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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. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes.

    PubMed Central

    Cawley, D B; Simpson, D L; Herschman, H R

    1981-01-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialogalactoorsomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated. Images PMID:6167984

  3. Asialoglycoprotein receptor mediates the toxic effects of an asialofetuin-diphtheria toxin fragment A conjugate on cultured rat hepatocytes

    SciTech Connect

    Cawley, D.B.; Simpson, D.L.; Herschman, H.R.

    1981-06-01

    We have constructed a toxic hybrid protein that is recognized by asialoglycoprotein (ASGP) receptors of cultured rat hepatocytes. The conjugate consists of fragment A of diphtheria toxin (DTA) linked by a disulfide bond to asialofetuin (ASF). This conjugate is highly toxic, inhibiting protein synthesis in primary rat hepatocytes at concentrations as low as 10 pM. The ASF-DTA conjugate was 600 and 1800 times as toxic as diphtheria toxin and DTA, respectively, on primary rat hepatocytes. The ASGP receptor recognizes galactose-terminated proteins. We tested a series of glycoproteins for their ability to block the action of the ASF-DTA conjugate. Fetuin and orosomucoid, two glycoproteins with terminal sialic acid on their oligosaccharide chains, did not block the action of the conjugate. Their galactose-terminated asialo derivatives, ASF and asialoorosomucoid, as expected, did block the action of the conjugate. The N-acetylglucosaminyl-terminated derivative (asialoagalactoorosomucoid) had no appreciable effect on the activity of the conjugate. We tested the ASF-DTA conjugate on six cell types; except for primary rat hepatocytes, none of them were affected by a high concentration (10 nM) of ASF-DTA conjugate. A fetuin-DTA conjugate was less toxic by a factor of 300 than the ASF-DTA conjugate and exerted its effects primarily through non-receptor-mediated mechanisms. The highly toxic ASF-DTA conjugate is cell-type specific, and its action is mediated by a well-characterized receptor, whose mechanism of receptor-ligand internalization has been extensively investigated.

  4. 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

  5. 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.

  6. Asialoglycoprotein receptor targeted delivery of doxorubicin nanoparticles for hepatocellular carcinoma.

    PubMed

    Pranatharthiharan, Sandhya; Patel, Mitesh D; Malshe, Vinod C; Pujari, Vaishali; Gorakshakar, Ajit; Madkaikar, Manisha; Ghosh, Kanjaksha; Devarajan, Padma V

    2017-11-01

    We report asialoglycoprotein receptor (ASGPR)-targeted doxorubicin hydrochloride (Dox) nanoparticles (NPs) for hepatocellular carcinoma (HCC). Polyethylene sebacate (PES)-Gantrez® AN 119 Dox NPs of average size 220 nm with PDI < 0.62 and ∼20% Dox loading were prepared by modified nanoprecipitation. ASGPR ligands, pullulan (Pul), arabinogalactan (AGn), and the combination (Pul-AGn), were anchored by adsorption. Ligand anchoring enabled high liver uptake with a remarkable hepatocyte:nonparenchymal cell ratio of 85:15. Furthermore, Pul-AGn NPs exhibited an additive effect implying incredibly high hepatocyte accumulation. Galactose-mediated competitive inhibition confirmed ASGPR-mediated uptake of ligand-anchored NPs in HepG2 cell lines. Subacute toxicity in rats confirmed the safety of the NP groups. However, histopathological evaluation suggested mild renal toxicity of AGn. Pul NPs revealed sustained reduction in tumor volume in PLC/PRF/5 liver tumor-bearing Nod/Scid mice up to 46 days. Extensive tumor necrosis, reduced collagen content, reduction in the HCC biomarker serum α-fetoprotein (p < 0.05), a mitotic index of 1.135 (day 46), and tumor treated/tumor control (T/C) values of <0.42 signified superior efficacy of Pul NPs. Furthermore, weight gain in the NP groups, and no histopathological alterations indicated that they were well tolerated by the mice. The high efficacy coupled with greater safety portrayed Pul Dox NPs as a promising nanocarrier for improved therapy of HCC.

  7. Asialoglycoprotein receptor-targeted radiopharmaceuticals for measurement of liver function.

    PubMed

    Yang, W; Zhang, X; Liu, Y

    2014-01-01

    The number of Asialoglycoprotein (ASGP) receptors on the hepatocytes of patients with liver disease is reduced and is thus considered a good indicator for the evaluation of liver function. ASGP receptor-targeted radiopharmaceuticals permit a non-invasive way to evaluate total and regional hepatic function and hepatic functional reserve visually and quantitatively. Over the past three decades, a variety of ASGP receptor-targeted probes have been developed with different molecular backbones (albumin, polymer, small-molecular-weight ligand), different glycol-residues (galactose, lactose, N-acetyl-galactosamine) and different chelating systems suitable for radiolabeling with SPECT isotopes ((99m)Tc, (111)In, (67)Ga, (131/125)I, (153)Sm) and PET isotopes ((68)Ga, (18)F). In this review, we present an overview of ASGP receptor-targeted radiopharmaceuticals, discuss their chemistry, biodistribution, catabolism and challenge as well as application for measurement of liver function.

  8. 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.

  9. 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

  10. Cholesterol anchored arabinogalactan for asialoglycoprotein receptor targeting: synthesis, characterization, and proof of concept of hepatospecific delivery.

    PubMed

    Pathak, Pankaj Omprakash; Nagarsenker, Mangal Shailesh; Barhate, Chandrashekhar Rishikant; Padhye, Sameer Govind; Dhawan, Vivek Vijay; Bhattacharyya, Dibyendu; Viswanathan, C L; Steiniger, Frank; Fahr, Alfred

    2015-05-18

    Asialoglycoprotein receptors (ASGPR) are hepatocyte bound receptors, which exhibit receptor mediated endocytosis (RME) for galactose specific moieties. Arabinogalactan (AG), a liver specific high galactose containing branched polysaccharide was hydrophobized using cholesterol (CHOL) as a lipid anchor via a two step reaction process to yield the novel polysaccharide lipid conjugated ligand (CHOL-AL-AG). CHOL-AL-AG was characterized by Fourier transform infra red (FTIR) spectroscopy, (1)H and (13)C nuclear magnetic spectroscopy (NMR), size exclusion chromatography (SEC) and differential scanning calorimetry (DSC). Conventional liposomes (CL) and surface modified liposomes (SML) containing CHOL-AL-AG were prepared using reverse phase evaporation technique. Effect of CHOL-AL-AG concentration on particle size and zeta potential of SML was evaluated. Surface morphology of CL and SML was studied using cryo-transmission electron microscopy (cryo-TEM). In vitro binding affinity of SML and CL was evaluated using Ricinus communis agglutinin (RCA) assay. Cellular uptake of SML and CL was determined on ASGPR expressing HepG2 cell lines by confocal laser scanning microscopy technique (CLSM). FTIR spectra revealed bands at 1736 cm(-1) and 1664 cm(-1) corresponding to ester and carbamate functional groups, respectively. Signals at δ 0.5-2.5 corresponding to the cholestene ring and δ 3-5.5 corresponding to the carbohydrate backbone were observed in (1)H NMR spectrum of the product. CHOL-AL-AG possessed a mean average molecular weight of 27 KDa as determined by size exclusion chromatography. An endothermic peak at 207 °C was observed in the DSC thermogram of CHOL-AL-AG, which was not observed in thermograms of reactants and intermediate product. Synthesized CHOL-AL-AG was successfully incorporated in liposomes to yield SML. Both CL and SML possessed a mean particle size of ∼ 200 nm with polydispersity index of ∼ 0.25. The zeta potential of CLs was observed to be -17 m

  11. (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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Efficient Liver Targeting by Polyvalent Display of a Compact Ligand for the Asialoglycoprotein Receptor.

    PubMed

    Sanhueza, Carlos A; Baksh, Michael M; Thuma, Benjamin; Roy, Marc D; Dutta, Sanjay; Préville, Cathy; Chrunyk, Boris A; Beaumont, Kevin; Dullea, Robert; Ammirati, Mark; Liu, Shenping; Gebhard, David; Finley, James E; Salatto, Christopher T; King-Ahmad, Amanda; Stock, Ingrid; Atkinson, Karen; Reidich, Benjamin; Lin, Wen; Kumar, Rajesh; Tu, Meihua; Menhaji-Klotz, Elnaz; Price, David A; Liras, Spiros; Finn, M G; Mascitti, Vincent

    2017-03-08

    A compact and stable bicyclic bridged ketal was developed as a ligand for the asialoglycoprotein receptor (ASGPR). This compound showed excellent ligand efficiency, and the molecular details of binding were revealed by the first X-ray crystal structures of ligand-bound ASGPR. This analogue was used to make potent di- and trivalent binders of ASGPR. Extensive characterization of the function of these compounds showed rapid ASGPR-dependent cellular uptake in vitro and high levels of liver/plasma selectivity in vivo. Assessment of the biodistribution in rodents of a prototypical Alexa647-labeled trivalent conjugate showed selective hepatocyte targeting with no detectable distribution in nonparenchymal cells. This molecule also exhibited increased ASGPR-directed hepatocellular uptake and prolonged retention compared to a similar GalNAc derived trimer conjugate. Selective release in the liver of a passively permeable small-molecule cargo was achieved by retro-Diels-Alder cleavage of an oxanorbornadiene linkage, presumably upon encountering intracellular thiol. Therefore, the multicomponent construct described here represents a highly efficient delivery vehicle to hepatocytes.

  17. Expression of a functional asialoglycoprotein receptor in human renal proximal tubular epithelial cells.

    PubMed

    Seow, Ying-ying T; Tan, Michelle G K; Woo, Keng Thye

    2002-07-01

    The asialoglycoprotein receptor (ASGPR) is a C lectin which binds and endocytoses serum glycoproteins. In humans, the ASGPR is shown mainly to occur in hepatocytes, but does occur extrahepatically in thyroid, in small and large intestines, and in the testis. In the kidney, there has been evidence both for and against its existence in mesangial cells. Standard light microscopy examination of renal tissue stained with an antibody against the ASGPR was performed. The mRNA expression for the ASGPR H1 and H2 subunits in primary human renal proximal tubular epithelial cells (RPTEC), in the human proximal tubular epithelial cell line HK2, and in human renal cortex was investigated using reverse-transcribed nested polymerase chain reaction. ASGPR protein expression as well as ligand binding and uptake were also examined using confocal microscopy and flow cytometry (fluorescence-activated cell sorting). Light microscopy of paraffin renal biopsy sections stained with a polyclonal antibody against the ASGPR showed proximal tubular epithelial cell staining of the cytoplasm and particularly in the basolateral region. Renal cortex and RPTEC specifically have mRNA for both H1 and H2 subunits of the ASGPR, but HK2 only expresses mRNA for H1. Using a monoclonal antibody, the presence of the ASGPR in RPTEC was shown by fluorescence-activated cell sorting and immunofluorescent staining. Specific binding and uptake of fluorescein isothiocyanate labelled asialofetuin which is a specific ASGPR ligand was also demonstrated in RPTEC. Primary renal proximal tubular epithelial cells have a functional ASGPR, consisting of the H1 and H2 subunits, that is capable of specific ligand binding and uptake. Copyright 2002 S. Karger AG, Basel

  18. Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor.

    PubMed

    Schmidt, Karsten; Prakash, Thazha P; Donner, Aaron J; Kinberger, Garth A; Gaus, Hans J; Low, Audrey; Østergaard, Michael E; Bell, Melanie; Swayze, Eric E; Seth, Punit P

    2017-03-17

    Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Characterizing the effect of GalNAc and phosphorothioate backbone on binding of antisense oligonucleotides to the asialoglycoprotein receptor

    PubMed Central

    Schmidt, Karsten; Prakash, Thazha P.; Donner, Aaron J.; Kinberger, Garth A.; Gaus, Hans J.; Low, Audrey; Østergaard, Michael E.; Bell, Melanie; Swayze, Eric E.

    2017-01-01

    Abstract Targeted delivery of antisense oligonucleotides (ASO) to hepatocytes via the asialoglycoprotein receptor (ASGR) has improved the potency of ASO drugs ∼30-fold in the clinic (1). In order to fully characterize the effect of GalNAc valency, oligonucleotide length, flexibility and chemical composition on ASGR binding, we tested and validated a fluorescence polarization competition binding assay. The ASGR binding, and in vitro and in vivo activities of 1, 2 and 3 GalNAc conjugated single stranded and duplexed ASOs were studied. Two and three GalNAc conjugated single stranded ASOs bind the ASGR with the strongest affinity and display optimal in vitro and in vivo activities. 1 GalNAc conjugated ASOs showed 10-fold reduced ASGR binding affinity relative to three GalNAc ASOs but only 2-fold reduced activity in mice. An unexpected observation was that the ASGR also appears to play a role in the uptake of unconjugated phosphorothioate modified ASOs in the liver as evidenced by the loss of activity of GalNAc conjugated and unconjugated ASOs in ASGR knockout mice. Our results provide insights into how backbone charge and chemical composition assist in the binding and internalization of highly polar anionic single stranded oligonucleotides into cells and tissues. PMID:28158620

  20. 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.

  1. 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.

  2. Asialoglycoprotein receptor targeted gene delivery using galactosylated polyethylenimine-graft-poly(ethylene glycol): in vitro and in vivo studies.

    PubMed

    Kim, Eun-Mi; Jeong, Hwan-Jeong; Park, In-Kyu; Cho, Chong-Su; Moon, Hyung-Bae; Yu, Dae-Yeul; Bom, Hee-Seung; Sohn, Myung-Hee; Oh, In-Joon

    2005-11-28

    The asialoglycoprotein receptor (ASGP-R) on the hepatocyte membrane is a specific targeting marker for gene and drug delivery. Polyethylenimine (PEI) is a polycationic nonviral vector that is used for gene transfer. We have synthesized galactosylated polyethylenimine-graft-poly(ethylene glycol) (GPP) for performing gene delivery to the hepatocytes. The present study reports on the in vitro and in vivo data that was achieved in hepatoma bearing transgenic mice. The cytotoxicity was decreased with the increasing PEG content. The particle size of the complex was increased with the increasing PEG at an N/P ratio of 3.0, while the zeta potentials were decreased. The (99m)Tc labeled complexes were transfected into HepG2 and HeLa cells, while the GFP reporter genes were mainly expressed in the HepG2 cells. The in vivo data was achieved in ALB/c-Ha-ras transgenic mice. (99m)Tc labeled GPP(50)/DNA was injected into the mice via the tail vein, and the gamma images were acquired at 5, 15 and 30 min. The (99m)Tc labeled complexes were mainly localized in the heart and liver, and they were excreted through the kidneys. The GFP gene was mainly expressed in the proliferating cells at the tumor periphery. This result was confirmed by PCNA staining. The GPP(50)/DNA complexes were bound to ASGP-R of the proliferating hepatocytes in vitro and in vivo. The present results demonstrate the feasibility of nonviral gene transfer using galactosylated PEI-PEG in vivo.

  3. 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.

  4. 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.

  5. Different zonal distribution of the asialoglycoprotein receptor, the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein and the lipoprotein-remnant receptor of rat liver parenchymal cells.

    PubMed

    Voorschuur, A H; Kuiper, J; Neelissen, J A; Boers, W; Van Berkel, T J

    1994-11-01

    Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from

  6. 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.

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

    PubMed Central

    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. PMID:23892317

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. Tachykinin receptors mediating airway marcomolecular secretion

    SciTech Connect

    Gentry, S.E. )

    1991-01-01

    Three tachykinin receptor types, termed NK1, NK2, and NK3, can be distinguished by the relative potency of various peptides in eliciting tissue responses. Airway macromolecular secretion is stimulated by the tachykinin substance P (SP). The purposes of this study were to determine the tachykinin receptor subtype responsible for this stimulation, and to examine the possible involvement of other neurotransmitters in mediating this effect. Ferret tracheal explants maintained in organ culture were labeled with {sup 3}H-glucosamine, a precursor of high molecular weight glycoconjugates (HMWG) which are released by airway secretory cells. Secretion of labeled HMWG then was determined in the absence and presence of the tachykinins SP, neurokinin A (NKA), neurokinin B (NKB), physalaemin (PHY), and eledoisin (ELE). To evaluate the possible contribution of other mediators, tachykinin stimulation was examined in the presence of several receptor blockers.

