the skin of the upper beak are most likely the magnetoreceptors that cause the change in headings observed after pulse treatment.

  17. Muscarinic receptors mediate the endocrine-disrupting effects of an organophosphorus insecticide in zebrafish.

    PubMed

    Santos da Rosa, João Gabriel; Alcântara Barcellos, Heloísa Helena de; Fagundes, Michele; Variani, Cristiane; Rossini, Mainara; Kalichak, Fabiana; Koakoski, Gessi; Acosta Oliveira, Thiago; Idalencio, Renan; Frandoloso, Rafael; Piato, Angelo L; José Gil Barcellos, Leonardo

    2017-04-03

    The glucocorticoid cortisol, the end product of hypothalamus-pituitary-interrenal axis in zebrafish (Danio rerio), is synthesized via steroidogenesis and promotes important physiological regulations in response to a stressor. The failure of this axis leads to inability to cope with environmental challenges preventing adaptive processes in order to restore homeostasis. Pesticides and agrichemicals are widely used, and may constitute an important class of environmental pollutants when reach aquatic ecosystems and nontarget species. These chemical compounds may disrupt hypothalamus-pituitary-interrenal axis by altering synthesis, structure or function of its constituents. We present evidence that organophosphorus exposure disrupts stress response by altering the expression of key genes of the neural steroidogenesis, causing downregulation of star, hsp70, and pomc genes. This appears to be mediated via muscarinic receptors, since the muscarinic antagonist scopolamine blocked these effects.

  18. Nonsense-mediated mRNA decay modulates immune receptor levels to regulate plant antibacterial defense.

    PubMed

    Gloggnitzer, Jiradet; Akimcheva, Svetlana; Srinivasan, Arunkumar; Kusenda, Branislav; Riehs, Nina; Stampfl, Hansjörg; Bautor, Jaqueline; Dekrout, Bettina; Jonak, Claudia; Jiménez-Gómez, José M; Parker, Jane E; Riha, Karel

    2014-09-10

    Nonsense-mediated mRNA decay (NMD) is a conserved eukaryotic RNA surveillance mechanism that degrades aberrant mRNAs. NMD impairment in Arabidopsis is linked to constitutive immune response activation and enhanced antibacterial resistance, but the underlying mechanisms are unknown. Here we show that NMD contributes to innate immunity in Arabidopsis by controlling the turnover of numerous TIR domain-containing, nucleotide-binding, leucine-rich repeat (TNL) immune receptor-encoding mRNAs. Autoimmunity resulting from NMD impairment depends on TNL signaling pathway components and can be triggered through deregulation of a single TNL gene, RPS6. Bacterial infection of plants causes host-programmed inhibition of NMD, leading to stabilization of NMD-regulated TNL transcripts. Conversely, constitutive NMD activity prevents TNL stabilization and impairs plant defense, demonstrating that host-regulated NMD contributes to disease resistance. Thus, NMD shapes plant innate immunity by controlling the threshold for activation of TNL resistance pathways.

  19. Kappa opioid receptor activation alleviates experimental autoimmune encephalomyelitis and promotes oligodendrocyte-mediated remyelination.

    PubMed

    Du, Changsheng; Duan, Yanhui; Wei, Wei; Cai, Yingying; Chai, Hui; Lv, Jie; Du, Xiling; Zhu, Jian; Xie, Xin

    2016-04-04

    Multiple sclerosis (MS) is characterized by autoimmune damage to the central nervous system. All the current drugs for MS target the immune system. Although effective in reducing new lesions, they have limited effects in preventing the progression of disability. Promoting oligodendrocyte-mediated remyelination and recovery of neurons are the new directions of MS therapy. The endogenous opioid system, consisting of MOR, DOR, KOR and their ligands, has been suggested to participate in the pathogenesis of MS. However, the exact receptor and mechanism remain elusive. Here we show that genetic deletion of KOR exacerbates experimental autoimmune encephalomyelitis, whereas activating KOR with agonists alleviates the symptoms. KOR does not affect immune cell differentiation and function. Instead, it promotes oligodendrocyte differentiation and myelination both in vitro and in vivo. Our study suggests that targeting KOR might be an intriguing way to develop new MS therapies that may complement the existing immunosuppressive approaches.

  20. Methods for recording and measuring tonic GABAA receptor-mediated inhibition

    PubMed Central

    Bright, Damian P.; Smart, Trevor G.

    2013-01-01

    Tonic inhibitory conductances mediated by GABAA receptors have now been identified and characterized in many different brain regions. Most experimental studies of tonic GABAergic inhibition have been carried out using acute brain slice preparations but tonic currents have been recorded under a variety of different conditions. This diversity of recording conditions is likely to impact upon many of the factors responsible for controlling tonic inhibition and can make comparison between different studies difficult. In this review, we will firstly consider how various experimental conditions, including age of animal, recording temperature and solution composition, are likely to influence tonic GABAA conductances. We will then consider some technical considerations related to how the tonic conductance is measured and subsequently analyzed, including how the use of current noise may provide a complementary and reliable method for quantifying changes in tonic current. PMID:24367296

  1. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms

    PubMed Central

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.

    2002-01-01

    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  2. The overexpressed human 46-kDa mannose 6-phosphate receptor mediates endocytosis and sorting of. beta. -glucuronidase

    SciTech Connect

    Watanabe, H.; Grubb, J.H.; Sly, W.S. )

    1990-10-01

    The authors studied the function of the human small (46-kDa) mannose 6-phosphate receptor (SMPR) in transfected mouse L cells that do not express the larger insulin-like growth factor II/mannose 6-phosphate receptor. Cells overexpressing human SMPR were studied for enzyme binding to cell surface receptors, for binding to intracellular receptors in permeabilized cells, and for receptor-mediated endocytosis of recombinant human {beta}-glucuronidase. Specific binding to human SMPR in permeabilized cells showed a pH optimum between pH 6.0 and pH 6.5. Binding was significant in the present of EDTA but was enhanced by added divalent cations. Up to 2.3{percent} of the total functional receptor could be detected on the cell surface by enzyme binding. They present experiments showing that at very high levels of overexpression, and at pH 6.5, human SMPR mediated the endocytosis of {beta}-glucuronidase. At pH 7.5, the rate of endocytosis was only 14{percent} the rate seen at pH 6.5. Cells overexpressing human SMPR also showed reduced secretion of newly synthesized {beta}-glucuronidase when compared to cells transfected with vector only, suggesting that overexpressed human SMPR can participate in sorting of newly synthesized {beta}-glucuronidase and partially correct the sorting defect in mouse L cells that do not express the insulin-like growth factor II/mannose 6-phosphate receptor.