  16. Receptor-mediated signaling at plasmodesmata.

    PubMed

    Faulkner, Christine

    2013-01-01

    Plasmodesmata (PD) generate continuity between plant cells via the cytoplasm, endoplasmic reticulum (ER) and plasma membrane (PM), allowing movement of different classes of molecules between cells. Proteomic data indicates that the PD PM hosts many receptors and receptor kinases, as well as lipid raft and tetraspanin enriched microdomain associated proteins, suggesting the hypothesis that the PD PM is specialized with respect to both composition and function. PD-located receptor proteins and receptor kinases are responsible for perception of microbe associated molecular patterns at PD and initiate signaling that mediates changes to PD flux. In addition, developmentally relevant receptor kinases have different interactions dependent upon whether located at the PD PM or the cellular PM. The implications of these findings are that receptor-mediated signaling in PD membranes differs from that in the cellular PM and, in light the identification of PD-located proteins associated with membrane microdomains and the role of membrane microdomains in analogous signaling processes in animals, suggests that the PD PM contains specialized signaling platforms.

  17. 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

  18. 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

  19. Mechanics of receptor-mediated endocytosis

    NASA Astrophysics Data System (ADS)

    Gao, Huajian; Shi, Wendong; Freund, Lambert B.

    2005-07-01

    Most viruses and bioparticles endocytosed by cells have characteristic sizes in the range of tens to hundreds of nanometers. The process of viruses entering and leaving animal cells is mediated by the binding interaction between ligand molecules on the viral capid and their receptor molecules on the cell membrane. How does the size of a bioparticle affect receptor-mediated endocytosis? Here, we study how a cell membrane containing diffusive mobile receptors wraps around a ligand-coated cylindrical or spherical particle. It is shown that particles in the size range of tens to hundreds of nanometers can enter or exit cells via wrapping even in the absence of clathrin or caveolin coats, and an optimal particles size exists for the smallest wrapping time. This model can also be extended to include the effect of clathrin coat. The results seem to show broad agreement with experimental observations. Author contributions: H.G. and L.B.F. designed research; H.G., W.S., and L.B.F. performed research; and H.G., W.S., and L.B.F. wrote the paper.Abbreviations: CNT, carbon nanotube; SWNT, single-walled nanotube.

  20. 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.

  1. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish.

  2. SPHINGOSINE-1-PHOSPHATE RECEPTORS MEDIATE NEUROMODULATORY FUNCTIONS IN THE CNS

    PubMed Central

    Sim-Selley, Laura J.; Goforth, Paulette B.; Mba, Mba U.; Macdonald, Timothy L.; Lynch, Kevin R.; Milstien, Sheldon; Spiegel, Sarah; Satin, Leslie S.; Welch, Sandra P.; Selley, Dana E.

    2009-01-01

    Sphingosine-1-phosphate is a ubiquitous, lipophilic cellular mediator that acts in part by activation of G-protein-coupled receptors. Modulation of S1P signaling is an emerging pharmacotherapeutic target for immunomodulatory drugs. Although multiple S1P receptor types exist in the CNS, little is known about their function. Here we report that S1P stimulated G- protein activity in the CNS, and results from [35S]GTPγS autoradiography using the S1P1-selective agonist SEW2871 and the S1P1/3-selective antagonist VPC44116 show that in several regions a majority of this activity is mediated by S1P1 receptors. S1P receptor activation inhibited glutamatergic neurotransmission as determined by electrophysiological recordings in cortical neurons in vitro, and this effect was mimicked by SEW2871 and inhibited by VPC44116. Moreover, central administration of S1P produced in vivo effects resembling the actions of cannabinoids, including thermal antinociception, hypothermia, catalepsy and hypolocomotion, but these actions were independent of CB1 receptors. At least one of the central effects of S1P, thermal antinociception, is also at least partly S1P1 receptor mediated because it was produced by SEW2871 and attenuated by VPC44116. These results indicate that CNS S1P receptors are part of a physiologically relevant and widespread neuromodulatory system, and that the S1P1 receptor contributes to S1P-mediated antinociception. PMID:19493165

  3. 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.

  4. Receptor-mediated Endocytosis in the Caenorhabditis elegans Oocyte

    PubMed Central

    Grant, Barth; Hirsh, David

    1999-01-01

    The Caenorhabditis elegans oocyte is a highly amenable system for forward and reverse genetic analysis of receptor-mediated endocytosis. We describe the use of transgenic strains expressing a vitellogenin::green fluorescent protein (YP170::GFP) fusion to monitor yolk endocytosis by the C. elegans oocyte in vivo. This YP170::GFP reporter was used to assay the functions of C. elegans predicted proteins homologous to vertebrate endocytosis factors using RNA-mediated interference. We show that the basic components and pathways of endocytic trafficking are conserved between C. elegans and vertebrates, and that this system can be used to test the endocytic functions of any new gene. We also used the YP170::GFP assay to identify rme (receptor-mediated endocytosis) mutants. We describe a new member of the low-density lipoprotein receptor superfamily, RME-2, identified in our screens for endocytosis defective mutants. We show that RME-2 is the C. elegans yolk receptor. PMID:10588660

  5. 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.

  6. Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP.

    PubMed

    Wu, Dick; Bacaj, Taulant; Morishita, Wade; Goswami, Debanjan; Arendt, Kristin L; Xu, Wei; Chen, Lu; Malenka, Robert C; Südhof, Thomas C

    2017-04-20

    Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca(2+) influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca(2+) induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca(2+)-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca(2+)-dependent presynaptic vesicle exocytosis, also blocked Ca(2+)-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca(2+)-sensors for Ca(2+)-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.

  7. Receptor Complex Mediated Regulation of Symplastic Traffic.

    PubMed

    Stahl, Yvonne; Faulkner, Christine

    2016-05-01

    Plant receptor kinases (RKs) and receptor proteins (RPs) are involved in a plethora of cellular processes, including developmental decisions and immune responses. There is increasing evidence that plasmodesmata (PD)-localized RKs and RPs act as nexuses that perceive extracellular signals and convey them into intra- and intercellular responses by regulating the exchange of molecules through PD. How RK/RP complexes regulate the specific and nonspecific traffic of molecules through PD, and how these receptors are specifically targeted to PD, have been elusive but underpin comprehensive understanding of the function and regulation of the symplast. In this review we gather the current knowledge of RK/RP complex function at PD and how they might regulate intercellular traffic. Copyright © 2015 Elsevier Ltd. All rights reserved.

  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. Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons.

    PubMed

    Paternain, A V; Morales, M; Lerma, J

    1995-01-01

    Although both protein and mRNAs for kainate receptor subunits are abundant in several brain regions, the responsiveness of AMPA receptors to kainate has made it difficult to demonstrate the presence of functional kainate-type receptors in native cells. Recently, however, we have shown that many hippocampal neurons in culture express glutamate receptors of the kainate type. The large nondesensitizing response that kainate induces at AMPA receptors precludes detection and analysis of smaller, rapidly desensitizing currents induced by kainate at kainate receptors. Consequently, the functional significance of these strongly desensitizing glutamate receptors remains enigmatic. We report here that the family of new noncompetitive antagonists of AMPA receptors (GYKI 52466 and 53655) minimally affects kainate-induced responses at kainate receptors while completely blocking AMPA receptor-mediated currents, making it possible to separate the responses mediated by each receptor. These compounds will allow determination of the role played by kainate receptors in synaptic transmission and plasticity in the mammalian brain, as well as evaluation of their involvement in neurotoxicity.

  10. 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.

  11. 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.

  12. Modulation of Toll-interleukin 1 receptor mediated signaling.

    PubMed

    Li, Xiaoxia; Qin, Jinzhong

    2005-04-01

    Toll-like receptors (TLRs) belong to the Toll-interleukin 1 receptor superfamily, which is defined by a common intracellular Toll-IL-1 receptor (TIR) domain. A group of TIR domain containing adaptors (MyD88, TIRAP, TRIF and TRAM), are differentially recruited to the Toll-IL-1 receptors, contributing to the specificity of signaling. The IL-1 mediated signaling pathway serves as a "prototype" for other family members. Genetic and biochemical studies reveal that IL-1R uses adaptor molecule MyD88 to mediate a very complex pathway, involving a cascade of kinases organized by multiple adapter molecules into signaling complexes, leading to activation of the transcription factor NFkappaB. Several Toll-like receptors utilize variations of the "prototype" pathway by employing different adaptor molecules. Double-stranded RNA triggered, TLR3-mediated signaling is independent of MyD88, IRAK4, and IRAK. The adapter molecule TRIF is utilized by TLR3 to mediate the activation of NFkappaB and IRF3. LPS-induced, TLR4-mediated signaling employs multiple TIR-domain containing adaptors, MyD88/TIRAP to mediate NFkappaB activation, TRIF/TRAM for IRF3 activation. Recent studies have also begun to unravel how these pathways are negatively regulated. SIGIRR (also known as TIR8), a member of TIR superfamily that does not activate the transcription factors NFkappaB and IRF3, instead negatively modulates responses. Cells from SIGIRR-null mice show enhanced activation in response to either IL-1 or certain Toll ligands. In addition to SIGIRR, several other negative regulators have been shown to inhibit the TIR signaling, including ST2, IRAKM, MyD88s, SOCS1, and Triad3A. The coordinated positive and negative regulation of the TIR signaling ensures the appropriate modulation of the innate and inflammatory responses.

  13. Cell receptors: definition, mechanisms and regulation of receptor-mediated endocytosis.

    PubMed

    Féger, J; Gil-Falgon, S; Lamaze, C

    1994-12-01

    Receptors allow the cells to recognize specific ligands and to receive extracellular messages. They can be classified into five families: 1) receptors for lipidic or lipophilic ligands; 2) the seven transmembrane receptors which mediate their messages by transduction through the activation of G-proteins, effectors and second messengers to amplify the response; 3) receptors which present an enzymatic activity on their transmembrane domains; 4) channel-receptors, transmembrane oligomeric molecules which let ions flow into the cell and 5) receptors which role is to internalize ligands, whatever their various functions. In parallel a concept of membrane plasticity was developed: vesicles are constantly formed from the plasma membrane, addressing complexes of ligand-receptors to specific intracellular compartments. This receptor-mediated endocytosis of ligand plays a critical role in regulating the number of a given receptor at the plasma membrane and in the cellular uptake of nutrients, growth factors and hormones. Many pathways exist for these transports but little is known about the signals which select the ligands or the receptors and direct them to their appropriate intracellular destination.

  14. Dose-response approaches for nuclear receptor-mediated ...

    EPA Pesticide Factsheets

    A public workshop, organized by a Steering Committee of scientists from government, industry, universities, and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of subthreshold doses for a number of receptor-mediated MOPs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance framework (HRF). Case studies addressed activation of the AHR; the CAR/PXR, and the PPARa. This paper describes the workshop process, key issues discussed, and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the

  15. Dose-response approaches for nuclear receptor-mediated ...

    EPA Pesticide Factsheets

    A public workshop, organized by a Steering Committee of scientists from government, industry, universities, and research organizations, was held at the National Institute of Environmental Health Sciences (NIEHS) in September, 2010. The workshop explored the dose-response implications of toxicant modes of action (MOA) mediated by nuclear receptors. The dominant paradigm in human health risk assessment has been linear extrapolation without a threshold for cancer, and estimation of sub-threshold doses for non-cancer and (in appropriate cases) cancer endpoints. However, recent publications question the application of dose-response modeling approaches with a threshold. The growing body of molecular toxicology information and computational toxicology tools has allowed for exploration of the presence or absence of subthreshold doses for a number of receptor-mediated MOPs. The workshop explored the development of dose-response approaches for nuclear receptor-mediated liver cancer, within a MOA Human Relevance framework (HRF). Case studies addressed activation of the AHR; the CAR/PXR, and the PPARa. This paper describes the workshop process, key issues discussed, and conclusions. The value of an interactive workshop approach to apply current MOA/HRF frameworks was demonstrated. The results may help direct research on the MOA and dose-response of receptor-based toxicity, since there are commonalities for many receptors in the basic pathways involved for late steps in the

  16. Analgesic Effects Mediated by Muscarinic Receptors: Mechanisms and Pharmacological Approaches.

    PubMed

    De Angelis, Federica; Tata, Ada Maria

    2016-01-01

    Chronic pain represents a research field on great clinical relevance and social impactful. It is associated to a variety of pathological events causing un altered excitability of peripheral nerves derived by tissue damage depending on physical, biological and chemical injury. In the last years much attention has been paid in the identification of novel molecules involved in mediating pain sensation useful as therapeutic tools for the development of new analgesic drugs. Muscarinic receptors are widely distributed both in the central and peripheral nervous system. It is known that muscarinic agonists cause analgesic effects via spinal and supraspinal mechanisms. Considering that the analgesia induced by cholinergic agonists is comparable to that observed with morphine, the identification of receptor subtypes involved and the identification of the muscarinic ligands capable of selectively activate these receptors, is of considerable interest for potential therapeutic application. In the present review we describe the role of muscarinic receptors in mediating central and peripheral pain and the mechanisms downstream these receptors responsible of the modulation of nociceptive stimuli. Moreover the therapeutic perspectives and the identification of potential drugs binding muscarinic receptors involved in pain modulation will also be discussed.

  17. 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

  18. 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

  19. Low and high affinity receptors mediate cellular uptake of heparanase

    PubMed Central

    Ben-Zaken, Olga; Shafat, Itay; Gingis-Velitski, Svetlana; Bangio, Haim; Kelson, Idil Kasuto; Alergand, Tal; Amor, Yehudit; Maya, Ruth Ben-Yakar; Vlodavsky, Israel; Ilan, Neta

    2008-01-01

    Heparanase is an endoglycosidase which cleaves heparan sulfate and hence participates in degradation and remodeling of the extracellular matrix. Importantly, heparanase activity correlated with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix barrier. Heparanase has been characterized as a glycoprotein, yet glycan biochemical analysis was not performed to date. Here, we applied the Qproteome™ GlycoArray kit to perform glycan analysis of heparanase, and compared the kit results with the more commonly used biochemical analyses. We employed fibroblasts isolated from patients with I-cell disease (mucolipidosis II), fibroblasts deficient of low density lipoprotein receptor-related protein and fibroblasts lacking mannose 6-phosphate receptor, to explore the role of mannose 6-phosphate in heparanase uptake. Iodinated heparanase has been utilized to calculate binding affinity. We provide evidence for hierarchy of binding to cellular receptors as a function of heparanase concentration. We report the existence of a high affinity, low abundant (i.e., low density lipoprotein receptor-related protein, mannose 6-phosphate receptor), as well as a low affinity, high abundant (i.e., heparan sulfate proteoglycan) receptors that mediate heparanase binding, and suggest that these receptors cooperate to establish high affinity binding sites for heparanase, thus maintaining extracellular retention of the enzyme tightly regulated. PMID:17981072

  20. Receptor-mediated regulation of neuropeptide gene expression in astrocytes.

    PubMed

    Schwartz, J P; Nishiyama, N; Wilson, D; Taniwaki, T

    1994-06-01

    One of the functions of glial receptors is to regulate synthesis and release of a variety of neuropeptides and growth factor peptides, which in turn act on neurons or other glia. Because of the potential importance of these interactions in injured brain, we have examined the role of two different receptors in the regulation of astrocyte neuropeptide synthesis. Stimulation of beta-adrenergic receptors on type 1 astrocytes resulted in increased mRNA and protein for the proenkephalin (PE) and somatostatin genes. This receptor also increased expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). The potential role of opiate receptors was examined in several ways. Treatment of newborn rats for 7 days with the opiate antagonist naltrexone, prior to preparation of astrocytes, had no effect on PE mRNA or met-enkephalin content but resulted in a significant increase in NGF content. However, treatment of astrocytes in culture with met-enkephalin, morphine, or naltrexone had no effect on any of these parameters. No opiate binding could be detected, using either etorphine or bremazocine, to membranes of astrocytes prepared from cortex, cerebellum, striatum, or hippocampus of 1-day, 7-day, or 14-day postnatal rats. Thus we conclude that type 1 astrocytes do not express opiate receptors and that the in vivo effects of naltrexone are mediated indirectly via some other cell type/receptor.

  1. Metabotropic glutamate receptor-mediated signaling in neuroglia

    PubMed Central

    Loane, David J.; Stoica, Bogdan A.; Faden, Alan I.