  3. Src, a Molecular Switch Governing Gain Control of Synaptic Transmission Mediated by N-methyl-D-Aspartate Receptors

    NASA Astrophysics Data System (ADS)

    Yu, Xian-Min; Salter, Michael W.

    1999-07-01

    The N-methyl-D-aspartate (NMDA) receptor is a principal subtype of glutamate receptor mediating fast excitatory transmission at synapses in the dorsal horn of the spinal cord and other regions of the central nervous system. NMDA receptors are crucial for the lasting enhancement of synaptic transmission that occurs both physiologically and in pathological conditions such as chronic pain. Over the past several years, evidence has accumulated indicating that the activity of NMDA receptors is regulated by the protein tyrosine kinase, Src. Recently it has been discovered that, by means of up-regulating NMDA receptor function, activation of Src mediates the induction of the lasting enhancement of excitatory transmission known as long-term potentiation in the CA1 region of the hippocampus. Also, Src has been found to amplify the up-regulation of NMDA receptor function that is produced by raising the intracellular concentration of sodium. Sodium concentration increases in neuronal dendrites during high levels of firing activity, which is precisely when Src becomes activated. Therefore, we propose that the boost in NMDA receptor function produced by the coincidence of activating Src and raising intracellular sodium may be important in physiological and pathophysiological enhancement of excitatory transmission in the dorsal horn of the spinal cord and elsewhere in the central nervous system.

  4. Cholera Toxin Inhibits the T-Cell Antigen Receptor-Mediated Increases in Inositol Trisphosphate and Cytoplasmic Free Calcium

    NASA Astrophysics Data System (ADS)

    Imboden, John B.; Shoback, Dolores M.; Pattison, Gregory; Stobo, John D.

    1986-08-01

    The addition of monoclonal antibodies to the antigen receptor complex on the malignant human T-cell line Jurkat generates increases in inositol trisphosphate and in the concentration of cytoplasmic free calcium. Exposure of Jurkat cells to cholera toxin for 3 hr inhibited these receptor-mediated events and led to a selective, partial loss of the antigen receptor complex from the cellular surface. None of the effects of cholera toxin on the antigen receptor complex were mimicked by the B subunit of cholera toxin or by increasing intracellular cAMP levels with either forskolin or 8-bromo cAMP. These results suggest that a cholera toxin substrate can regulate signal transduction by the T-cell antigen receptor.

  5. Cubilin, the endocytic receptor for intrinsic factor-vitamin B(12) complex, mediates high-density lipoprotein holoparticle endocytosis.

    PubMed

    Hammad, S M; Stefansson, S; Twal, W O; Drake, C J; Fleming, P; Remaley, A; Brewer, H B; Argraves, W S

    1999-08-31

    Receptors that endocytose high-density lipoproteins (HDL) have been elusive. Here yolk-sac endoderm-like cells were used to identify an endocytic receptor for HDL. The receptor was isolated by HDL affinity chromatography and identified as cubilin, the recently described endocytic receptor for intrinsic factor-vitamin B(12). Cubilin antibodies inhibit HDL endocytosis by the endoderm-like cells and in mouse embryo yolk-sac endoderm, a prominent site of cubilin expression. Cubilin-mediated HDL endocytosis is inhibitable by HDL(2), HDL(3), apolipoprotein (apo)A-I, apoA-II, apoE, and RAP, but not by low-density lipoprotein (LDL), oxidized LDL, VLDL, apoC-I, apoC-III, or heparin. These findings, coupled with the fact that cubilin is expressed in kidney proximal tubules, suggest a role for this receptor in embryonic acquisition of maternal HDL and renal catabolism of filterable forms of HDL.

  6. Pharmacologic specificity of nicotinic receptor-mediated relaxation of muscarinic receptor precontracted human gastric clasp and sling muscle fibers within the gastroesophageal junction.

    PubMed

    Braverman, Alan S; Vegesna, Anil K; Miller, Larry S; Barbe, Mary F; Tiwana, Mansoor; Hussain, Kashif; Ruggieri, Michael R

    2011-07-01

    Relaxation of gastric clasp and sling muscle fibers is involved the transient lower esophageal sphincter relaxations underlying the pathophysiology of gastroesophageal reflux disease (GERD). These fibers do not contribute tone to the high-pressure zone in GERD patients, indicating their role in pathophysiology. This study identifies some mediators of the nicotine-induced relaxation of muscarinic receptor precontracted gastric clasp and sling fibers. Muscle strips from organ donors precontracted with bethanechol were relaxed with nicotine and then rechallenged after washing and adding inhibitors tetrodotoxin (TTX), the nitric-oxide synthase inhibitor L-nitro-arginine methyl ester (L-NAME), the β-adrenoceptor antagonist propranolol, the glycine receptor antagonist strychnine or ginkgolide B, and the GABA(A) receptor antagonist bicuculline or 2-(3-carboxypropyl)-3-amino-6-(4 methoxyphenyl)pyridazinium bromide [(gabazine) SR95531]. TTX only inhibited clasp fiber relaxations. L-NAME and propranolol inhibited, and ginkgolide B was ineffective in both. SR95531 was ineffective in clasp fibers and partially effective in sling fibers. Strychnine and bicuculline prevented relaxations with low potency, indicating actions not on glycine or GABA(A) receptors but more consistent with nicotinic receptor blockade. Bethanechol-precontracted fibers were relaxed by the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine and by the β-adrenergic agonist isoproterenol (clasp fibers only) but not by the glycine receptor agonist taurine or glycine or the GABA(A) agonist muscimol. These data indicate that nicotinic receptor activation mediates relaxation via release of nitric oxide in clasp and sling fibers, norepinephrine acting on β-adrenoceptors in clasp fibers, and GABA acting on GABA(A) receptors in sling fibers. Agents that selectively prevent these relaxations may be useful in the treatment of GERD.

  7. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    PubMed

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  8. Current injection and receptor-mediated excitation produce similar maximal firing rates in hypoglossal motoneurons.

    PubMed

    Wakefield, Hilary E; Fregosi, Ralph F; Fuglevand, Andrew J

    2016-03-01

    The maximum firing rates of motoneurons (MNs), activated in response to synaptic drive, appear to be much lower than that elicited by current injection. It could be that the decrease in input resistance associated with increased synaptic activity (but not current injection) might blunt overall changes in membrane depolarization and thereby limit spike-frequency output. To test this idea, we recorded, in the same cells, maximal firing responses to current injection and to synaptic activation. We prepared 300 μm medullary slices in neonatal rats that contained hypoglossal MNs and used whole-cell patch-clamp electrophysiology to record their maximum firing rates in response to triangular-ramp current injections and to glutamate receptor-mediated excitation. Brief pressure pulses of high-concentration glutamate led to significant depolarization, high firing rates, and temporary cessation of spiking due to spike inactivation. In the same cells, we applied current clamp protocols that approximated the time course of membrane potential change associated with glutamate application and with peak current levels large enough to cause spike inactivation. Means (SD) of maximum firing rates obtained in response to glutamate application were nearly identical to those obtained in response to ramp current injection [glutamate 47.1 ± 12.0 impulses (imp)/s, current injection 47.5 ± 11.2 imp/s], even though input resistance was 40% less during glutamate application compared with current injection. Therefore, these data suggest that the reduction in input resistance associated with receptor-mediated excitation does not, by itself, limit the maximal firing rate responses in MNs.

  9. Androgen and estrogen receptor mediated mechanisms of testosterone action in male rat pelvic autonomic ganglia

    PubMed Central

    Purves-Tyson, T.D.; Arshi, M.S.; Handelsman, D. J.; Cheng, Y.; Keast, J. R.

    2007-01-01

    Although male reproductive function is primarily androgen dependent, many studies suggest that estrogens have direct actions on the male reproductive organs. Pelvic autonomic neurons provide the motor control of the internal reproductive organs and the penis and various properties of these neurons are affected by endogenous androgens. However, the possible role of estrogens at this site has not been examined. Here we have investigated the significance of estrogens produced by aromatisation of testosterone in the physiological actions of androgens on adult male rat pelvic ganglion neurons. RT-PCR studies showed that aromatase and both estrogen receptors (ERα and ERβ) are expressed in these ganglia. Western blotting also showed that aromatase is expressed in male pelvic ganglia. Using immunohistochemical visualisation, ERα was predominantly expressed by nitric oxide synthase (NOS)-positive parasympathetic pelvic ganglion neurons. In vivo studies showed that the decrease in pelvic ganglion soma size caused by gonadectomy could be prevented by administration of testosterone (T) or dihydrotestosterone (DHT), but not 17β-estradiol (E2), showing that this maintenance action of testosterone is mediated entirely by androgenic mechanisms. However, in vitro studies of cultured pelvic ganglion neurons revealed that T, DHT and E each stimulated the growth of longer and more complex neurites in both noradrenergic and cholinergic NOS-expressing neurons. The effects of T were attenuated by either androgen or estrogen receptor antagonists, or by inhibition of aromatase. Together these studies demonstrate that estrogens are likely to be synthesised in the male pelvic ganglia, produced from testosterone by local aromatase. The effects of androgens on axonal growth are likely to be at least partly mediated by estrogenic mechanisms, which may be important for understanding disease-, aging- and injury-induced plasticity in this part of the nervous system. PMID:17629410

  10. The TM2 6′ Position of GABAA Receptors Mediates Alcohol Inhibition

    PubMed Central

    Howard, Rebecca J.; Trudell, James R.; Harris, R. Adron

    2012-01-01

    Ionotropic GABAA receptors (GABAARs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABAARs. We used Xenopus laevis oocytes to investigate the 6′ position in the second transmembrane region of GABAARs as a site influencing alcohol inhibition. We asked whether modification of the 6′ position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6′ position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6′ position. We verified a role for the 6′ position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABAARs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABAAR homolog, revealing that the 6′ position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6′ positions in both α2 and β2 GABAAR subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels. PMID:22072732

  11. cap alpha. /sub 2/-Adrenergic receptor-mediated sensitization of forskolin-stimulated cyclic AMP production

    SciTech Connect

    Jones, S.B.; Toews, M.L.; Turner, J.T.; Bylund, D.B.

    1987-03-01

    Preincubation of HT29 human colonic adenocarcinoma cells with ..cap alpha../sub 2/-adrenergic agonists resulted in a 10- to 20-fold increase in forskolin-stimulated cyclic AMP production as compared to cells preincubated without agonist. Similar results were obtained using either a (/sup 3/H)adenine prelabeling assay or a cyclic AMP radioimmunoassay to measure cyclic AMP levels. This phenomenon, which is termed sensitization, is ..cap alpha../sub 2/-adrenergic receptor-mediated and rapid in onset and reversal. Yohimbine, an ..cap alpha../sub 2/-adrenergic receptor-selective antagonist, blocked norepinephrine-induced sensitization, whereas prazosin (..cap alpha../sub 1/-adrenergic) and sotalol (..beta..-adrenergic) did not. The time for half-maximal sensitization was 5 min and the half-time for reversal was 10 min. Only a 2-fold sensitization of cyclic AMP production stimulated by vasoactive intestinal peptide was observed, indicating that sensitization is relatively selective for forskolin. Sensitization reflects an increased production of cyclic AMP and not a decreased degradation of cyclic AMP, since incubation with a phosphodiesterase inhibitor and forskolin did not mimic sensitization. Increasing the levels of cyclic AMP during the preincubation had no effect on sensitization, indicating that sensitization is not caused by decreased cyclic AMP levels during the preincubation. This rapid and dramatic sensitization of forskolin-stimulated cyclic AMP production is a previously unreported effect that can be added to the growing list of ..cap alpha../sub 2/-adrenergic responses that are not mediated by a decrease in cyclic AMP.