    2011-01-01

    Metabotropic glutamate (mGlu) receptors are G-protein-coupled receptors, which include eight subtypes that have been classified into three groups (I–III) based upon sequence homology, signal transduction mechanism and pharmacological profile. Although most studied with regard to neuronal function and modulation, mGlu receptors are also expressed by neuroglia-including astrocytes, microglia and oligodendrocytes. Activation of mGlu receptors on neuroglia under both physiologic and pathophysiologic conditions mediates numerous actions that are essential for intrinsic glial cell function, as well as for glial–neuronal interactions. Astrocyte mGlu receptors play important physiological roles in regulating neurotransmission and maintaining neuronal homeostasis. However, mGlu receptors on astrocytes and microglia also serve to modulate cell death and neurological function in a variety of pathophysiological conditions such as acute and chronic neurodegenerative disorders. The latter effects are complex and bi-directional, depending on which mGlu receptor sub-types are activated. PMID:22662309

  2. Evidence that somatostatin sst2 receptors mediate striatal dopamine release

    PubMed Central

    Hathway, G J; Humphrey, P P A; Kendrick, K M

    1999-01-01

    Somatostatin (SRIF) is a cyclic tetradecapeptide present in medium-sized aspiny interneurones in the rat striatum. We have previously shown that exogenous SRIF potently stimulates striatal dopamine (DA) release via a glutamate-dependent mechanism. We now report the ability of the selective sst2 receptor agonist, BIM-23027, to mimic this effect of SRIF.In vivo microdialysis studies were performed in anaesthetized male Wistar rats. In most experiments, compounds were administered by retrodialysis into the striatum for 15 min periods, 90 min and 225 min after sampling commenced, with levels of neurotransmitters being measured by HPLC with electrochemical and fluorescence detection.BIM-23027 (50 and 100 nM) stimulated DA release with extracellular levels increasing by up to 18 fold.Prior retrodialysis of BIM-23027 (50 nM) abolished the effects of subsequent administration of SRIF (100 nM).The agonist effects of both BIM-23027 and SRIF were abolished by the selective sst2 receptor antagonist, L-Tyr8-CYN-154806 (100 nM).The AMPA/kainate receptor antagonist, DNQX (100 μM), abolished the agonist effects of BIM-23027 as previously shown for SRIF.This study provides evidence that the sst2 receptor mediates the potent dopamine-releasing actions observed with SRIF in the rat striatum. Dopamine release evoked by both peptides appears to be mediated indirectly via a glutamatergic pathway. Other subtype-specific somatostatin receptor ligands were unable to elicit any effects and therefore we conclude that no other somatostatin receptor types are involved in mediating the dopamine-releasing actions of SRIF in the striatum. PMID:10578151

  3. 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.

  4. Pharmacology of inflammatory pain: local alteration in receptors and mediators.

    PubMed

    Holzer, Peter; Holzer-Petsche, Ulrike

    2009-01-01

    Inflammation is commonly associated with hyperalgesia. Ideally, this change should abate once inflammation is resolved, but this is not necessarily the case because phenotypic changes in the tissue can persist, as appears to be the case in post-infectious irritable bowel syndrome. Basically, all primary afferent neurons supplying the gut can be sensitized in response to pro-inflammatory mediators, and the mechanisms whereby hypersensitivity is initiated and maintained are, thus, of prime therapeutic interest. There is a multitude of molecular nocisensors that can be responsible for the hypersensitivity of afferent neurons. These entities include: (i) receptors and sensors at the peripheral terminals of afferent neurons that are relevant to stimulus transduction, (ii) ion channels that govern the excitability and conduction properties of afferent neurons, and (iii) transmitters and transmitter receptors that mediate communication between primary afferents and second-order neurons in the spinal cord and brainstem. Persistent increases in the sensory gain may result from changes in the expression of transmitters, receptors or ion channels; changes in the subunit composition and biophysical properties of receptors and ion channels; or changes in the structure, connectivity and survival of afferent neurons. Particular therapeutic potential is attributed to targets that are selectively expressed by afferent neurons and whose number and function are altered in abdominal hypersensitivity. Emerging targets of therapeutic relevance include distinct members of the transient receptor potential (TRP) channel family (TRPV1, TRPV4, TRPA1), acid-sensing ion channels, protease-activated receptors, corticotropin-releasing factor receptors and sensory neuron-specific sodium channels. Copyright 2010 S. Karger AG, Basel.

  5. 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

  6. 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

  7. Receptor-Mediated Transport of Insulin across Endothelial Cells

    NASA Astrophysics Data System (ADS)

    King, George L.; Johnson, Sandra M.

    1985-03-01

    Hormones such as insulin are transported from the interior to the exterior of blood vessels. Whether endothelial cells, which line the inner walls of blood vessels have a role in this transport of hormones is not clear, but it is known that endothelial cells can internalize and release insulin rapidly with little degradation. The transport of iodine-125-labeled insulin was measured directly through the use of dual chambers separated by a horizontal monolayer of cultured bovine aortic endothelial cells. In this setting, endothelial cells took up and released the labeled insulin, thereby transporting it across the cells. The transport of insulin across the endothelial cells was temperature sensitive and was inhibited by unlabeled insulin and by antibody to insulin receptor in proportion to the ability of these substances to inhibit insulin binding to its receptor. More than 80 percent of the transported insulin was intact. These data suggest that insulin is rapidly transported across endothelial cells by a receptor-mediated process.

  8. 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

  9. 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.

  10. 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. Copyright © 2013 Wiley Periodicals, Inc.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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.

  18. Commensal-Epithelial Signaling Mediated via Formyl Peptide Receptors

    PubMed Central

    Wentworth, Christy C.; Jones, Rheinallt M.; Kwon, Young Man; Nusrat, Asma; Neish, Andrew S.

    2010-01-01

    Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH2-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a Gi-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia. PMID:21037077

  19. Commensal-epithelial signaling mediated via formyl peptide receptors.

    PubMed

    Wentworth, Christy C; Jones, Rheinallt M; Kwon, Young Man; Nusrat, Asma; Neish, Andrew S

    2010-12-01

    Commensal bacteria and/or their products engender beneficial effects to the mammalian gut, including stimulating physiological cellular turnover and enhancing wound healing, without activating overt inflammation. In the present study, we observed commensal bacteria-mediated activation of the noninflammatory extracellular signal-regulated kinase[ERK]/mitogen-activated protein kinase and Akt signaling pathways in gut epithelial cells and delineated a mechanism for this bacterially activated signaling. All tested strains of commensal bacteria induced ERK phosphorylation without stimulating pro-inflammatory phospho-IκB or pro-apoptotic phospho-c-Jun NH(2)-terminal kinase, with Lactobacillus species being most potent. This pattern of signaling activation was recapitulated using the peptide N-formyl-Met-Leu-Phe, a bacterial product known to stimulate signaling events in mammalian phagocytes. Sensing of N-formyl-Met-Leu-Phe by gut epithelial cells occurs via recently characterized formyl peptide receptors located in the plasma membrane. Both commensal bacteria and N-formyl-Met-Leu-Phe application to the apical surface of polarized gut epithelial cells resulted in specific formyl peptide receptor activation. In addition, pretreatment of model epithelia and murine colon with Boc2 (a specific peptide antagonist) or pertussis toxin (a G(i)-protein inhibitor) abolished commensal-mediated ERK phosphorylation. Taken together, these data show that commensal bacteria specifically activate the ERK/mitogen-activated protein kinase pathway in an formyl peptide receptor-dependent manner, delineating a mechanism by which commensal bacteria contribute to cellular signaling in gut epithelia.

  20. NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

    PubMed

    Matta, Jose A; Gu, Shenyan; Davini, Weston B; Lord, Brian; Siuda, Edward R; Harrington, Anthony W; Bredt, David S

    2017-04-25

    Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and β subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4β2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  1. 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

  2. Hemoglobin uptake by Paracoccidioides spp. is receptor-mediated.

    PubMed

    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

    2014-05-01

    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.

  3. 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.

  4. Cannabinoid CB1 receptors mediate the effects of dipyrone.

    PubMed

    Crunfli, Fernanda; Vilela, Fabiana C; Giusti-Paiva, Alexandre

    2015-03-01

    Dipyrone is a non-steroidal anti-inflammatory drug used primarily as an analgesic and antipyretic. Some hypothesize that dipyrone activity can modulate other pathways, including endocannabinoid signalling. Thus, the aim of the present study was to evaluate the possible role of endocannabinoids in mediating dipyrone activity. This study is based on the tetrad effects of cannabinoids, namely an antinociceptive and cataleptic state, hypolocomotion and hypothermia. Dipyrone (500 mg/kg, i.p.) treatment decreased locomotor activity, increased the latency to a thermal analgesic response and induced a cataleptic and hypothermic state. These reactions are similar to the tetrad effects caused by the cannabinoid agonist WIN 55,212-2 (3 mg/kg, i.p.). The cannabinoid CB1 receptor antagonist AM251 (10 mg/kg, i.p.) reversed the effects of dipyrone on locomotor activity, the cataleptic response and thermal analgesia. Both AM251 (10 mg/kg, i.p.) and the transient receptor potential vanilloid 1 (TRPV1) antagonist capsazepine (10 mg/kg, i.p.) accentuated the reduction in body temperature caused by dipyrone. However, the CB2 receptor antagonist AM630 did not alter the hypothermic response to dipyrone. These results indicate involvement of the endocannabinoid system, especially CB1 receptors, in the analgesic and cataleptic effects of dipyrone, as well as hypolocomotion. However, cannabinoid receptors and TRPV1 were not involved in the hypothermic effects of dipyrone. We hypothesize that the mechanism of action of dipyrone may involve inhibition of cyclo-oxygenase and fatty acid amide hydrolase, which together provide additional arachidonic acid as substrate for endocannabinoid synthesis or other related molecules. This increase in endocannabinoid availability enhances CB1 receptor stimulation, contributing to the observed effects. © 2014 Wiley Publishing Asia Pty Ltd.

  5. Bombesin receptor-mediated imaging and cytotoxicity: review and current status

    PubMed Central

    Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

    2010-01-01

    The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

  6. VEGF mediates commissural axon chemoattraction through its receptor Flk1

    PubMed Central

    de Almodovar, Carmen Ruiz; Fabre, Pierre J.; Knevels, Ellen; Coulon, Cathy; Segura, Inmaculada; Haddick, Patrick C.G.; Aerts, Liesbeth; Delattin, Nicolas; Strasser, Geraldine; Oh, Won-Jong; Lange, Christian; Vinckier, Stefan; Haigh, Jody; Fouquet, Coralie; Henderson, Christopher; Gu, Chengua; Alitalo, Kari; Castellani, Valerie; Tessier-Lavigne, Marc; Chedotal, Alain; Charron, Frederic; Carmeliet, Peter

    2013-01-01

    Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons towards the midline. However, the combined inhibition of their activity in commissural axon turning assays does not completely abrogate turning towards floor plate tissue, suggesting that additional guidance cues are present. Here, we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, while Flk1-blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand / receptor pair controlling commissural axon guidance. PMID:21658588

  7. 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.

  8. Extranuclear Signaling Effects Mediated by the Estrogen Receptor

    DTIC Science & Technology

    2008-03-01

    of the author( s ) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other...Mediated by the Estrogen Receptor 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-05-1-0241 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR( S ) Erin...O’Neill, B.S. 5d. PROJECT NUMBER 5e. TASK NUMBER E-Mail: eionson@uchicago.edu 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME( S ) AND

  9. Crosstalk between purinergic receptors and lipid mediators in leishmaniasis.

    PubMed

    Chaves, Mariana M; Canetti, Cláudio; Coutinho-Silva, Robson

    2016-09-05

    Leishmaniasis is a neglected tropical disease affecting millions of people around the world caused by organisms of the genus Leishmania. Parasite escape mechanisms of the immune system confer the possibility of resistance and dissemination of the disease. A group of molecules that has become a target for Leishmania survival strategies are lipid mediators. Among them, leukotriene B4 (LTB4) has been described as a pro-inflammatory molecule capable of activating cells of the immune system to combat Leishmania. In an opposite way, prostaglandin E2 (PGE2) is a lipid mediator described as a deactivator of macrophages and neutrophils. The balance of these two molecules can be generated by extracellular nucleotides, such as adenosine 5'-triphosphate (ATP) and adenosine (Ado), which activate the purinergic receptors system. Herein, we discuss the role of extracellular nucleotides and the resulting balance of LTB4 and PGE2 in Leishmania fate, survival or death.

  10. 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.

  11. 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

  12. GIPC interacts with the beta1-adrenergic receptor and regulates beta1-adrenergic receptor-mediated ERK activation.

    PubMed

    Hu, Liaoyuan A; Chen, Wei; Martin, Negin P; Whalen, Erin J; Premont, Richard T; Lefkowitz, Robert J

    2003-07-11

    Beta1-adrenergic receptors, expressed at high levels in the human heart, have a carboxyl-terminal ESKV motif that can directly interact with PDZ domain-containing proteins. Using the beta1-adrenergic receptor carboxyl terminus as bait, we identified the novel beta1-adrenergic receptor-binding partner GIPC in a yeast two-hybrid screen of a human heart cDNA library. Here we demonstrate that the PDZ domain-containing protein, GIPC, co-immunoprecipitates with the beta1-adrenergic receptor in COS-7 cells. Essential for this interaction is the Ser residue of the beta1-adrenergic receptor carboxyl-terminal ESKV motif. Our data also demonstrate that beta1-adrenergic receptor stimulation activates the mitogen-activated protein kinase, ERK1/2. beta1-adrenergic receptor-mediated ERK1/2 activation was inhibited by pertussis toxin, implicating Gi, and was substantially decreased by the expression of GIPC. Expression of GIPC had no observable effect on beta1-adrenergic receptor sequestration or receptor-mediated cAMP accumulation. This GIPC effect was specific for the beta1-adrenergic receptor and was dependent on an intact PDZ binding motif. These data suggest that GIPC can regulate beta1-adrenergic receptor-stimulated, Gi-mediated, ERK activation while having no effect on receptor internalization or Gs-mediated cAMP signaling.

  13. A functionalized superparamagnetic iron oxide colloid as a receptor directed MR contrast agent

    SciTech Connect

    Josephson, L.; Groman, E.V.; Menz, E.; Lewis, J.M.; Bengele, H. )

    1990-01-01

    We have synthesized a surface functionalized superparamagnetic iron oxide colloid whose clearance from the vascular compartment was inhibited by asialofetuin but not fetuin. Unlike other particulate or colloidal magnetic resonance (MR) contrast agents, the agent of the current communication is not withdrawn from the vascular compartment by cells of the macrophage-monocyte phagocytic system, as indicated by its selective increase in hepatic relaxation rates. Because of this we refer to this colloid as a hepatic selective (HS) MR contrast agent. At 20 mumol Fe/kg the HS MR agent darkened MR images of liver. The HS MR agent exhibited no acute toxicity when injected into rats at 1800 mumol Fe/kg. Based on these observations, surface functionalized superparamagnetic iron oxide colloids may be the basis of MR contrast agents internalized by receptor mediated endocytosis generally, and by the asialoglycoprotein receptor in particular.

  14. Fast Modulation of μ-Opioid Receptor (MOR) Recycling Is Mediated by Receptor Agonists*

    PubMed Central

    Roman-Vendrell, Cristina; Yu, Y. Joy; Yudowski, Guillermo Ariel

    2012-01-01

    The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca2+-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance. PMID:22378794

  15. 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.

  16. Fast modulation of μ-opioid receptor (MOR) recycling is mediated by receptor agonists.

    PubMed

    Roman-Vendrell, Cristina; Yu, Y Joy; Yudowski, Guillermo Ariel

    2012-04-27

    The μ-opioid receptor (MOR) is a member of the G protein-coupled receptor family and the main target of endogenous opioid neuropeptides and morphine. Upon activation by ligands, MORs are rapidly internalized via clathrin-coated pits in heterologous cells and dissociated striatal neurons. After initial endocytosis, resensitized receptors recycle back to the cell surface by vesicular delivery for subsequent cycles of activation. MOR trafficking has been linked to opioid tolerance after acute exposure to agonist, but it is also involved in the resensitization process. Several studies describe the regulation and mechanism of MOR endocytosis, but little is known about the recycling of resensitized receptors to the cell surface. To study this process, we induced internalization of MOR with [D-Ala(2), N-Me-Phe(4), Gly(5)-ol]-enkephalin (DAMGO) and morphine and imaged in real time single vesicles recycling receptors to the cell surface. We determined single vesicle recycling kinetics and the number of receptors contained in them. Then we demonstrated that rapid vesicular delivery of recycling MORs to the cell surface was mediated by the actin-microtubule cytoskeleton. Recycling was also dependent on Rab4, Rab11, and the Ca(2+)-sensitive motor protein myosin Vb. Finally, we showed that recycling is acutely modulated by the presence of agonists and the levels of cAMP. Our work identifies a novel trafficking mechanism that increases the number of cell surface MORs during acute agonist exposure, effectively reducing the development of opioid tolerance.