  12. Transient receptor potential ankyrin-1 has a major role in mediating visceral pain in mice.

    PubMed

    Cattaruzza, Fiore; Spreadbury, Ian; Miranda-Morales, Marcela; Grady, Eileen F; Vanner, Stephen; Bunnett, Nigel W

    2010-01-01

    The excitatory ion channel transient receptor potential ankyrin-1 (TRPA1) is prominently expressed by primary afferent neurons and is a mediator of inflammatory pain. Inflammatory agents can directly activate [e.g., hydroxynonenal (HNE), prostaglandin metabolites] or indirectly sensitize [e.g., agonists of protease-activated receptor (PAR(2))] TRPA1 to induce somatic pain and hyperalgesia. However, the contribution of TRPA1 to visceral pain is unknown. We investigated the role of TRPA1 in visceral hyperalgesia by measuring abdominal visceromotor responses (VMR) to colorectal distention (CRD) after intracolonic administration of TRPA1 agonists [mustard oil (MO), HNE], sensitizing agents [PAR(2) activating peptide (PAR(2)-AP)], and the inflammatory agent trinitrobenzene sulfonic acid (TNBS) in trpa1(+/+) and trpa1(-/-) mice. Sensory neurons innervating the colon, identified by retrograde tracing, coexpressed immunoreactive TRPA1, calcitonin gene-related peptide, and substance P, expressed TRPA1 mRNA and responded to MO with depolarizing currents. Intracolonic MO and HNE increased VMR to CRD and induced immunoreactive c-fos in spinal neurons in trpa1+/+ but not in trpa1(-/-) mice. Intracolonic PAR(2)-AP induced mechanical hyperalgesia in trpa1+/+ but not in trpa1(-/-) mice. TNBS-induced colitis increased in VMR to CRD and induced c-fos in spinal neurons in trpa1(+/+) but not in trpa1(-/-) mice. Thus TRPA1 is expressed by colonic primary afferent neurons. Direct activation of TRPA1 causes visceral hyperalgesia, and TRPA1 mediates PAR(2)-induced hyperalgesia. TRPA1 deletion markedly reduces colitis-induced mechanical hyperalgesia in the colon. Our results suggest that TRPA1 has a major role in visceral nociception and may be a therapeutic target for colonic inflammatory pain.

  13. The TM2 6' position of GABA(A) receptors mediates alcohol inhibition.

    PubMed

    Johnson, W David; Howard, Rebecca J; Trudell, James R; Harris, R Adron

    2012-02-01

    Ionotropic GABA(A) receptors (GABA(A)Rs), which mediate inhibitory neurotransmission in the central nervous system, are implicated in the behavioral effects of alcohol and alcoholism. Site-directed mutagenesis studies support the presence of discrete molecular sites involved in alcohol enhancement and, more recently, inhibition of GABA(A)Rs. We used Xenopus laevis oocytes to investigate the 6' position in the second transmembrane region of GABA(A)Rs as a site influencing alcohol inhibition. We asked whether modification of the 6' position by substitution with larger residues or methanethiol labeling [using methyl methanethiosulfonate (MMTS)] of a substituted cysteine, reduced GABA action and/or blocked further inhibition by alcohols. Labeling of the 6' position in either α2 or β2 subunits reduced responses to GABA. In addition, methanol and ethanol potentiation increased after MMTS labeling or substitution with tryptophan or methionine, consistent with elimination of an inhibitory site for these alcohols. Specific alcohols, but not the anesthetic etomidate, competed with MMTS labeling at the 6' position. We verified a role for the 6' position in previously tested α2β2 as well as more physiologically relevant α2β2γ2s GABA(A)Rs. Finally, we built a novel molecular model based on the invertebrate glutamate-gated chloride channel receptor, a GABA(A)R homolog, revealing that the 6' position residue faces the channel pore, and modification of this residue alters volume and polarity of the pore-facing cavity in this region. These results indicate that the 6' positions in both α2 and β2 GABA(A)R subunits mediate inhibition by short-chain alcohols, which is consistent with the presence of multiple counteracting sites of action for alcohols on ligand-gated ion channels.

  14. RIPK1 promotes death receptor-independent caspase-8-mediated apoptosis under unresolved ER stress conditions

    PubMed Central

    Estornes, Y; Aguileta, M A; Dubuisson, C; De Keyser, J; Goossens, V; Kersse, K; Samali, A; Vandenabeele, P; Bertrand, M J M

    2014-01-01

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and results in the activation of the unfolded protein response (UPR), which aims at restoring ER homeostasis. However, when the stress is too severe the UPR switches from being a pro-survival response to a pro-death one, and the molecular mechanisms underlying ER stress-mediated death have remained incompletely understood. In this study, we identified receptor interacting protein kinase 1 (RIPK1)—a kinase at the crossroad between life and death downstream of various receptors—as a new regulator of ER stress-induced death. We found that Ripk1-deficient MEFs are protected from apoptosis induced by ER stressors, which is reflected by reduced caspase activation and PARP processing. Interestingly, the pro-apoptotic role of Ripk1 is independent of its kinase activity, is not regulated by its cIAP1/2-mediated ubiquitylation, and does not rely on the direct regulation of JNK or CHOP, two reportedly main players in ER stress-induced death. Instead, we found that ER stress-induced apoptosis in these cells relies on death receptor-independent activation of caspase-8, and identified Ripk1 upstream of caspase-8. However, in contrast to RIPK1-dependent apoptosis downstream of TNFR1, we did not find Ripk1 associated with caspase-8 in a death-inducing complex upon unresolved ER stress. Our data rather suggest that RIPK1 indirectly regulates caspase-8 activation, in part via interaction with the ER stress sensor inositol-requiring protein 1 (IRE1). PMID:25476903

  15. Stress-induced decrease of uterine blood flow in sheep is mediated by alpha 1-adrenergic receptors.

    PubMed

    Dreiling, Michelle; Bischoff, Sabine; Schiffner, Rene; Rupprecht, Sven; Kiehntopf, Michael; Schubert, Harald; Witte, Otto W; Nathanielsz, Peter W; Schwab, Matthias; Rakers, Florian

    2016-09-01

    Prenatal maternal stress can be transferred to the fetus via a catecholamine-dependent decrease of uterine blood flow (UBF). However, it is unclear which group of adrenergic receptors mediates this mechanism of maternal-fetal stress transfer. We hypothesized that in sheep, alpha 1-adrenergic receptors may play a key role in catecholamine mediated UBF decrease, as these receptors are mainly involved in peripheral vasoconstriction and are present in significant number in the uterine vasculature. After chronic instrumentation at 125 ± 1 days of gestation (dGA; term 150 dGA), nine pregnant sheep were exposed at 130 ± 1 dGA to acute isolation stress for one hour without visual, tactile, or auditory contact with their flockmates. UBF, blood pressure (BP), heart rate (HR), stress hormones, and blood gases were determined before and during this isolation challenge. Twenty-four hours later, experiments were repeated during alpha 1-adrenergic receptor blockage induced by a continuous intravenous infusion of urapidil. In both experiments, ewes reacted to isolation with an increase in serum norepinephrine, cortisol, BP, and HR as typical signs of activation of sympatho-adrenal and the hypothalamic-pituitary-adrenal axis. Stress-induced UBF decrease was prevented by alpha 1-adrenergic receptor blockage. We conclude that UBF decrease induced by maternal stress in sheep is mediated by alpha 1-adrenergic receptors. Future studies investigating prevention strategies of impact of prenatal maternal stress on fetal health should consider selective blockage of alpha 1-receptors to interrupt maternal-fetal stress transfer mediated by utero-placental malperfusion.