  17. 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

  18. Transient receptor potential-like channels mediate metabotropic glutamate receptor EPSCs in rat dopamine neurones

    PubMed Central

    Bengtson, C Peter; Tozzi, Alessandro; Bernardi, Giorgio; Mercuri, Nicola B

    2004-01-01

    Transient receptor potential (TRP) channels form cationic channels activated by diverse factors including mechanical stimuli, changes in osmolarity, pH and temperature, as well as the exogenous irritant, capsaicin. Metabotropic glutamate receptors have also recently been linked to TRP channel activation in neurones of the substantia nigra, hippocampus and cerebellum, suggesting a novel role for such channels in synaptic communication via endogenous neurotransmitters. We tested this for dopamine neurones in rat brain slices by characterizing the current–voltage relationship and pharmacology of EPSCs mediated by group I metabotropic glutamate receptor subtype 1 (mGluR1). Slow inward currents (273 ± 35 pA peak amplitude, 381 ± 25 ms latency, holding potential (Vh) =−73 mV) representing evoked mGluR1 EPSCs were isolated in the presence of antagonists of AMPA, NMDA, GABAA, GABAB, muscarinic and glycine receptors. CPCCOEt (100 μm), an mGluR1 antagonist, blocked the residual EPSC in all recordings. mGluR1-activated EPSCs reversed polarity near −10 mV, consistent with the involvement of a cationic channel. Extracellular application of the non-selective TRP channel blockers SKF 96365, flufenamic acid and ruthenium red caused reversible inhibition of mGluR1-activated EPSCs. These characteristics parallel those of mGluR1 activation with an agonist and indicate the involvement of a TRP-like channel in mGluR1-mediated EPSCs. PMID:14724196

  19. 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.

  20. Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors.

    PubMed

    van Koppen, C J; Meyer zu Heringdorf, D; Alemany, R; Jakobs, K H

    2001-04-27

    Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.

  1. 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

  2. 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.

  3. 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.

  4. 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

  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. 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

  7. 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.

  8. Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

    PubMed Central

    Ohm, Ingrid Kristine; Alfsnes, Katrine; Belland Olsen, Maria; Ranheim, Trine; Sandanger, Øystein; Dahl, Tuva Børresdatter; Aukrust, Pål; Finsen, Alexandra Vanessa; Yndestad, Arne; Vinge, Leif Erik

    2014-01-01

    Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and –C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions. PMID:25126740

  9. Toll-like receptor 9 mediated responses in cardiac fibroblasts.

    PubMed

    Ohm, Ingrid Kristine; Alfsnes, Katrine; Belland Olsen, Maria; Ranheim, Trine; Sandanger, Øystein; Dahl, Tuva Børresdatter; Aukrust, Pål; Finsen, Alexandra Vanessa; Yndestad, Arne; Vinge, Leif Erik

    2014-01-01

    Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and -C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.

  10. 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

  11. 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.

  12. 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.

  13. 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

  14. Receptors mediating contraction of isolated human vas deferens.

    PubMed

    Canevari, A; Pacifico, P; Cugurra, F; Giglio, C; Nadalini, V F

    1986-09-01

    A large body of evidences has suggested the role of adrenergic, opioidergic and other peptidergic receptors in the mediation of animal vas deferens motility. Different animal species showed different neurochemical patterns, so it is to be expected that human vas deferens has its own specific response to several substances, in relation to its peculiar function. In this study we report on the effects of monoaminergic (norepinephrine, dopamine, serotonin, isoproterenol, cholinomimetic drugs) and opioidergic (morphine, buprenorphin, beta-endorphin, met-enkephalin and dynorphin) agonists on isolated human vas deferens motility. Norepinephrine and dopamine provoked complex patterns of motility while opioids did not affect the field electroinduced contractions. The implications of this finding are discussed in relation to human vas deferens function.

  15. Receptor mediated cellular uptake of low molecular weight dendritic polyglycerols.

    PubMed

    Calderón, Marcelo; Reichert, Stephanie; Welker, Pia; Licha, Kai; Kratz, Felix; Haag, Rainer

    2014-01-01

    The development of effective polymer-based nanocarriers which are able to target diseased tissues still remains a great challenge in current research. Dendritic polyglycerols have emerged as novel polymeric scaffolds that have demonstrated a great potential for diverse biomedical applications. These architectures have already proven their usefulness in therapeutic approaches related to multivalency, given by the synergy between the nanosized dimensions combined with the high density of functional groups. However, a continuous effort is necessary to modify and tailor polyglycerol architectures to fit the future demands of biomedical applications. The present work deals with the development of a general synthetic strategy that allows the linkage of low molecular weight dendritic polyglycerols to fluorescent dyes and cell targeting ligands. The receptor mediated cellular uptake of the polyglycerol conjugates highlight their potential to acts as new targeted nanocarriers that should be able to decrease non-specific cellular uptake.

  16. 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.

  17. 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.

  18. Hepatoprotective actions of melatonin: Possible mediation by melatonin receptors

    PubMed Central

    Mathes, Alexander M

    2010-01-01

    Melatonin, the hormone of darkness and messenger of the photoperiod, is also well known to exhibit strong direct and indirect antioxidant properties. Melatonin has previously been demonstrated to be a powerful organ protective substance in numerous models of injury; these beneficial effects have been attributed to the hormone’s intense radical scavenging capacity. The present report reviews the hepatoprotective potential of the pineal hormone in various models of oxidative stress in vivo, and summarizes the extensive literature showing that melatonin may be a suitable experimental substance to reduce liver damage after sepsis, hemorrhagic shock, ischemia/reperfusion, and in numerous models of toxic liver injury. Melatonin’s influence on hepatic antioxidant enzymes and other potentially relevant pathways, such as nitric oxide signaling, hepatic cytokine and heat shock protein expression, are evaluated. Based on recent literature demonstrating the functional relevance of melatonin receptor activation for hepatic organ protection, this article finally suggests that melatonin receptors could mediate the hepatoprotective actions of melatonin therapy. PMID:21182223

  19. 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

  20. Direct Visualization of Estrogen Receptor-Mediated Transcription in Living Cells

    DTIC Science & Technology

    2007-10-01

    Receptor-Mediated Transcription in Living Cells PRINCIPAL INVESTIGATOR: Paul M. Yen, M.D...reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of...Sep 2007 4. TITLE AND SUBTITLE Direct Visualization of Estrogen Receptor-Mediated Transcription in Living Cells 5a. CONTRACT NUMBER 5b. GRANT

  1. 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.

  2. 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

  3. 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

  4. 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...

  5. 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...

  6. Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses.

    PubMed

    Muku, Gulsum E; Lahoti, Tejas S; Murray, Iain A; Podolsky, Michael A; Smith, Kayla J; Hubbard, Troy D; Kuzu, Guray; Gowda, Krishne; Amin, Shantu G; Perdew, Gary H

    2017-09-11

    The Ah receptor (AHR) has been shown to exhibit both inflammatory and anti-inflammatory activity in a context-specific manner. In vivo macrophage-driven acute inflammation models were utilized here to test whether the selective Ah receptor modulator 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA360) would reduce inflammation. Exposure to SGA360 was capable of significantly inhibiting lipopolysaccharide (LPS)-mediated endotoxic shock in a mouse model, both in terms of lethality and attenuating inflammatory signaling in tissues. Topical exposure to SGA360 was also able to mitigate joint edema in a monosodium urate (MSU) crystal gout mouse model. Inhibition was dependent on the expression of the high-affinity allelic AHR variant in both acute inflammation models. Upon peritoneal MSU crystal exposure SGA360 pretreatment inhibited neutrophil and macrophage migration into the peritoneum. RNA-seq analysis revealed that SGA360 attenuated the expression of numerous inflammatory genes and genes known to be directly regulated by AHR in thioglycolate-elicited primary peritoneal macrophages treated with LPS. In addition, expression of the high-affinity allelic AHR variant in cultured macrophages was necessary for SGA360-mediated repression of inflammatory gene expression. Mechanistic studies revealed that SGA360 failed to induce nuclear translocation of the AHR and actually enhanced cytoplasmic localization. LPS treatment of macrophages enhanced the occupancy of the AHR and p65 to the Ptgs2 promoter, whereas SGA360 attenuated occupancy. AHR ligand activity was detected in peritoneal exudates isolated from MSU-treated mice, thus suggesting that the anti-inflammatory activity of SGA360 is mediated at least in part through AHR antagonism of endogenous agonist activity. These results underscore an important role of the AHR in participating in acute inflammatory signaling and warrants further investigations into possible clinical applications

  7. 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

  8. Morphine inhibits an alpha9-acetylcholine nicotinic receptor-mediated response by a mechanism which does not involve opioid receptors.

    PubMed

    Lioudyno, M I; Verbitsky, M; Holt, J C; Elgoyhen, A B; Guth, P S

    2000-11-01

    Nicotinic acetylcholine (nACh) receptors are known to be targets for modulation by a number of substances, including the opiates. It is known that acetylcholine (ACh) coexists with opioid peptides in cochlear efferent neurons, and such a colocalization has been proposed for the vestibular system. In the present study we test the hypothesis that morphine, an opioid receptor agonist with a broad spectrum of selectivity, modulates alpha9nACh receptor-mediated responses in frog vestibular hair cells. Morphine dose-dependently and reversibly inhibited ACh-induced currents as recorded by the perforated patch-clamp method. In the presence of morphine the ACh dose-response curve was shifted to the right in a parallel fashion, suggesting a competitive interaction. However, naloxone did not antagonize the inhibition produced by morphine. To test the hypothesis that morphine could interact with the alpha9nACh receptor without the involvement of opioid receptors, experiments were performed using Xenopus laevis oocytes injected with the alpha9nACh receptor cRNA. The currents activated by ACh in Xenopus oocytes, a system that lacks opioid receptors, were also dose-dependently inhibited by morphine. We conclude that morphine inhibits the alpha9nACh receptor-mediated response in hair cells and Xenopus oocytes through a mechanism which does not involve opioid receptors but may be a direct block of the alpha9nACh receptor.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

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

    PubMed

    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

    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. 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. 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) ). 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. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The

  14. 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

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

    PubMed

    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

    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. 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. Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion.

  16. 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

  17. The Measles Virus Receptor SLAMF1 Can Mediate Particle Endocytosis.

    PubMed

    Gonçalves-Carneiro, Daniel; McKeating, Jane A; Bailey, Dalan

    2017-04-01

    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 glycoproteins found

  18. 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

  19. 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.

  20. 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.

  1. Cardiomyocyte ryanodine receptor degradation by chaperone-mediated autophagy.

    PubMed

    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

    2013-05-01

    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. 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. 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.

  2. 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

  3. The melatonin-MT1 receptor axis modulates mutant huntingtin-mediated toxicity

    PubMed Central

    Wang, Xin; Sirianni, Ana; Pei, Zhijuan; Cormier, Kerry; Smith, Karen; Jiang, Jiying; Zhou, Shuanhu; Wang, Hui; Zhao, Rong; Yano, Hiroko; Kim, Jeong Eun; Li, Wei; Kristal, Bruce S.; Ferrante, Robert J.; Friedlander, Robert M.

    2011-01-01

    Melatonin mediates neuroprotection in several experimental models of neurodegeneration. It is not yet known, however, whether melatonin provides neuroprotection in genetic models of Huntington’s disease (HD). We report that melatonin delays disease onset and mortality in a transgenic mouse model of HD. Moreover, mutant huntingtin (htt)-mediated toxicity in cells, mice, and humans is associated with loss of the type 1 melatonin receptor (MT1). We observe high levels of MT1 receptor in mitochondria from the brains of wild-type mice but much less in brains from HD mice. Moreover, we demonstrate that melatonin inhibits mutant htt-induced caspase activation and preserves MT1 receptor expression. This observation is critical, since melatonin-mediated protection is dependent upon the presence and activation of the MT1 receptor. In summary, we delineate a pathologic process whereby mutant htt-induced loss of the mitochondrial MT1 receptor enhances neuronal vulnerability and potentially accelerates the neurodegenerative process. PMID:21994366

  4. 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

  5. Chronic intermittent ethanol exposure enhances NMDA-receptor-mediated synaptic responses and NMDA receptor expression in hippocampal CA1 region.

    PubMed

    Nelson, T E; Ur, C L; Gruol, D L

    2005-06-28

    In previous studies, we found that chronic intermittent ethanol (CIE) treatment-a model of ethanol consumption in which animals are exposed to and withdrawn from intoxicating levels of ethanol on a daily basis-produces neuroadaptive changes in hippocampal area CA1 excitatory synaptic transmission and plasticity. Synaptic responses mediated by N-methyl-D-aspartate (NMDA) receptors are known to be sensitive to ethanol and could play an important role in the neuroadaptive changes induced by CIE treatment. To address this issue, we compared electrophysiological recordings of pharmacologically isolated NMDA-receptor-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 region of hippocampal slices prepared from control rats and rats exposed to 2 weeks of CIE treatment administered by vapor inhalation. We found that fEPSPs induced by NMDA receptor activation were unaltered in slices prepared shortly after cessation of CIE treatment (i.e., < or = 1 day of withdrawal from CIE). However, following 7 days of withdrawal from CIE treatment, NMDA-receptor-mediated fEPSPs were augmented relative to age-matched controls. Western blot analysis of NMDA receptor subunit expression showed that, at 7 days of withdrawal, the level of protein for NR2A and NR2B subunits was elevated in the CA1 region of hippocampal slices from CIE-treated animals compared with slices from age-matched controls. These results are consistent with an involvement of NMDA-receptor-mediated synaptic responses in the neuroadaptive effects of CIE on hippocampal physiology and suggest that such changes may contribute to ethanol-induced changes in processes dependent on NMDA-receptor-mediated synaptic responses such as learning and memory, neural development, hyperexcitability and seizures, and neurotoxicity.

  6. Interaction between muscarinic receptor subtype signal transduction pathways mediating bladder contraction

    PubMed Central

    BRAVERMAN, ALAN S.; TALLARIDA, RONALD J.; RUGGIERI, MICHAEL R.

    2012-01-01

    M3 muscarinic receptors mediate cholinergic-induced contraction in most smooth muscles. However, in the denervated rat bladder, M2 receptors participate in contraction because M3-selective antagonists [para-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD) and 4-DAMP] have low affinities. However, the affinity of the M2-selective antagonist methoctramine in the denervated bladder is consistent with M3 receptor mediating contraction. It is possible that two pathways interact to mediate contraction: one mediated by the M2 receptor and one by the M3 receptor. To determine whether an interaction exists, the inhibitory potencies of combinations of methoctramine and p-F-HHSiD for reversing cholinergic contractions were measured. In normal bladders, all combinations gave additive effects. In denervated bladders, synergistic effects were seen with the 10:1 and 1:1 (methoctramine:p-F-HHSiD wt/wt) combinations. After application of the sarcoplasmic reticulum ATPase inhibitor thapsigargin to normal tissue, the 10:1 and 1:1 ratios became synergistic, mimicking denervated tissue. Thus in normal bladders both M2 and M3 receptors can induce contraction. In the denervated bladder, the M2 and the M3 receptors interact in a facilitatory manner to mediate contraction. PMID:12185001

  7. 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

  8. 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.

  9. 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…

  10. 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…

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

    USDA-ARS?s Scientific Manuscript database

    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...

  12. 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

  13. Activation of membrane estrogen receptors attenuates opioid receptor-like1 receptor-mediated antinociception via an ERK-dependent non-genomic mechanism.