  16. Influence of sodium substitutes on 5-HT-mediated effects at mouse 5-HT3 receptors

    PubMed Central

    Barann, M; Schmidt, K; Göthert, M; Urban, B W; Bönisch, H

    2004-01-01

    The influence of sodium ion substitutes on the 5-hydroxytryptamine (5-HT)-induced flux of the organic cation [14C]guanidinium through the ion channel of the mouse 5-HT3 receptor and on the competition of 5-HT with the selective 5-HT3 receptor antagonist [3H]GR 65630 was studied, unless stated otherwise, in mouse neuroblastoma N1E-115 cells. Under physiological conditions (135 mM sodium), 5-HT induced a concentration-dependent [14C]guanidinium influx with an EC50 (1.3 μM) similar to that in electrophysiological studies. The stepwise replacement of sodium by increasing concentrations of the organic cation hydroxyethyl trimethylammonium (choline) concentration dependently caused both a rightward shift of the 5-HT concentration–response curve and an increase in the maximum effect of 5-HT. Complete replacement of sodium resulted in a 34-fold lower potency of 5-HT and an almost two times higher maximal response. A low potency of 5-HT in choline buffer was also observed in other 5-HT3 receptor-expressing rodent cell lines (NG 108-15 or NCB 20). Replacement of Na+ by Li+ left the potency and maximal effects of 5-HT almost unchanged. Replacement by tris (hydroxymethyl) methylamine (Tris), tetramethylammonium (TMA) or N-methyl-D-glucamine (NMDG) caused an increase in maximal response to 5-HT similar to that caused by choline. The potency of 5-HT was only slightly reduced by Tris, to a high degree decreased by TMA (comparable to the decrease by choline), but not influenced by NMDG. The potency of 5-HT in inhibiting [3H]GR65630 binding to intact cells was 35-fold lower when sodium was completely replaced by choline, but remained unchanged after replacement by NMDG. The results are compatible with the suggestion that choline competes with 5-HT for the 5-HT3 receptor; the increase in maximal response may be partly due to a choline-mediated delay of the 5-HT-induced desensitization. For studies of 5-HT-evoked [14C]guanidinium flux through 5-HT3 receptor channels, NMDG appears

  17. Autocrine EGF receptor activation mediates endothelial cell migration and vascular morphogenesis induced by VEGF under interstitial flow

    SciTech Connect

    Semino, Carlos E. . E-mail: semino@mit.edu; Kamm, Roger D.; Lauffenburger, Douglas A.

    2006-02-01

    We show here that autocrine ligand activation of epidermal growth factor (EGF) receptor in combination with interstitial flow is critically involved in the morphogenetic response of endothelial cells to VEGF stimulation. Human umbilical vein endothelial cell (HUVEC) monolayers cultured on a collagen gel and exposed to low interstitial flow in the absence of EGF and VEGF remained viable and mitotic but exhibited little evidence of vascular morphogenesis. Addition of VEGF produced a flow-dependent morphogenetic response within 48 to 72 h, characterized by branched capillary-like structures. The response was substantially abolished by inhibitors related to the autocrine EGF receptor pathway including Galardin, AG1478, PD98059, and an EGF receptor-blocking antibody, indicating that regulation of the morphogenetic process operates via autocrine EGF receptor activation. Moreover, we observed that in our system the EGF receptor was always activated independently of the interstitial flow, and, in addition, the EGF receptor inhibitors used above reduced the phosphorylation state of the receptor, correlating with inhibition of capillary morphogenesis. Finally, 5'bromo-2'-deoxyuridine (BrdU) labeling identified dividing cells at the monolayer but not in the extending capillary-like structures. EGF pathway inhibitors Galardin and AG1478 did not reduce BrdU incorporation in the monolayer, indicating that the EGF-receptor-mediated morphogenetic behavior is mainly due to cell migration rather than proliferation. Based on these results, we propose a two-step model for in vitro capillary morphogenesis in response to VEGF stimulation with interstitial fluid flow: monolayer maintenance by mitotic activity independent of EGF receptors and a migratory response mediated by autocrine EGF receptor activation wherein cells establish capillary-like structures.

  18. Allosteric interactions between the oxytocin receptor and the β2-adrenergic receptor in the modulation of ERK1/2 activation are mediated by heterodimerization.

    PubMed

    Wrzal, Paulina K; Devost, Dominic; Pétrin, Darlaine; Goupil, Eugénie; Iorio-Morin, Christian; Laporte, Stéphane A; Zingg, Hans H; Hébert, Terence E

    2012-01-01

    The oxytocin receptor (OTR) and the β(2)-adrenergic receptor (β(2)AR) are key regulators of uterine contraction. These two receptors are targets of tocolytic agents used to inhibit pre-term labor. Our recent study on the nature of OTR- and β(2)AR-mediated ERK1/2 activation in human hTERT-C3 myometrial cells suggested the presence of an OTR/β(2)AR hetero-oligomeric complex (see companion article). The goal of this study was to investigate potential allosteric interactions between OTR and β(2)AR and establish the nature of the interactions between these receptors in myometrial cells. We found that OTR-mediated ERK1/2 activation was attenuated significantly when cells were pretreated with the β(2)AR agonist isoproterenol or two antagonists, propranolol or timolol. In contrast, pretreatment of cells with a third β(2)AR antagonist, atenolol resulted in an increase in OTR-mediated ERK1/2 activation. Similarly, β(2)AR-mediated ERK1/2 activation was strongly attenuated by pretreatment with the OTR antagonists, atosiban and OTA. Physical interactions between OTR and β(2)AR were demonstrated using co-immunoprecipitation, bioluminescence resonance energy transfer (BRET) and protein-fragment complementation (PCA) assays in HEK 293 cells, the latter experiments indicating the interactions between the two receptors were direct. Our analyses suggest physical interactions between OTR and β(2)AR in the context of a new heterodimer pair lie at the heart of the allosteric effects.