    PubMed

    Small, K M; Nag, S; Mokha, S S

    2013-01-01

    To our knowledge, the present data are the first to demonstrate that activation of membrane estrogen receptors (mERs) abolishes opioid receptor-like 1 (ORL1) receptor-mediated analgesia via extracellular signal-regulated kinase (ERK)-dependent non-genomic mechanisms. Estrogen was shown previously to both attenuate ORL1-mediated antinociception and down-regulate the ORL1 gene expression. The present study investigated whether non-genomic mechanisms contribute to estrogen-induced attenuation of ORL1-mediated antinociception by the mERs GPR30, Gq-coupled mER, ERα, and ERβ. E2BSA [β-estradiol-6-(O-carboxymethyl)oxime: bovine serum albumin] (0.5mM), a membrane impermeant analog of estradiol, injected intrathecally immediately prior to orphanin FQ (OFQ;10 nmol), the endogenous ligand for the ORL1 receptor, abolished OFQ's antinociceptive effect in both male and ovariectomized (OVX) female rats, assessed using the heat-induced tail-flick assay. This effect was not altered by protein synthesis inhibitor, anisomycin (125 μg), given intrathecally 15 min prior to E2BSA and OFQ. Intrathecal application of selective receptor agonists permitted the relative contributions of various estrogen receptors in mediating this blockade of the antinociceptive response of OFQ. Activation of GPR30, Gq-mER, ERα, but not ERβ abolished ORL1-mediated antinociception in males and OVX females. E2BSA produced a parallel and significant increase in the phosphorylation of ERK 2 only in OVX females, and pre-treatment with MEK/ERK 1/2 inhibitor, U0126 (10 μg), blocked the mER-mediated abolition of ORL1-mediated antinociception in OVX females. Taken together, the data are consistent with the interpretations that mER activation attenuates ORL1-mediated antinociception through a non-genomic, ERK 2-dependent mechanism in females. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  14. Pharmacological significance of the interplay between angiotensin receptors: MAS receptors as putative final mediators of the effects elicited by angiotensin AT1 receptors antagonists.

    PubMed

    Pernomian, Larissa; Pernomian, Laena; Gomes, Mayara S; da Silva, Carlos H T P

    2015-12-15

    The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. 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

  16. 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...

  17. 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...

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

    USDA-ARS?s Scientific Manuscript database

    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 ...

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

    USDA-ARS?s Scientific Manuscript database

    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...

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. Buprenorphine-induced antinociception is mediated by mu-opioid receptors and compromised by concomitant activation of opioid receptor-like receptors.

    PubMed

    Lutfy, Kabirullah; Eitan, Shoshana; Bryant, Camron D; Yang, Yu C; Saliminejad, Nazli; Walwyn, Wendy; Kieffer, Brigitte L; Takeshima, Hiroshi; Carroll, F Ivy; Maidment, Nigel T; Evans, Christopher J

    2003-11-12

    Buprenorphine is a mixed opioid receptor agonist-antagonist used clinically for maintenance therapy in opiate addicts and pain management. Dose-response curves for buprenorphine-induced antinociception display ceiling effects or are bell shaped, which have been attributed to the partial agonist activity of buprenorphine at opioid receptors. Recently, buprenorphine has been shown to activate opioid receptor-like (ORL-1) receptors, also known as OP4 receptors. Here we demonstrate that buprenorphine, but not morphine, activates mitogen-activated protein kinase and Akt via ORL-1 receptors. Because the ORL-1 receptor agonist orphanin FQ/nociceptin blocks opioid-induced antinociception, we tested the hypothesis that buprenorphine-induced antinociception might be compromised by concomitant activation of ORL-1 receptors. In support of this hypothesis, the antinociceptive effect of buprenorphine, but not morphine, was markedly enhanced in mice lacking ORL-1 receptors using the tail-flick assay. Additional support for a modulatory role for ORL-1 receptors in buprenorphine-induced antinociception was that coadministration of J-113397, an ORL-1 receptor antagonist, enhanced the antinociceptive efficacy of buprenorphine in wild-type mice but not in mice lacking ORL-1 receptors. The ORL-1 antagonist also eliminated the bell-shaped dose-response curve for buprenorphine-induced antinociception in wild-type mice. Although buprenorphine has been shown to interact with multiple opioid receptors, mice lacking micro-opioid receptors failed to exhibit antinociception after buprenorphine administration. Our results indicate that the antinociceptive effect of buprenorphine in mice is micro-opioid receptor-mediated yet severely compromised by concomitant activation of ORL-1 receptors.

  7. 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

  8. 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

  9. Role for Phospholipase D in Receptor-Mediated Endocytosis

    PubMed Central

    Shen, Yingjie; Xu, Lizhong; Foster, David A.

    2001-01-01

    In response to epidermal growth factor (EGF), the EGF receptor is endocytosed and degraded. A substantial lag period exists between endocytosis and degradation, suggesting that endocytosis is more than a simple negative feedback. Phospholipase D (PLD), which has been implicated in vesicle formation in the Golgi, is activated in response to EGF and other growth factors. We report here that EGF receptor endocytosis is dependent upon PLD and the PLD1 regulators, protein kinase C α and RalA. EGF-induced receptor degradation is accelerated by overexpression of either wild-type PLD1 or PLD2 and retarded by overexpression of catalytically inactive mutants of either PLD1 or PLD2. EGF-induced activation of mitogen-activated protein kinase, which is dependent upon receptor endocytosis, is also dependent upon PLD. These data suggest a role for PLD in signaling that facilitates receptor endocytosis. PMID:11134345

  10. Legume LysM receptors mediate symbiotic and pathogenic signalling.

    PubMed

    Kelly, Simon; Radutoiu, Simona; Stougaard, Jens

    2017-10-01

    Legume-rhizobia symbiosis is coordinated through the production and perception of signal molecules by both partners with legume LysM receptor kinases performing a central role in this process. Receptor complex formation and signalling outputs derived from these are regulated through ligand binding and further modulated by a diverse variety of interactors. The challenge now is to understand the molecular mechanisms of these reported interactors. Recently attributed roles of LysM receptors in the perception of rhizobial exopolysaccharide, distinguishing between pathogens and symbionts, and assembly of root and rhizosphere communities expand on the importance of these receptors. These studies also highlight challenges, such as identification of cognate ligands, formation of responsive receptor complexes and separation of downstream signal transduction pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. Receptor-mediated signalling in plants: molecular patterns and programmes

    PubMed Central

    Tör, Mahmut; Lotze, Michael T.; Holton, Nicholas

    2009-01-01

    A highly evolved surveillance system in plants is able to detect a broad range of signals originating from pathogens, damaged tissues, or altered developmental processes, initiating sophisticated molecular mechanisms that result in defence, wound healing, and development. Microbe-associated molecular pattern molecules (MAMPs), damage-associated molecular pattern molecules (DAMPs), virulence factors, secreted proteins, and processed peptides can be recognized directly or indirectly by this surveillance system. Nucleotide binding-leucine rich repeat proteins (NB-LRR) are intracellular receptors and have been targeted by breeders for decades to elicit resistance to crop pathogens in the field. Receptor-like kinases (RLKs) or receptor like proteins (RLPs) are membrane bound signalling molecules with an extracellular receptor domain. They provide an early warning system for the presence of potential pathogens and activate protective immune signalling in plants. In addition, they act as a signal amplifier in the case of tissue damage, establishing symbiotic relationships and effecting developmental processes. The identification of several important ligands for the RLK-type receptors provided an opportunity to understand how plants differentiate, how they distinguish beneficial and detrimental stimuli, and how they co-ordinate the role of various types of receptors under varying environmental conditions. The diverse roles of extra-and intracellular plant receptors are examined here and the recent findings on how they promote defence and development is reviewed. PMID:19628572

  20. 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

  1. 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.

  2. 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

  3. Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors.

    PubMed

    Abdelhamid, Ramy E; Kovacs, Katalin J; Pasley, Jeffrey D; Nunez, Myra G; Larson, Alice A

    2013-09-01

    The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26 °C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Forced swim-induced musculoskeletal hyperalgesia is mediated by CRF2 receptors but not by TRPV1 receptors

    PubMed Central

    Abdelhamid, Ramy E.; Kovacs, Katalin J.; Pasley, Jeff D.; Nunez, Myra G.; Larson, Alice A.

    2013-01-01

    The exacerbation of musculoskeletal pain by stress in humans is modeled by the musculoskeletal hyperalgesia in rodents following a forced swim. We hypothesized that stress-sensitive corticotropin releasing factor (CRF) receptors and transient receptor vanilloid 1 (TRPV1) receptors are responsible for the swim stress-induced musculoskeletal hyperalgesia. We confirmed that a cold swim (26°C) caused a transient, morphine-sensitive decrease in grip force responses reflecting musculoskeletal hyperalgesia in mice. Pretreatment with the CRF2 receptor antagonist astressin 2B, but not the CRF1 receptor antagonist NBI-35965, attenuated this hyperalgesia. Desensitizing the TRPV1 receptor centrally or peripherally using desensitizing doses of resiniferatoxin (RTX) failed to prevent the musculoskeletal hyperalgesia produced by cold swim. SB-366791, a TRPV1 antagonist, also failed to influence swim-induced hyperalgesia. Together these data indicate that swim stress-induced musculoskeletal hyperalgesia is mediated, in part, by CRF2 receptors but is independent of the TRPV1 receptor. PMID:23624287

  5. 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

  6. 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.

  7. Glutamate mediates platelet activation through the AMPA receptor

    PubMed Central

    Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

    2008-01-01

    Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

  8. 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.

  9. 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

  10. EP₃ receptors mediate PGE₂-induced hypothalamic paraventricular nucleus excitation and sympathetic activation.

    PubMed

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Nakamura, Yoshiko; Nakamura, Kazuhiro; Felder, Robert B

    2011-10-01

    Prostaglandin E(2) (PGE(2)), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE(2) acts on at least four E-class prostanoid (EP) receptors known as EP(1), EP(2), EP(3), and EP(4). Since PGE(2) production within the brain is ubiquitous, the different functions of PGE(2) depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE(2) remain unknown. We examined this question using PGE(2), the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP(1), EP(3), and EP(4). In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE(2), sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP(3) receptor antagonist; the EP(1) and EP(4) receptor antagonists had little or no effect. ICV PGE(2) or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP(3) receptor antagonist. Real-time PCR detected EP(3) receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP(3) receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE(2) or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP(3) receptor antagonist. These data suggest that EP(3) receptors mediate the central excitatory effects of PGE(2) on PVN neurons and sympathetic discharge.

  11. EP3 receptors mediate PGE2-induced hypothalamic paraventricular nucleus excitation and sympathetic activation

    PubMed Central

    Zhang, Zhi-Hua; Yu, Yang; Wei, Shun-Guang; Nakamura, Yoshiko; Nakamura, Kazuhiro

    2011-01-01

    Prostaglandin E2 (PGE2), an important mediator of the inflammatory response, acts centrally to elicit sympathetic excitation. PGE2 acts on at least four E-class prostanoid (EP) receptors known as EP1, EP2, EP3, and EP4. Since PGE2 production within the brain is ubiquitous, the different functions of PGE2 depend on the expression of these prostanoid receptors in specific brain areas. The type(s) and location(s) of the EP receptors that mediate sympathetic responses to central PGE2 remain unknown. We examined this question using PGE2, the relatively selective EP receptor agonists misoprostol and sulprostone, and the available selective antagonists for EP1, EP3, and EP4. In urethane-anesthetized rats, intracerebroventricular (ICV) administration of PGE2, sulprostone or misoprostol increased renal sympathetic nerve activity, blood pressure, and heart rate. These responses were significantly reduced by ICV pretreatment with the EP3 receptor antagonist; the EP1 and EP4 receptor antagonists had little or no effect. ICV PGE2 or misoprostol increased the discharge of neurons in the hypothalamic paraventricular nucleus (PVN). ICV misoprostol increased the c-Fos immunoreactivity of PVN neurons, an effect that was substantially reduced by the EP3 receptor antagonist. Real-time PCR detected EP3 receptor mRNA in PVN, and immunohistochemical studies revealed sparsely distributed EP3 receptors localized in GABAergic terminals and on a few PVN neurons. Direct bilateral PVN microinjections of PGE2 or sulprostone elicited sympathoexcitatory responses that were significantly reduced by the EP3 receptor antagonist. These data suggest that EP3 receptors mediate the central excitatory effects of PGE2 on PVN neurons and sympathetic discharge. PMID:21803943

  12. 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.

  13. 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.

  14. EP2 receptor mediates PGE2-induced cystogenesis of human renal epithelial cells.

    PubMed

    Elberg, Gerard; Elberg, Dorit; Lewis, Teresa V; Guruswamy, Suresh; Chen, Lijuan; Logan, Charlotte J; Chan, Michael D; Turman, Martin A

    2007-11-01

    Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.

  15. 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

  16. 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.

  17. 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

  18. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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

  6. SIGIRR inhibits interleukin-1 receptor- and toll-like receptor 4-mediated signaling through different mechanisms.

    PubMed

    Qin, Jinzhong; Qian, Youcun; Yao, Jianhong; Grace, Cui; Li, Xiaoxia

    2005-07-01

    The Toll-interleukin-1 receptor (TIR) domain-containing orphan receptor SIGIRR (single immunoglobulin interleukin-1 receptor-related protein) acts as a negative regulator of interleukin (IL)-1 and lipopolysaccharide (LPS) signaling. Endogenous SIGIRR transiently interacted with IL-1 receptor and the receptor-proximal signaling components (MyD88, IRAK, and tumor necrosis factor receptor-associated factor 6) upon IL-1 stimulation, indicating that SIGIRR interacts with the IL-1 receptor complex in a ligand-dependent manner. Similar interaction was also observed between SIGIRR and Toll-like receptor 4 receptor complex upon LPS stimulation. To identify the domains of SIGIRR required for its interaction with the Toll-like receptor 4 and IL-1 receptor complexes, several SIGIRR deletion mutants were generated, including DeltaN (lacking the extracellular immunoglobulin (Ig) domain with deletion of amino acids 1-119), DeltaC (lacking the C-terminal domain with deletion of amino acids 313-410), and DeltaTIR (lacking the TIR domain with deletion of amino acids 161-313). Whereas both the extracellular Ig domain and the intracellular TIR domains are important for SIGIRR to inhibit IL-1 signaling, only the TIR domain is necessary for SIGIRR to inhibit LPS signaling. The extracellular Ig domain exerts its inhibitory role in IL-1 signaling by interfering with the heterodimerization of IL-1 receptor and IL-1RAcP, whereas the intracellular TIR domain inhibits both IL-1 and LPS signaling by attenuating the recruitment of receptor-proximal signaling components to the receptor. These results indicate that SIGIRR inhibits IL-1 and LPS signaling pathways through differential mechanisms.

  7. 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

  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. 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.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Lotus japonicus Cytokinin Receptors Work Partially Redundantly to Mediate Nodule Formation[W][OPEN

    PubMed Central

    Held, Mark; Hou, Hongwei; Miri, Mandana; Huynh, Christian; Ross, Loretta; Hossain, Md Shakhawat; Sato, Shusei; Tabata, Satoshi; Perry, Jillian; Wang, Trevor L.; Szczyglowski, Krzysztof

    2014-01-01

    Previous analysis of the Lotus histidine kinase1 (Lhk1) cytokinin receptor gene has shown that it is required and also sufficient for nodule formation in Lotus japonicus. The L. japonicus mutant carrying the loss-of-function lhk1-1 allele is hyperinfected by its symbiotic partner, Mesorhizobium loti, in the initial absence of nodule organogenesis. At a later time point following bacterial infection, lhk1-1 develops a limited number of nodules, suggesting the presence of an Lhk1-independent mechanism. We have tested a hypothesis that other cytokinin receptors function in at least a partially redundant manner with LHK1 to mediate nodule organogenesis in L. japonicus. We show here that L. japonicus contains a small family of four cytokinin receptor genes, which all respond to M. loti infection. We show that within the root cortex, LHK1 performs an essential role but also works partially redundantly with LHK1A and LHK3 to mediate cell divisions for nodule primordium formation. The LHK1 receptor is also presumed to partake in mediating a feedback mechanism that negatively regulates bacterial infections at the root epidermis. Interestingly, the Arabidopsis thaliana AHK4 receptor gene can functionally replace Lhk1 in mediating nodule organogenesis, indicating that the ability to perform this developmental process is not determined by unique, legume-specific properties of LHK1. PMID:24585837

  17. 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.

  18. 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.

  19. 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

  20. 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.

  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 Central

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

    2015-01-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age- matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. PMID:24434437

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

    PubMed Central

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    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. PMID:26833785

  4. Comparison of lentiviral and sleeping beauty mediated αβ T cell receptor gene transfer.

    PubMed

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm's tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies.

  5. Comparison of Lentiviral and Sleeping Beauty Mediated αβ T Cell Receptor Gene Transfer

    PubMed Central

    Field, Anne-Christine; Vink, Conrad; Gabriel, Richard; Al-Subki, Roua; Schmidt, Manfred; Goulden, Nicholas; Stauss, Hans; Thrasher, Adrian; Morris, Emma; Qasim, Waseem

    2013-01-01

    Transfer of tumour antigen-specific receptors to T cells requires efficient delivery and integration of transgenes, and currently most clinical studies are using gamma retroviral or lentiviral systems. Whilst important proof-of-principle data has been generated for both chimeric antigen receptors and αβ T cell receptors, the current platforms are costly, time-consuming and relatively inflexible. Alternative, more cost-effective, Sleeping Beauty transposon-based plasmid systems could offer a pathway to accelerated clinical testing of a more diverse repertoire of recombinant high affinity T cell receptors. Nucleofection of hyperactive SB100X transposase-mediated stable transposition of an optimised murine-human chimeric T cell receptor specific for Wilm’s tumour antigen from a Sleeping Beauty transposon plasmid. Whilst transfer efficiency was lower than that mediated by lentiviral transduction, cells could be readily enriched and expanded, and mediated effective target cells lysis in vitro and in vivo. Integration sites of transposed TCR genes in primary T cells were almost randomly distributed, contrasting the predilection of lentiviral vectors for transcriptionally active sites. The results support exploitation of the Sleeping Beauty plasmid based system as a flexible and adaptable platform for accelerated, early-phase assessment of T cell receptor gene therapies. PMID:23840834

  6. Salicylate, an aspirin metabolite, specifically inhibits the current mediated by glycine receptors containing α1-subunits

    PubMed Central

    Lu, Y-G; Tang, Z-Q; Ye, Z-Y; Wang, H-T; Huang, Y-N; Zhou, K-Q; Zhang, M; Xu, T-L; Chen, L

    2009-01-01

    Background and purpose: Aspirin or its metabolite sodium salicylate is widely prescribed and has many side effects. Previous studies suggest that targeting neuronal receptors/ion channels is one of the pathways by which salicylate causes side effects in the nervous system. The present study aimed to investigate the functional action of salicylate on glycine receptors at a molecular level. Experimental approach: Whole-cell patch-clamp and site-directed mutagenesis were deployed to examine the effects of salicylate on the currents mediated by native glycine receptors in cultured neurones of rat inferior colliculus and by glycine receptors expressed in HEK293T cells. Key results: Salicylate effectively inhibited the maximal current mediated by native glycine receptors without altering the EC50 and the Hill coefficient, demonstrating a non-competitive action of salicylate. Only when applied simultaneously with glycine and extracellularly, could salicylate produce this antagonism. In HEK293T cells transfected with either α1-, α2-, α3-, α1β-, α2β- or α3β-glycine receptors, salicylate only inhibited the current mediated by those receptors that contained the α1-subunit. A single site mutation of I240V in the α1-subunit abolished inhibition by salicylate. Conclusions and implications: Salicylate is a non-competitive antagonist specifically on glycine receptors containing α1-subunits. This action critically involves the isoleucine-240 in the first transmembrane segment of the α1-subunit. Our findings may increase our understanding of the receptors involved in the side effects of salicylate on the central nervous system, such as seizures and tinnitus. PMID:19594751

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

    PubMed Central

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

    2008-01-01

    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 FcγRI, but not its associated γ chain, and was not supported by other FcγR family members (FcγRIIA, FcγRIIB and FcγRIII). 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, FcγRI-mediated enhancement of phage transduction in mammalian cells, and suggest new approaches to improve bacteriophage-mediated gene transfer. PMID:18191979

  8. 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.

  9. 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

  10. 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.

  11. Knockdown of microplitis mediator odorant receptor involved in the sensitive detection of two chemicals.

    PubMed

    Li, Ke-Ming; Ren, Li-Yan; Zhang, Yong-Jun; Wu, Kong-Ming; Guo, Yu-Yuan

    2012-03-01

    Odorant receptors are thought to play critical roles in the perception of chemosensory stimuli by insects. The primary method to address the functions of odorant receptors in insects is to use in vitro binding assays between the receptors and potential chemical stimuli. We injected MmedOrco dsRNA into the abdominal cavity of a braconid wasp, Microplitis mediator, and assayed for expression of this gene 72 h after treatment (RNAi). Quantitative real-time PCR demonstrated that the level of mRNA expression in MmedOrco dsRNA-treated M. mediator was significantly reduced (>90%) when compared with water-treated controls. Furthermore, electroantennogram (EAG) responses of M. mediator to two chemical attractants, nonanal and farnesene, were also reduced significantly (~70%) in RNAi-treated M. mediator when compared to controls. RNAi-treated M. mediator also responded by walking/flying at a lower rate to both chemicals when compared with controls in a Y-tube olfactometer bioassay, which provides direct evidence that MmedOrco plays an important role in perception of nonanal and farnesene in M. mediator.

  12. Androgen Receptor-Mediated Escape Mechanisms from Androgen Ablation Therapy

    DTIC Science & Technology

    2006-10-01

    well as mechanisms associated with prostate cancer growth and expansion, we may be able to develop therapies that prolong lives. Understanding the...and G. A. Coetzee. 2004. Androgen receptor signaling: mechanism of interleukin-6 inhibition. Cancer Res. 64:2619–2626. 19. Jia, L., and G. A. Coetzee...the prostate specific antigen locus: steroidal and non-steroidal mechanisms . Mol. Cancer Res. 1:385–392. 21. Johnstone, R. W. 2002. Histone

  13. 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

  14. 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

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

    PubMed Central

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

    1982-01-01

    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). Images PMID:6952251

  16. Taste Receptors Mediate Sinonasal Immunity and Respiratory Disease.

    PubMed

    Douglas, Jennifer E; Cohen, Noam A

    2017-02-17

    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.

  17. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons

    PubMed Central

    Corey, Elizabeth A.; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W.

    2010-01-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors (GPCRs) on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a GPCR-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kγ and cloned a gene for a PI3K with amino acid homology with PI3Kβ. The lobster olfactory PI3K co-immunoprecipitates with the G protein α and β subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kγ and β isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction. PMID:20132480

  18. 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).

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. Somatostatin receptor-mediated arachidonic acid mobilization: evidence for partial agonism of synthetic peptides

    PubMed Central

    Alderton, Forbes; Fan, Tai-Ping D; Humphrey, Patrick P A

    2001-01-01

    Somatostatin and the stable octapeptide analogues, octreotide and angiopeptin, were examined for their ability to stimulate the release of tritium from [3H]-arachidonic acid pre-loaded CHO-K1 cells expressing human recombinant sst2, sst3 or sst5 receptors. Somatostatin stimulated tritium release (pEC50) through the sst2 (7.8±0.1) and sst5 (7.3±0.2), but not the sst3 receptor. Octreotide behaved as a full (sst2 receptor) or partial agonist (sst5 receptor), whereas angiopeptin behaved as a weak partial agonist at both receptor types. Maximum responses to somatostatin through both receptor types were significantly reduced by pertussis toxin, whereas pEC50 estimates were unaffected. Inhibition of MEK1 or Src, but not PKA, PI 3-kinases or tyrosine kinases, by reportedly selective inhibitors reduced sst2-mediated responses by somatostatin, but not angiopeptin. A selective inhibitor of PKC (Ro-31-8220) reduced both somatostatin and angiopeptin responses. These data provide further evidence for partial agonist activity of synthetic peptides of somatostatin. Furthermore, the somatostatin receptor signalling mechanisms which mediate arachidonic acid mobilization appear to be multiple and complex. PMID:11159729

  5. Arsenic as an endocrine disruptor: arsenic disrupts retinoic acid receptor-and thyroid hormone receptor-mediated gene regulation and thyroid hormone-mediated amphibian tail metamorphosis.

    PubMed

    Davey, Jennifer C; Nomikos, Athena P; Wungjiranirun, Manida; Sherman, Jenna R; Ingram, Liam; Batki, Cavus; Lariviere, Jean P; Hamilton, Joshua W

    2008-02-01

    Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors. The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation. Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01-5 microM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element-luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element-luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1- 4.0 microM As. As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are critical for both normal human development and adult

  6. 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

  7. 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

  8. 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

  9. Androgen Receptor-Mediated Escape Mechanisms from Androgen Ablation Therapy

    DTIC Science & Technology

    2005-10-01

    03- catenin in lipogenesis can be assigned. In addition to being highly expressed in adipose tissue, both PPAR8 and PPARy are thought to serve...ProliferationPrlfato? a) Androgen Dependent PrCa b) Androgen Independent PrCa Figure 3 a) ER TR VDR VDR Wnt -*P-cat - *lo AR Wn Ic AR: RAR, RAR, RXR RXR* PPAR8 PPARy ...97) Tcf-4 PPARy Activation Increased PPAR7 detected in colonic cancer cells (85) Table 11. Nuclear Receptor Modulation of Wnt//J-catenin/Tcf

  10. 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.

  11. 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.

  12. 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.

  13. Estrogen-mediated neuroprotection in the cortex may require NMDA receptor activation.

    PubMed

    Connell, B J; Crosby, K M; Richard, M J P; Mayne, M B; Saleh, T M

    2007-04-25

    Several studies have suggested that a potential mechanism for estrogen-mediated neuroprotection following experimental stroke is a result of modulating glutamate-mediated excitotoxicity. Our laboratory has shown that in male rats, estrogen injection (systemic or direct intracortical injection) resulted in an immediate depolarization of cortical neurons. Therefore, the present study was designed to investigate whether the estrogen-induced depolarization of cortical neurons was required in mediating the early events associated with this neuroprotection. We tested this hypothesis by co-injecting selective antagonists of the NMDA (MK-801) or AMPA (DNQX) glutamatergic receptors with estrogen. Systemic injection of estrogen significantly attenuated the MK-801-induced decrease in infarct volume following middle cerebral artery occlusion (MCAO). Similarly, when estrogen and MK-801 were co-injected directly into the cortex, no neuroprotection was observed. However, when estrogen or MK-801 was injected centrally 10 min prior to the injection of the other drug, significant neuroprotection was observed. This led us to hypothesize that estrogen-mediated neuroprotection required an initial activation of NMDA receptors. Furthermore, our results suggest that this estrogen-mediated neuroprotection was also associated with a significant increase in m-calpain and activation of an endoplasmic reticulum (ER) specific caspase-12. Finally, the results of current clamp experiments showed that estrogen significantly depolarized cortical neurons as well as enhanced NMDA-induced depolarization. Taken together, these results suggest that estrogen pretreatment may activate NMDA receptors resulting in modification of ER-associated molecular mechanisms involved in neuroprotection following MCAO.

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. 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. © 2015 Authors; published by Portland Press Limited.

  20. Niacin mediates lipolysis in adipose tissue through its G-protein coupled receptor HM74A.

    PubMed

    Zhang, Youyan; Schmidt, Robert J; Foxworthy, Patricia; Emkey, Renee; Oler, Jennifer K; Large, Thomas H; Wang, He; Su, Eric W; Mosior, Marion K; Eacho, Patrick I; Cao, Guoqing

    2005-08-26

    A G-protein coupled receptor to niacin (nicotinic acid) was identified recently but the physiological/pharmacological role of the receptor remains poorly defined. We present our studies to demonstrate that HM74A, but not HM74, binds niacin at high affinities and effectively mediates Gi signaling events in human embryonic kidney HEK293 cells as well as in 3T3L1 adipocytes expressing HM74A. Furthermore, HM74A, but not HM74, expressed in differentiated 3T3L1 adipocytes effectively mediated inhibition of lipolysis by niacin. Our results provided direct evidence indicating that HM74A, but not HM74, was sufficient to mediate anti-lipolytic effect of niacin in adipose tissue.

  1. 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.

  2. Pathogen-Mediated Inflammatory Atherosclerosis Is Mediated in Part via Toll-Like Receptor 2-Induced Inflammatory Responses

    PubMed Central

    Hayashi, Chie; Madrigal, Andres G.; Liu, Xinyan; Ukai, Takashi; Goswami, Sulip; Gudino, Cynthia V.; Gibson, III, 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 apolipoprotien 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-γ and the pro-inflammatory cytokines IL-1β, IL-6 and tumor necrosis factor-α 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. PMID:20505314

  3. The minor population of M3-receptors mediate contraction of human detrusor muscle in vitro.

    PubMed

    Chess-Williams, R; Chapple, C R; Yamanishi, T; Yasuda, K; Sellers, D J

    2001-01-01

    1 The objective was to determine the role of muscarinic receptor subtypes in mediating contraction of the human detrusor smooth muscle in vitro. 2 Contractile responses of human detrusor muscle strips to carbachol were obtained in the absence and presence of a range of muscarinic antagonists (pirenzepine, methoctramine, 4-diphenylacetoxy-N-methyl piperidine methiodide (4-DAMP), tropicamide, oxybutynin and tolterodine). Affinity estimates (pKB values) were calculated for the antagonists and correlated with values at the cloned muscarinic receptor subtypes quoted in the literature. 3 Pirenzepine, methoctramine and tropicamide drugs that have high affinities at M1, M2 and M4-receptors, respectively, all had low affinities on the human detrusor (pKB values of 6.8, 6.9 and 6.5, respectively), whilst the M3-selective antagonist 4-DAMP had a high affinity (9.5). Schild plots for all four antagonists had slopes of unity indicating an action at a single receptor. Oxybutynin and tolterodine also acted as competitive antagonists with affinity estimates of 7.6 and 8.1, respectively. 4 When the antagonist affinities obtained on the bladder were plotted against the values published for these antagonists at the cloned muscarinic receptor subtypes, the best correlations were obtained for the m3- and m5-muscarinic receptor subtypes. 5 These data suggest that direct contractile responses of the human detrusor muscle to muscarinic receptor stimulation in vitro are mediated solely via the M3-muscarinic receptor subtype with no contribution from the major M2-receptor population.

  4. The unique cytoplasmic domain of human FcγRIIIA regulates receptor mediated function

    PubMed Central

    Li, Xiaoli; Baskin, Julie G.; Mangan, Erin K.; Su, Kaihong; Gibson, Andrew W.; Ji, Chuanyi; Edberg, Jeffrey C.; Kimberly, Robert P.

    2012-01-01

    Ligand specificity characterizes receptors for antibody and many other immune receptors, but the common use of the FcR-γ-chain as their signaling subunit challenges the concept that these receptors are functionally distinct. We hypothesized that elements for specificity might be determined by the unique cytoplasmic domain (CY) sequences of the ligand-binding α-chains of γ-chain associated receptors. Among Fcγ receptors (FcRs), a protein kinase C (PKC) phosphorylation consensus motif, [RSSTR], identified within the FcγRIIIa (CD16A) CY by in silico analysis, is specifically phosphorylated by PKCs, unlike other FcRs. Phosphorylated CD16A mediates a more robust calcium flux, tyrosine phosphorylation of Syk and pro-inflammatory cytokine production while non-phosphorylatable CD16A is more effective at activation of the Gab2/PI3K pathway, leading to enhanced degranulation. S100A4, a specific protein binding partner for CD16A-CY newly identified by yeast two-hybrid analysis, inhibits phosphorylation of CD16A-CY by PKC in vitro, and reduction of S100A4 levels in vivo enhances receptor phosphorylation upon cross-linking. Taken together, PKC-mediated phosphorylation of CD16A modulates distinct signaling pathways engaged by the receptor. Calcium activated binding of S100A4 to CD16A, promoted by the initial calcium flux, attenuates the phosphorylation of CY, and acting as a molecular switch, may both serve as a negative feedback on cytokine production pathways during sustained receptor engagement and favor a shift to degranulation, consistent with the importance of granule release following conjugate formation between CD16A+ effector cells and target cells. This switch mechanism points to new therapeutic targets and provides a frame for understanding novel receptor polymorphisms. PMID:23024279

  5. 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.