  19. Enhanced GABAA receptor-mediated activity following activation of NMDA receptors in Cajal-Retzius cells in the developing mouse neocortex

    PubMed Central

    Chan, Chun-Hung; Yeh, Hermes H

    2003-01-01

    Cajal-Retzius (CR) cells are among the earliest generated population of neurons in the developing neocortex and have been implicated in regulating cortical lamination. In rodents, CR cells are transient, being present only up to 2–3 weeks after birth. Although previous electrophysiological studies have demonstrated the presence of NMDA and GABAA receptors in CR cells, little is known about the functional properties of these receptors. Using whole-cell patch-clamp techniques in neocortical slices, we confirmed the presence of D-aminophosphonovaleric acid (APV)- and ifenprodil-sensitive NMDA receptors, and found that the functional expression of this receptor subtype is strain specific. The NMDA-induced response was consistently accompanied by overriding current transients that were blocked by APV and ifenprodil. In addition, bicuculline readily abolished these transients without affecting the NMDA-induced current response. The generation of these overriding current transients was dependent upon intracellular Ca2+ and was prevented by dialysis with the high-affinity Ca2+-chelator BAPTA. Overall, this study uncovered a synergistic interaction between these receptors, whereby activation of NMDA receptors leads to enhanced GABAA receptor-mediated activity through a Ca2+-dependent mechanism. PMID:12730335

  20. Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris

    PubMed Central

    Malafoglia, Valentina; Traversetti, Lorenzo; Del Grosso, Floriano; Scalici, Massimiliano; Lauro, Filomena; Russo, Valeria; Persichini, Tiziana; Salvemini, Daniela; Mollace, Vincenzo; Fini, Massimo; Raffaeli, William

    2016-01-01

    The ability of mammals to feel noxious stimuli lies in a heterogeneous group of primary somatosensory neurons termed nociceptors, which express specific membrane receptors, such as the Transient Receptor Potential (TRP) family. Here, we show that one of the most important nociceptive-like pathways is conserved in the freshwater coelenterate Hydra vulgaris, the most primitive organism possessing a nervous system. In particular, we found that H. vulgaris expresses TRPM3, a nociceptor calcium channel involved in the detection of noxious heat in mammals. Furthermore, we detected that both heat shock and TRPM3 specific agonist (i.e., pregnenolone sulfate) induce the modulation of the heat shock protein 70 (HSP70) and the nitric oxide synthase (NOS), two genes activated by TRP-mediated heat painful stimuli in mammals. As expected, these effects are inhibited by a TRPM3 antagonist (i.e., mefenamic acid). Interestingly, the TRPM3 agonist and heat shock also induce the expression of nuclear transcription erythroid 2-related factor (Nrf2) and superoxide dismutase (SOD), known markers of oxidative stress; noteworthy gene expression was also inhibited by the TRPM3 antagonist. As a whole, our results demonstrate the presence of conserved molecular oxidative/nociceptive-like pathways at the primordial level of the animal kingdom. PMID:26974325

  1. Transient Receptor Potential Melastatin-3 (TRPM3) Mediates Nociceptive-Like Responses in Hydra vulgaris.

    PubMed

    Malafoglia, Valentina; Traversetti, Lorenzo; Del Grosso, Floriano; Scalici, Massimiliano; Lauro, Filomena; Russo, Valeria; Persichini, Tiziana; Salvemini, Daniela; Mollace, Vincenzo; Fini, Massimo; Raffaeli, William; Muscoli, Carolina; Colasanti, Marco

    2016-01-01

    The ability of mammals to feel noxious stimuli lies in a heterogeneous group of primary somatosensory neurons termed nociceptors, which express specific membrane receptors, such as the Transient Receptor Potential (TRP) family. Here, we show that one of the most important nociceptive-like pathways is conserved in the freshwater coelenterate Hydra vulgaris, the most primitive organism possessing a nervous system. In particular, we found that H. vulgaris expresses TRPM3, a nociceptor calcium channel involved in the detection of noxious heat in mammals. Furthermore, we detected that both heat shock and TRPM3 specific agonist (i.e., pregnenolone sulfate) induce the modulation of the heat shock protein 70 (HSP70) and the nitric oxide synthase (NOS), two genes activated by TRP-mediated heat painful stimuli in mammals. As expected, these effects are inhibited by a TRPM3 antagonist (i.e., mefenamic acid). Interestingly, the TRPM3 agonist and heat shock also induce the expression of nuclear transcription erythroid 2-related factor (Nrf2) and superoxide dismutase (SOD), known markers of oxidative stress; noteworthy gene expression was also inhibited by the TRPM3 antagonist. As a whole, our results demonstrate the presence of conserved molecular oxidative/nociceptive-like pathways at the primordial level of the animal kingdom.

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

    PubMed Central

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

    2016-01-01

    Emotionally relevant experiences form strong and long-lasting memories by critically engaging the stress hormone/neurotransmitter noradrenaline, which mediates and modulates the consolidation of these memories. Noradrenaline acts through adrenergic receptors (ARs), of which β2-adrenergic receptors (βARs) are of particular importance. The differential anatomical and cellular distribution of βAR subtypes in the brain suggests that they play distinct roles in memory processing, although much about their specific contributions and mechanisms of action remains to be understood. Here we show that astrocytic rather than neuronal β2ARs in the hippocampus play a key role in the consolidation of a fear-based contextual memory. These hippocampal β2ARs, but not β1ARs, are coupled to the training-dependent release of lactate from astrocytes, which is necessary for long-term memory formation and for underlying molecular changes. This key metabolic role of astrocytic β2ARs may represent a novel target mechanism for stress-related psychopathologies and neurodegeneration. PMID:27402767

  3. Both neuropeptide Y knockdown and Y1 receptor inhibition modulate CART-mediated appetite control.

    PubMed

    Chu, Shu-Chen; Chen, Pei-Ni; Ho, Ying-Jui; Yu, Ching-Han; Hsieh, Yih-Shou; Kuo, Dong-Yih

    2015-01-01

    Amphetamine (AMPH)-induced appetite suppression has been attributed to its inhibition of neuropeptide Y (NPY)-containing neurons in the hypothalamus. This study examined whether hypothalamic cocaine- and amphetamine-regulated transcript (CART)-containing neurons and NPY Y1 receptor (Y1R) were involved in the action of AMPH. Rats were treated daily with AMPH for four days, and changes in feeding behavior and expression levels of NPY, CART, and POMC were assessed and compared. The results showed that both feeding behavior and NPY expression decreased during AMPH treatment, with the biggest reduction occurring on Day 2. By contrast, the expression of CART and melanocortin 3 receptor (MC3R), a member of the POMC neurotransmission, increased with the maximum response on Day 2, directly opposite to the NPY expression results. The intracerebroventricular infusion of NPY antisense or Y1R inhibitor both modulated AMPH-induced anorexia and the expression levels of MC3R and CART. The results suggest that in the hypothalamus both POMC- and CART-containing neurons participate in regulating NPY-mediated appetite control during AMPH treatment. These results may advance the knowledge of molecular mechanism of anorectic drugs.

  4. Accumbens shell AMPA receptors mediate expression of extinguished reward seeking through interactions with basolateral amygdala.

    PubMed

    Millan, E Zayra; McNally, Gavan P

    2011-07-01

    Extinction is the reduction in drug seeking when the contingency between drug seeking behavior and the delivery of drug reward is broken. Here, we investigated a role for the nucleus accumbens shell (AcbSh). Rats were trained to respond for 4% (v/v) alcoholic beer in one context (Context A) followed by extinction in a second context (Context B). Rats were subsequently tested in the training context, A (ABA), or the extinction context, B (ABB). Pre-test injections of the glutamate AMPA receptor antagonist, NBQX (1 µg) into AcbSh had no effect on renewal of alcoholic beer seeking when rats were returned to the training context (ABA). However, NBQX increased responding when rats were tested in the extinction context (ABB). In a second experiment, rats received training, extinction, and test in the same context. Pre-test injections of NBQX (0, 0.3, and 1 µg) into the AcbSh dose-dependently attenuated expression of extinction. We also found that NBQX in the AcbSh had no effect on initial acquisition of extinction or the motivation to respond for reward as measured by break point on a progressive ratio schedule. Finally, we show that pharmacological disconnection of a basolateral amygdala (BLA) → AcbSh pathway via NBQX in AcbSh combined with reversible inactivation of the contralateral BLA attenuates expression of extinction. Together, these results suggest that AcbSh AMPA receptors mediate expression of extinguished reward seeking through glutamatergic inputs from the BLA.

  5. Dietary Restriction Induced Longevity Is Mediated by Nuclear Receptor NHR-62 in Caenorhabditis elegans

    PubMed Central

    Heestand, Bree N.; Shen, Yidong; Liu, Wei; Magner, Daniel B.; Storm, Nadia; Meharg, Caroline; Habermann, Bianca; Antebi, Adam

    2013-01-01

    Dietary restriction (DR) extends lifespan in a wide variety of species, yet the underlying mechanisms are not well understood. Here we show that the Caenorhabditis elegans HNF4α-related nuclear hormone receptor NHR-62 is required for metabolic and physiologic responses associated with DR-induced longevity. nhr-62 mediates the longevity of eat-2 mutants, a genetic mimetic of dietary restriction, and blunts the longevity response of DR induced by bacterial food dilution at low nutrient levels. Metabolic changes associated with DR, including decreased Oil Red O staining, decreased triglyceride levels, and increased autophagy are partly reversed by mutation of nhr-62. Additionally, the DR fatty acid profile is altered in nhr-62 mutants. Expression profiles reveal that several hundred genes induced by DR depend on the activity of NHR-62, including a putative lipase required for the DR response. This study provides critical evidence of nuclear hormone receptor regulation of the DR longevity response, suggesting hormonal and metabolic control of life span. PMID:23935515

  6. The thrombospondin-1 receptor CD36 is an important mediator of ovarian angiogenesis and folliculogenesis

    PubMed Central

    2014-01-01

    Background Ovarian angiogenesis is a complex process that is regulated by a balance between pro- and anti-angiogenic factors. Physiological processes within the ovary, such as folliculogenesis, ovulation, and luteal formation are dependent upon adequate vascularization and anything that disrupts normal angiogenic processes may result in ovarian dysfunction, and possibly infertility. The objective of this study was to evaluate the role of the thrombospondin-1 (TSP-1) receptor CD36 in mediating ovarian angiogenesis and regulating ovarian function. Methods The role of CD36 was evaluated in granulosa cells in vitro and ovarian morphology and protein expression were determined in wild type and CD36 null mice. Results In vitro, CD36 inhibition increased granulosa cell proliferation and decreased apoptosis. Granulosa cells in which CD36 was knocked down also exhibited an increase in expression of survival and angiogenic proteins. Ovaries from CD36 null mice were hypervascularized, with increased expression of pro-angiogenic vascular endothelial growth factor (VEGF) and its receptor VEGFR-2. Ovaries from CD36 null mice contained an increase in the numbers of pre-ovulatory follicles and decreased numbers of corpora lutea. CD36 null mice also had fewer number of offspring compared to wild type controls. Conclusions The results from this study demonstrate that CD36 is integral to the regulation of ovarian angiogenesis by TSP-1 and the expression of these family members may be useful in the control of ovarian vascular disorders. PMID:24628875

  7. Receptor-mediated uptake and transport of macromolecules in the human placenta.

    PubMed

    Schneider, Henning; Miller, Richard K

    2010-01-01

    The human placenta is required to be the anchor, the conduit and the controller during pregnancy. The survival of the baby and its associated placenta is dependent upon the placenta shielding the embryo/fetus from harm, e.g., autoimmune disease - thrombophilia, antiphospholipid syndrome or infections, while simultaneously providing for the passage of critical nutrients (e.g., amino acids, vitamins) and beneficial immunoglobulins. In a number of instances, the movements of macromolecules into and through the placenta can result in the passage of the intact molecules into the fetal circulation or in the case of proteins - catabolism to amino acids which are utilized by the placenta and the fetus for continued growth and development. The transfer of two such macromolecules, immunoglobulin G (IgG) and vitamin B12 (cyanocobalamin or B12), are examined as to the unique receptor-mediated transfer capability of the human placenta, its transfer specificity as related to specific receptors and the role of endogeneous placental proteins (trancobalamins) in facilitating the recognition and transport of specifically B12. Brief comparisons will be made to other animal species and the differences in specific organ transfer capabilities.