  6. NPY/Y1 receptor-mediated vasoconstrictory and proliferative effects in pulmonary hypertension

    PubMed Central

    Crnkovic, S; Egemnazarov, B; Jain, P; Seay, U; Gattinger, N; Marsh, L M; Bálint, Z; Kovacs, G; Ghanim, B; Klepetko, W; Schermuly, R T; Weissmann, N; Olschewski, A; Kwapiszewska, G

    2014-01-01

    BACKGROUND AND PURPOSE Pulmonary arteries (PAs) are innervated, but little is known about the role of neuronal axis in pulmonary hypertension (PH). Here, we have examined the role of the neuropeptide Y (NPY) and its Y1 receptor in PH pathogenesis. EXPERIMENTAL APPROACH NPY was localized by immunofluorescence. Expression of NPY and Y1 receptor were determined by quantitative PCR. Cellular response to NPY stimulation was assessed by Western blotting, thymidine incorporation and calcium imaging. Wire myography and isolated perfused mouse lung were applied to study pulmonary vasoactive effects of NPY. Selective receptor antagonists were used to assess the contribution of receptor subtypes in mediating NPY effects. KEY RESULTS Samples from PH patients showed increased NPYergic innervation within the PA wall and higher Y1 receptor expression, compared with donors. However, NPY levels were unchanged in both PA and serum. In the chronic hypoxic mouse model, Y1 receptor were up-regulated, while expression of both NPY and Y1 receptor was increased in the lungs of monocrotaline and SU5416-hypoxia rats. On a functional level, NPY acutely increased intracellular calcium levels and enhanced vasoconstriction of lung vessels preconstricted with adrenaline. Furthermore, NPY stimulated proliferation of human pulmonary arterial smooth muscle cells and activated p38 and PKD pathways. Correspondingly, higher phosphorylation of PKD was observed in remodelled vessels from PH patients. The selective Y1 receptor antagonist, BIBO 3304, concentration-dependently inhibited vasoconstrictive and proliferative effects of NPY. CONCLUSIONS AND IMPLICATIONS NPY and Y1 receptor are possible mediators of both vasoconstriction and pulmonary vascular remodelling in PH. PMID:24779394

  7. 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

  8. Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

    PubMed Central

    Ruiz, A; Matute, C; Alberdi, E

    2010-01-01

    Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP3Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP3Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. PMID:21364659

  9. 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'.

  10. CB1 cannabinoid receptor-mediated aggressive behavior.

    PubMed

    Rodriguez-Arias, Marta; Navarrete, Francisco; Daza-Losada, Manuel; Navarro, Daniela; Aguilar, María A; Berbel, Pere; Miñarro, José; Manzanares, Jorge

    2013-12-01

    This study examined the role of cannabinoid CB1 receptors (CB1r) in aggressive behavior. Social encounters took place in grouped and isolated mice lacking CB1r (CB1KO) and in wild-type (WT) littermates. Cognitive impulsivity was evaluated in the delayed reinforcement task (DRT). Gene expression analyses of monoaminooxidase-A (MAO-A), catechol-o-methyl-transferase (COMT), 5-hydroxytriptamine transporter (5-HTT) and 5-HT1B serotonergic receptor (5HT1Br) in the median and dorsal raphe nuclei (MnR and DR, respectively) and in the amygdala (AMY) were performed by real time-PCR. Double immunohistochemistry studies evaluated COMT and CB1r co-localization in the raphe nuclei and in the cortical (ACo), basomedian (BMA) and basolateral (BLA) amygdaloid nuclei. The behavioral effects of the CB1r agonist ACEA (1 and 2 mg/kg) on aggression were also evaluated in isolated OF1 mice. CB1KO mice housed in groups showed higher levels of offensive aggression. Isolation increased aggressive behavior only in WT. In grouped CB1KO mice COMT gene expression was significantly higher in the MnR and DR, while MAO-A gene expression was lower in the MnR. Gene expression of 5HT1Br, COMT and MAO-A was higher in the amygdala of CB1KO mice. CB1r double-immunohistochemistry revealed cytoplasmic-labeled COMT-ir cells in the raphe nuclei and in the ACo, BMA and BLA. CB1r immunolabeling was observed only in ACo, BMA and BLA, where it was localized in axons and buttons. The density of labeled processes increased in BLA. Acute administration of the CB1 agonist ACEA (2 mg/kg) significantly decreased the aggression levels of OF1 mice. These results suggest that CB1r plays an important role in social interaction and aggressive behavior.

  11. 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).

  12. 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.

  13. 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.

  14. 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.

  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. 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

  17. TGF-beta receptor mediated telomerase inhibition, telomere shortening and breast cancer cell senescence.

    PubMed

    Cassar, Lucy; Nicholls, Craig; Pinto, Alex R; Chen, Ruping; Wang, Lihui; Li, He; Liu, Jun-Ping

    2017-01-01

    Human telomerase reverse transcriptase (hTERT) plays a central role in telomere lengthening for continuous cell proliferation, but it remains unclear how extracellular cues regulate telomerase lengthening of telomeres. Here we report that the cytokine bone morphogenetic protein-7 (BMP7) induces the hTERT gene repression in a BMPRII receptor- and Smad3-dependent manner in human breast cancer cells. Chonic exposure of human breast cancer cells to BMP7 results in short telomeres, cell senescence and apoptosis. Mutation of the BMPRII receptor, but not TGFbRII, ACTRIIA or ACTRIIB receptor, inhibits BMP7-induced repression of the hTERT gene promoter activity, leading to increased telomerase activity, lengthened telomeres and continued cell proliferation. Expression of hTERT prevents BMP7-induced breast cancer cell senescence and apoptosis. Thus, our data suggest that BMP7 induces breast cancer cell aging by a mechanism involving BMPRII receptor- and Smad3-mediated repression of the hTERT gene.

  18. Heterogeneity of prejunctional NPY receptor-mediated inhibition of cardiac neurotransmission

    PubMed Central

    Serone, Adrian P; Wright, Christine E; Angus, James A

    1999-01-01

    Neuropeptide Y (NPY) has been proposed as the candidate inhibitory peptide mediating interactions between sympathetic and vagal neurotransmission in several species, including man. Here, we have defined the NPY receptors involved in modulation of cardiac autonomic neurotransmission using receptor-selective agonists and antagonists in the rabbit and guinea-pig isolated right atria.In isolated atrial preparations, sympathetically-mediated tachycardia (ST; with atropine 1 μM) or vagally-mediated bradycardia (VB; with propranolol 0.1–1 μM) in response to electrical field stimulation (EFS, 1–4 pulses) were tested 0–30 min after incubation with single concentrations of vehicle, NPY (0.01–10 μM), the Y2 receptor agonist N-Acetyl-[Leu28,31]NPY(24–36) (termed N-A[L]NPY(24–36)) or the Y1 receptor agonist [Leu31,Pro34]NPY (LP). The effect of NPY on the concentration-chronotropic response curves to isoprenaline and bethanechol were also assessed.Guinea-pig atria: NPY and N-A[L]NPY(24–36) caused concentration-dependent inhibition of VB and ST to EFS. Both peptides caused maximal inhibition of VB and ST within 10 min incubation and this remained constant. LP caused a concentration-dependent, transient inhibition of ST which was antagonized by the Y1-receptor antagonist GR231118 (0.3 μM), with apparent competitive kinetics. Rabbit atria: NPY (1 or 10 μM) had no effect on VB at any time point, but both NPY and LP caused a transient (∼10 min) inhibition of sympathetic tachycardia. This inhibition could be prevented by 0.3 μM GR231118. N-A[L]NPY(24–36) had no effect on ST. NPY had no effect on the response to β-adrenoceptor stimulation by isoprenaline nor muscarinic-receptor stimulation by bethanechol in either species.Thus, in the guinea-pig, NPY causes a stable inhibition of both VB and ST to EFS via Y2 receptors and transient inhibition of ST via Y1 receptors. In contrast in the rabbit, NPY has no effect on the cardiac vagus and

  19. 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

  20. Stem cells are resistant to TRAIL receptor-mediated apoptosis.

    PubMed

    Szegezdi, Eva; O'Reilly, Aoife; Davy, Yeung; Vawda, Reaz; Taylor, Deanna L; Murphy, Mary; Samali, Afshin; Mehmet, Huseyin

    2009-01-01

    New therapeutic approaches aim to eradicate tumours by expression of tumouricidal proteins in the tumour stroma. One such anti-neoplastic protein is tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) because it induces apoptosis in cancerous cells, but not in non-transformed cells. Stem cells can migrate to, survive and proliferate in tumours. We examined the suitability of bone marrow-derived adult mesenchymal stem cells (bmMSC), foetal-MSC and umbilical cord matrix stem cells (Wharton's Jelly MSCs) as TRAIL-delivery vehicles. Although all MSC types expressed DR4 and/or DR5, none of them were sensitive to TRAIL-induced apoptosis. Selective activation of DR4 or DR5 with agonistic antibodies or DR5-selective TRAIL-mutant (D269H/E195R) revealed that the TRAIL receptors are inactive in MSCs. In fMSC DR5 was not fully inactivated, its activity however was minimal in comparison to the colon carcinoma cell, Colo205. The intracellular components of the TRAIL-apoptotic pathway, such as pro-caspase-8 and -9 were also expressed at very low; almost undetectable levels in all three MSC types. In conclusion, the MSC species examined are resistant to TRAIL and thus can be suitable tools for TRAIL delivery to tumours.

  1. Stem cells are resistant to TRAIL receptor-mediated apoptosis

    PubMed Central

    Szegezdi, Eva; O’Reilly, Aoife; Davy, Yeung; Vawda, Reaz; Taylor, Deanna L; Murphy, Mary; Samali, Afshin; Mehmet, Huseyin

    2009-01-01

    New therapeutic approaches aim to eradicate tumours by expression of tumouricidal proteins in the tumour stroma. One such anti-neoplastic protein is tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) because it induces apoptosis in cancerous cells, but not in non-transformed cells. Stem cells can migrate to, survive and proliferate in tumours. We examined the suitability of bone marrow-derived adult mesenchymal stem cells (bmMSC), foetal-MSC and umbilical cord matrix stem cells (Wharton’s Jelly MSCs) as TRAIL-delivery vehicles. Although all MSC types expressed DR4 and/or DR5, none of them were sensitive to TRAIL-induced apoptosis. Selective activation of DR4 or DR5 with agonistic antibodies or DR5-selective TRAIL-mutant (D269H/E195R) revealed that the TRAIL receptors are inactive in MSCs. In fMSC DR5 was not fully inactivated, its activity however was minimal in comparison to the colon carcinoma cell, Colo205. The intracellular components of the TRAIL-apoptotic pathway, such as pro-caspase-8 and -9 were also expressed at very low; almost undetectable levels in all three MSC types. In conclusion, the MSC species examined are resistant to TRAIL and thus can be suitable tools for TRAIL delivery to tumours. PMID:19604313

  2. 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

  3. Androgen receptor upregulation mediates radioresistance after ionizing radiation

    PubMed Central

    Spratt, Daniel E.; Evans, Michael J.; Davis, Brian J.; Doran, Michael G.; Lee, Man Xia; Shah, Neel; Wongvipat, John; Carnazza, Kathryn E.; Klee, George G.; Polkinghorn, William; Tindall, Donald J.; Lewis, Jason S.; Sawyers, Charles L.

    2015-01-01

    Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy (RT) in prostate cancer. ADT sensitizes prostate cancer to RT-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons adjuvant ADT provides further survival benefits. Here we show that androgen receptor (AR) expression and activity are durably upregulated following RT in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20 percent of patients after RT. Furthermore, these men were three fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that RT can upregulate AR signaling post-therapy to an extent that negatively impacts disease progression and/or survival. PMID:26432404

  4. 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.

  5. 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.

  6. Regulation of L-Selectin–mediated Rolling through Receptor Dimerization

    PubMed Central

    Li, Xuan; Steeber, Douglas A.; Tang, Mimi L.K.; Farrar, Michael A.; Perlmutter, Roger M.; Tedder, Thomas F.

    1998-01-01

    L-selectin binding activity for its ligand expressed by vascular endothelium is rapidly and transiently increased after leukocyte activation. To identify mechanisms for upregulation and assess how this influences leukocyte/endothelial cell interactions, cell-surface dimers of L-selectin were induced using the coumermycin–GyrB dimerization strategy for cross-linking L-selectin cytoplasmic domains in L-selectin cDNA-transfected lymphoblastoid cells. Coumermycin- induced L-selectin dimerization resulted in an approximately fourfold increase in binding of phosphomanan monoester core complex (PPME), a natural mimic of an L-selectin ligand, comparable to that observed after leukocyte activation. Moreover, L-selectin dimerization significantly increased (by ∼700%) the number of lymphocytes rolling on vascular endothelium under a broad range of physiological shear stresses, and significantly slowed their rolling velocities. Therefore, L-selectin dimerization may explain the rapid increase in ligand binding activity that occurs after leukocyte activation and may directly influence leukocyte migration to peripheral lymphoid tissues or to sites of inflammation. Inducible oligomerization may also be a common mechanism for rapidly upregulating the adhesive or ligand-binding function of other cell-surface receptors. PMID:9763619

  7. TRPC Channels Mediate a Muscarinic Receptor-Induced Afterdepolarization in Cerebral Cortex

    PubMed Central

    Yan, Hai-Dun; Villalobos, Claudio; Andrade, Rodrigo

    2009-01-01

    Activation of muscarinic cholinergic receptors on pyramidal cells of the cerebral cortex induces the appearance of a slow afterdepolarization that can sustain autonomous spiking after a brief excitatory stimulus. Accordingly, this phenomenon has been hypothesized to allow for the transient storage of memory traces in neuronal networks. Here we investigated the molecular basis underlying the muscarinic receptor-induced afterdepolarization using molecular biological and electrophysiological strategies. We find that the ability of muscarinic receptors to induce the inward aftercurrent underlying the slow afterdepolarization is inhibited by expression of a Gαq-11 dominant negative and is also markedly reduced in a phospholipase C β1 (PLCβ1) knock-out mouse. Furthermore, we show, using a genetically encoded biosensor, that activation of muscarinic receptor induces the breakdown of phosphatidylinositol 4,5-bisphosphate in pyramidal cells. These results indicate that the Gαq-11/PLCβ1 cascade plays a key role in the ability of muscarinic receptors to signal the inward aftercurrent. We have shown previously that the muscarinic afterdepolarization is mediated by a calcium-activated nonselective cation current, suggesting the possible involvement of TRPC channels. We find that expression of a TRPC dominant negative inhibits, and overexpression of wild-type TRPC5 or TRPC6 enhances, the amplitude of the muscarinic receptor-induced inward aftercurrent. Furthermore, we find that coexpression of TRPC5 and T-type calcium channels is sufficient to reconstitute a muscarinic receptor-activated inward aftercurrent in human embryonic kidney HEK-293 cells. These results indicate that TRPC channels mediate the muscarinic receptor-induced slow afterdepolarization seen in pyramidal cells of the cerebral cortex and suggest a possible role for TRPC channels in mnemonic processes. PMID:19675237

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. Size of single-wall carbon nanotube affects the folate receptor-mediated cancer cell targeting.

    PubMed

    Charbgoo, Fahimeh; Nikkhah, Maryam; Behmanesh, Mehrdad

    2017-08-30

    Advances in nanobiotechnology and targeting strategy could improve the delivery of therapeutic molecules into cancer cells, leading to improved treatment efficiency with minimal side effects on normal cells. To design an efficient nanocarrier, consideration of parameters that facilitate direct drug delivery into the target cells is important. We studied the effect of single-wall carbon nanotubes (SWNTs) size on their cell internalization level via the folate receptor-mediated pathway through folic acid targeting. Folate-SWNTs were covalently synthesized and characterized. Folate-SWNTs ≤ 450 nm had lower cell internalization level than folate-SWNTs >450 nm with a P value of ≤0.01. This indicated that using folate-SWNT with an average length of ≤450 nm was not suitable for receptor-mediated cancer cell targeting. Receptor-mediated uptake of folate-SWNTs is dependent on the nanoparticle length. However, sub-450 nm SWNTs could serve as a vehicle to transfer nucleic acids into the cells due to direct cell penetrance based on their needle-like structure. We find that SWNTs larger than 450 nm were suitable to target the cells through receptors. These results might provide a promising approach for designing more effective targeted delivery systems based on SWNTs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  13. 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.