  8. Calcium Sensing Receptor as a Novel Mediator of Adipose Tissue Dysfunction: Mechanisms and Potential Clinical Implications

    PubMed Central

    Bravo-Sagua, Roberto; Mattar, Pamela; Díaz, Ximena; Lavandero, Sergio; Cifuentes, Mariana

    2016-01-01

    Obesity is currently a serious worldwide public health problem, reaching pandemic levels. For decades, dietary and behavioral approaches have failed to prevent this disease from expanding, and health authorities are challenged by the elevated prevalence of co-morbid conditions. Understanding how obesity-associated diseases develop from a basic science approach is recognized as an urgent task to face this growing problem. White adipose tissue (WAT) is an active endocrine organ, with a crucial influence on whole-body homeostasis. WAT dysfunction plays a key role linking obesity with its associated diseases such as type 2 diabetes mellitus, cardiovascular disease, and some cancers. Among the regulators of WAT physiology, the calcium-sensing receptor (CaSR) has arisen as a potential mediator of WAT dysfunction. Expression of the receptor has been described in human preadipocytes, adipocytes, and the human adipose cell lines LS14 and SW872. The evidence suggests that CaSR activation in the visceral (i.e., unhealthy) WAT is associated with an increased proliferation of adipose progenitor cells and elevated adipocyte differentiation. In addition, exposure of adipose cells to CaSR activators in vitro elevates proinflammatory cytokine expression and secretion. An increased proinflammatory environment in WAT plays a key role in the development of WAT dysfunction that leads to peripheral organ fat deposition and insulin resistance, among other consequences. We propose that CaSR may be one relevant therapeutic target in the struggle to confront the health consequences of the current worldwide obesity pandemic. PMID:27660614

  9. Human Milk Components Modulate Toll-Like Receptor-Mediated Inflammation.

    PubMed

    He, YingYing; Lawlor, Nathan T; Newburg, David S

    2016-01-01

    Toll-like receptor (TLR) signaling is central to innate immunity. Aberrant expression of TLRs is found in neonatal inflammatory diseases. Several bioactive components of human milk modulate TLR expression and signaling pathways, including soluble toll-like receptors (sTLRs), soluble cluster of differentiation (sCD) 14, glycoproteins, small peptides, and oligosaccharides. Some milk components, such as sialyl (α2,3) lactose and lacto-N-fucopentaose III, are reported to increase TLR signaling; under some circumstances this might contribute toward immunologic balance. Human milk on the whole is strongly anti-inflammatory, and contains abundant components that depress TLR signaling pathways: sTLR2 and sCD14 inhibit TLR2 signaling; sCD14, lactadherin, lactoferrin, and 2'-fucosyllactose attenuate TLR4 signaling; 3'-galactosyllactose inhibits TLR3 signaling, and β-defensin 2 inhibits TLR7 signaling. Feeding human milk to neonates decreases their risk of sepsis and necrotizing enterocolitis. Thus, the TLR regulatory components found in human milk hold promise as benign oral prophylactic and therapeutic treatments for the many gastrointestinal inflammatory disorders mediated by abnormal TLR signaling.

  10. Local heating of human skin causes hyperemia without mediation by muscarinic cholinergic receptors or prostanoids.

    PubMed

    Golay, Sandrine; Haeberli, Christian; Delachaux, Anne; Liaudet, Lucas; Kucera, Paul; Waeber, Bernard; Feihl, François

    2004-11-01

    Local changes in surface temperature have a powerful influence on the perfusion of human skin. Heating increases local skin blood flow, but the mechanisms and mediators of this response (thermal hyperemia response) are incompletely elucidated. In the present study, we examined the possible dependence of the thermal hyperemia response on stimulation of muscarinic cholinergic receptors and on production of vasodilator prostanoids. In 13 male healthy subjects aged 20-30 yr, a temperature-controlled chamber was positioned on the volar face of one forearm and used to raise surface temperature from 34 to 41 degrees C. The time course of the resulting thermal hyperemia response was recorded with a laser-Doppler imager. In one experiment, each of eight subjects received an intravenous bolus of the antimuscarinic agent glycopyrrolate (4 microg/kg) on one visit and saline on the other. The thermal hyperemia response was determined within the hour after the injections. Glycopyrrolate effectively inhibited the skin vasodilation induced by iontophoresis of acetylcholine but did not influence the thermal hyperemia response. In a second experiment, conducted in five other subjects, 1 g of the cyclooxygenase inhibitor aspirin administered orally totally abolished the vasodilation induced in the skin by anodal current but also failed to modify the thermal hyperemia response. The present study excludes the stimulation of muscarinic receptors and the production of vasodilator prostaglandins as essential and nonredundant mechanisms for the vasodilation induced by local heating in human forearm skin.

  11. Pregnane X-receptor promotes stem cell-mediated colon cancer relapse.

    PubMed

    Planque, Chris; Rajabi, Fatemeh; Grillet, Fanny; Finetti, Pascal; Bertucci, François; Gironella, Meritxell; Lozano, Juan José; Beucher, Bertrand; Giraud, Julie; Garambois, Véronique; Vincent, Charles; Brown, Daniel; Caillo, Ludovic; Kantar, Jovana; Pelegrin, André; Prudhomme, Michel; Ripoche, Jérémie; Bourgaux, Jean François; Ginestier, Christophe; Castells, Antoni; Hollande, Frédéric; Pannequin, Julie; Pascussi, Jean Marc

    2016-08-30

    Colorectal cancer lethality usually results from post-treatment relapse in the majority of stage II-IV patients, due to the enhanced resistance of Cancer Stem Cells (CSCs). Here, we show that the nuclear receptor Pregnane X Receptor (PXR, NR1I2), behaves as a key driver of CSC-mediated tumor recurrence. First, PXR is specifically expressed in CSCs, where it drives the expression of genes involved in self-renewal and chemoresistance. Clinically, high levels of PXR correlate with poor recurrence-free survival in a cohort of >200 stage II/III colorectal cancer patients treated with chemotherapy, for whom finding biomarkers of treatment outcome is an urgent clinical need. shRNA silencing of PXR increased the chemo-sensitivity of human colon CSCs, reduced their self-renewal and tumor-initiating potential, and drastically delayed tumor recurrence in mice following chemotherapy. This study uncovers PXR as a key factor for CSC self-renewal and chemoresistance and targeting PXR thus represents a promising strategy to minimize colorectal cancer relapse by selectively sensitizing CSCs to chemotherapy.

  12. Pregnane X-receptor promotes stem cell-mediated colon cancer relapse

    PubMed Central

    Grillet, Fanny; Finetti, Pascal; Bertucci, François; Gironella,