  14. Extracellular acidosis impairs P2Y receptor-mediated Ca(2+) signalling and migration of microglia.

    PubMed

    Langfelder, Antonia; Okonji, Emeka; Deca, Diana; Wei, Wei-Chun; Glitsch, Maike D

    2015-04-01

    Microglia are the resident macrophage and immune cell of the brain and are critically involved in combating disease and assaults on the brain. Virtually all brain pathologies are accompanied by acidosis of the interstitial fluid, meaning that microglia are exposed to an acidic environment. However, little is known about how extracellular acidosis impacts on microglial function. The activity of microglia is tightly controlled by 'on' and 'off' signals, the presence or absence of which results in generation of distinct phenotypes in microglia. Activation of G protein coupled purinergic (P2Y) receptors triggers a number of distinct behaviours in microglia, including activation, migration, and phagocytosis. Using pharmacological tools and fluorescence imaging of the murine cerebellar microglia cell line C8B4, we show that extracellular acidosis interferes with P2Y receptor-mediated Ca(2+) signalling in these cells. Distinct P2Y receptors give rise to signature intracellular Ca(2+) signals, and Ca(2+) release from stores and Ca(2+) influx are differentially affected by acidotic conditions: Ca(2+) release is virtually unaffected, whereas Ca(2+) influx, mediated at least in part by store-operated Ca(2+) channels, is profoundly inhibited. Furthermore, P2Y1 and P2Y6-mediated stimulation of migration is inhibited under conditions of extracellular acidosis, whereas basal migration independent of P2Y receptor activation is not. Taken together, our results demonstrate that an acidic microenvironment impacts on P2Y receptor-mediated Ca(2+) signalling, thereby influencing microglial responses and responsiveness to extracellular signals. This may result in altered behaviour of microglia under pathological conditions compared with microglial responses in healthy tissue. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. 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. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. 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

  17. 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.

  18. Kainate Receptors Mediate Signaling in Both Transient and Sustained OFF Bipolar Cell Pathways in Mouse Retina

    PubMed Central

    Looger, Loren L.; Tomita, Susumu

    2014-01-01

    A fundamental question in sensory neuroscience is how parallel processing is implemented at the level of molecular and circuit mechanisms. In the retina, it has been proposed that distinct OFF cone bipolar cell types generate fast/transient and slow/sustained pathways by the differential expression of AMPA- and kainate-type glutamate receptors, respectively. However, the functional significance of these receptors in the intact circuit during light stimulation remains unclear. Here, we measured glutamate release from mouse bipolar cells by two-photon imaging of a glutamate sensor (iGluSnFR) expressed on postsynaptic amacrine and ganglion cell dendrites. In both transient and sustained OFF layers, cone-driven glutamate release from bipolar cells was blocked by antagonists to kainate receptors but not AMPA receptors. Electrophysiological recordings from bipolar and ganglion cells confirmed the essential role of kainate receptors for signaling in both transient and sustained OFF pathways. Kainate receptors mediated responses to contrast modulation up to 20 Hz. Light-evoked responses in all mouse OFF bipolar pathways depend on kainate, not AMPA, receptors. PMID:24790183

  19. 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.

  20. 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

  1. Molecular determinants of glycine receptor αβ subunit sensitivities to Zn2+-mediated inhibition

    PubMed Central

    Miller, Paul S; Beato, Marco; Harvey, Robert J; Smart, Trevor G

    2005-01-01

    Glycine receptors exhibit a biphasic sensitivity profile in response to Zn2+-mediated modulation, with low Zn2+ concentrations potentiating (< 10 μm), and higher Zn2+ concentrations inhibiting submaximal responses to glycine. Here, a substantial 30-fold increase in sensitivity to Zn2+-mediated inhibition was apparent for the homomeric glycine receptor (GlyR) α1 subunit compared to either GlyR α2 or α3 subtypes. Swapping the divergent histidine (H107) residue in GlyR α1, which together with the conserved H109 forms part of an intersubunit Zn2+-binding site, for the equivalent asparagine residue present in GlyR α2 and α3, reversed this phenotype. Co-expression of heteromeric GlyR α1 or α2 with the ancillary β subunit yielded receptors that maintained their distinctive sensitivities to Zn2+ inhibition. However, GlyR α2β heteromers were consistently 2-fold more sensitive to inhibition compared to the GlyR α2 homomer. Comparative studies to elucidate the specific residue in the β subunit responsible for this differential sensitivity revealed instead threonine 133 in the α1 subunit as a new vital component for Zn2+-mediated inhibition. Further studies on heteromeric receptors demonstrated that a mutated β subunit could indeed affect Zn2+-mediated inhibition but only from one side of the intersubunit Zn2+-binding site, equivalent to the GlyR α1 H107 face. This strongly suggests that the α subunit is responsible for Zn2+-mediated inhibition and that this is effectively transduced, asymmetrically, from the side of the Zn2+-binding site where H109 and T133 are located. PMID:15905212

  2. Beta2-adrenergic receptor signaling mediates corneal epithelial wound repair.

    PubMed

    Ghoghawala, Shahed Y; Mannis, Mark J; Pullar, Christine E; Rosenblatt, Mark I; Isseroff, R Rivkah

    2008-05-01

    Beta-adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting With the use of an integrated pharmacologic and genetic approach, the authors investigated how the beta-AR impacts corneal epithelial healing. Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in beta2-AR(+/+) and beta2-AR(-/-) mice. Signaling pathways were evaluated by immunoblotting. results. The beta-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the beta-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P < 0.05). The same treatment protocols had no effect on AMCE cells derived from beta2-AR(-/-) mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In beta2-AR(+/+) animals, the beta-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas beta-AR antagonist (timolol) treatment increased the rate of healing by 16% (P < 0.05) compared with saline-treated controls. In contrast, in the beta2-AR(-/-) mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact beta2-AR for the modulation of corneal epithelial wound repair.

  3. 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

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

    PubMed Central

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

    2013-01-01

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

  5. Chronic psychoemotional stress impairs cannabinoid-receptor-mediated control of GABA transmission in the striatum.

    PubMed

    Rossi, Silvia; De Chiara, Valentina; Musella, Alessandra; Kusayanagi, Hajime; Mataluni, Giorgia; Bernardi, Giorgio; Usiello, Alessandro; Centonze, Diego

    2008-07-16

    Exposure to stressful events has a myriad of consequences in animals and in humans, and triggers synaptic adaptations in many brain areas. Stress might also alter cannabinoid-receptor-mediated transmission in the brain, but no physiological study has addressed this issue so far. In the present study, we found that social defeat stress, induced in mice by exposure to aggression, altered cannabinoid CB(1)-receptor-mediated control of synaptic transmission in the striatum. In fact, the presynaptic inhibition of GABAergic IPSCs induced by the cannabinoid CB(1) receptor agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol] was reduced after a single stressful episode and fully abolished after 3 and 7 d of stress exposure. Repeated psychoemotional stress also impaired the sensitivity of GABA synapses to endocannabinoids mobilized by group I metabotropic glutamate receptor stimulation, whereas the cannabinoid CB(1)-mediated control of glutamate transmission was unaffected by repeated exposure to an aggressor. Corticosteroids released in response to the activation of the hypothalamic-pituitary-adrenal axis played a major role in the synaptic defects observed in stressed animals, because these alterations were fully prevented by pharmacological blockade of glucocorticoid receptors and were mimicked by corticosterone injections. The recovery of stress-induced synaptic defects was favored when stressed mice were given access to a running wheel or to sucrose consumption, which function as potent natural rewards. A similar rescuing effect was obtained by a single injection of cocaine, a psychostimulant with strong rewarding properties. Targeting cannabinoid CB(1) receptors or endocannabinoid metabolism might be a valuable option to treat stress-associated neuropsychiatric conditions.

  6. 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

  7. 5-HT1-like receptor-mediated contraction in the human internal mammary artery.

    PubMed

    Yildiz, O; Ciçek, S; Ay, I; Tatar, H; Tuncer, M

    1996-07-01

    We wished to characterize the 5-hydroxytryptamine (5-HT) receptors mediating vasoconstriction in the human internal mammary artery (IMA). Segments of the IMA obtained from patients undergoing coronary by-pass surgery were suspended in an organ bath and exposed to 5-HT and sumatriptan (SUM), a 5-HT1-like receptor agonist, in the presence and absence of potassium chloride (KCl) and angiotensin II. 5-HT induced concentration-dependent contractions in all quiescent and pre-contracted preparations. SUM induced small contractions in 70% of quiescent IMA rings, whereas it elicited marked and concentration-dependent contractions in all of the preparations given a moderate tone by a threshold concentration of KCl and angiotensin II. The efficacy of SUM was higher in precontracted arteries. Concentration-effect curves (CEC) of 5-HT and SUM were not affected by the 5-HT3-receptor antagonist tropisetron (1 microM). The nonselective antagonist, methiothepin (30 nM), shifted the CEC of SUM to the right. 5-HT2A-receptor antagonist, ketanserin (1 microM) inhibited responses to 5-HT, whereas it affected only the responses to the smaller concentrations of SUM. When methiothepin (30 nM) was applied in the presence of ketanserin (1 microM), a further inhibition in the responses to 5-HT was observed. These results suggest that 5-HT1-like receptors mediate the contractile action of SUM and contribute to that of 5-HT in IMA.

  8. 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. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. 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.

  10. 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.

  11. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  12. Pyrimidine-based compounds modulate CXCR2-mediated signaling and receptor turnover.

    PubMed

    Ha, Helen; Neamati, Nouri

    2014-07-07

    Chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. Because of its role in inflammation, it has been implicated in a number of immune-mediated inflammatory diseases such as psoriasis, arthritis, COPD, cystic fibrosis, asthma, and various types of cancer. CXCR2 and its ligands are up-regulated in cancer cells as well as the tumor microenvironment, promoting tumor growth, angiogenesis, and invasiveness. Although pharmaceutical companies have pursued the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents within the last decades, there are currently no clinically approved CXCR2 inhibitors. Using a high-throughput, cell-based assay specific for CXCR2, we screened an in-house library of structurally diverse compounds and identified a class of pyrimidine-based compounds that alter CXCR2-mediated second messenger signaling. Our lead compound, CX797, inhibited IL8-mediated cAMP signaling and receptor degradation while specifically up-regulating IL8-mediated β-arrestin-2 recruitment. CX797 also inhibited IL8-mediated cell migration. Mechanistic comparison of CX797 and a previously reported CXCR2 inhibitor, SB265610, show these two classes of compounds have a distinct mechanism of action on CXCR2.

  13. 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

  14. 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.

  15. Opposite function of dopamine D1 and NMDA receptors in striatal cannabinoid-mediated signaling

    PubMed Central

    Daigle, Tanya L.; Wetsel, William C.; Caron, Marc G.

    2011-01-01

    It is well established that the cannabinoid and dopamine systems interact at various levels to regulate basal ganglia function. While it is well known that acute administration of cannabinoids to mice can modify dopamine-dependent behaviors, an understanding of the intraneuronal signaling pathways employed by these agents in the striatum is not well understood. Here we use knockout (KO) mouse models to examine the regulation of striatal ERK1/2 signaling by behaviorally relevant doses of cannabinoids. This cellular pathway has been implicated as a central mediator of drug reward and synaptic plasticity. In C57BL/6J mice, acute administration of cannabinoid agonists, HU-210 and Δ9-THC, promotes a dose- and time-dependent decrease in the phosphorylation of ERK1/2 in dorsal striatum. Co-administration of the CB1 cannabinoid receptor (CB1R) antagonist AM251 with HU-210 prevents ERK1/2 inactivation, indicating a requirement for activation of this receptor. In dopamine D1 receptor (D1R) KO animals treated with HU-210, the magnitude of the HU-210-dependent decrease in striatal ERK1/2 signaling is greater than in wild-type controls. In contrast, the HU-210 administration to NMDA receptor knockdown mice (NR1-Kd) was ineffective at promoting striatal ERK1/2 inactivation. Genetic deletion of other potential ERK1/2 mediators, the dopamine D2 receptors (D2R)s or βarrestin-1 or -2, did not affect HU-210-induced modulation of ERK1/2 signaling in the striatum. These results support the hypothesis that dopamine D1 receptors and NMDA receptors act in an opposite manner to regulate striatal CB1R signal transduction. PMID:22034973

  16. 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

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

    PubMed

    Okada, Y; Maeno, E; Shimizu, T; Dezaki, K; Wang, J; Morishima, S

    2001-04-01

    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 TNF receptor and Fas, induces AVD and thereafter biochemical apoptotic events in human lymphoid (U937), human epithelial (HeLa), mouse neuroblastoma x 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 abolished by

  18. 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.

  19. Glucocorticoid receptor mediated the propofol self-administration by dopamine D1 receptor in nucleus accumbens.

    PubMed

    Wu, Binbin; Liang, Yuyuan; Dong, Zhanglei; Chen, Zhichuan; Zhang, Gaolong; Lin, Wenxuan; Wang, Sicong; Wang, Benfu; Ge, Ren-Shan; Lian, Qingquan

    2016-07-22

    Propofol, a widely used anesthetic, can cause addictive behaviors in both human and experimental animals. In the present study, we examined the involvement of glucocorticoid receptor (GR) signaling in the molecular process by which propofol may cause addiction. The propofol self-administration model was established by a fixed ratio 1 (FR1) schedule of reinforced dosing over successive 14days in rats. On day 15, the rats were treated with dexamethasone, a GR agonist (10-100μg/kg), or RU486, a GR antagonist (10-100μg/kg) at 1h prior to the last training. The animal behaviors were recorded automatically by the computer. The expression of dopamine D1 receptor in the nucleus accumbens (NAc) was examined by Western blot and the concentrations of plasma corticosterone were measured by enzyme-linked immunosorbent assay (ELISA). To further examine the specificity of GR in the process, mineralocorticoid receptor (MR) antagonist, spironolactone, and dexamethasone plus MR agonist, aldosterone, were also tested. Administration of dexamethasone (100μg/kg) or RU486 (⩾10mg/kg) significantly attenuated the rate of propofol maintained active nose-poke responses and infusions, which were accompanied by reductions in both plasma corticosterone level and the expression of D1 receptor in the NAc. Neither spironolactone alone nor dexamethasone combined with aldosterone affected the propofol-maintaining self-administrative behavior, indicating GR, but not MR, modulates the propofol reward in rats. In addition, neither the food-maintaining sucrose responses under FR1 schedule nor the locomotor activity was affected by any doses of dexamethasone or RU486 tested. These findings provide evidence that GR signaling may play an important role in propofol reward. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  20. 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.

  1. 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.

  2. Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor.

    PubMed Central

    Eppstein, D A; Marsh, Y V; van der Pas, M; Felgner, P L; Schreiber, A B

    1985-01-01

    Recombinant murine gamma interferon (rMuIFN-gamma) was found to bind reversibly to a specific high-affinity surface receptor on L929 cells; neither murine alpha or beta nor human gamma IFN competed for receptor binding. Encapsulation of the rMuIFN-gamma in either negatively or positively charged liposomes reduced its immediate ability to bind to this surface receptor. Disruption of liposome integrity with detergent resulted in full ability of the rMuIFN-gamma to bind to the membrane receptor. Incubation of the liposomal IFN in serum-containing medium resulted in significant leakage so that the IFN was able to bind to its surface receptor. Assessment of the biological activity of the rMuIFN-gamma preparations revealed that full antiviral activity was observed in vitro with the liposomal IFN preparations without their prior disruption by detergent. The antiviral activity observed with either free or liposomal IFN was neutralized completely by antibodies against rMuIFN-gamma. Both free and liposomal rMuIFN-gamma, in conjunction with bacterial lipopolysaccharide, were also able to activate murine peritoneal macrophages to the tumoricidal state. Again, this activity of both free and liposomal IFN could be neutralized completely by antibody. These results indicate that although rMuIFN-gamma can be effectively incorporated into liposomes, it must ultimately leak out of the liposome in order to mediate its biological effects; these effects are triggered after the IFN binds to its cell surface receptors. PMID:3159018

  3. 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

  4. 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

  5. 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.

  6. 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

  7. IL-1 receptors mediate persistent, but not acute, airway hyperreactivity to ozone in guinea pigs.

    PubMed

    Verhein, Kirsten C; Jacoby, David B; Fryer, Allison D

    2008-12-01

    Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M(2) muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1beta in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1beta mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure.

  8. IL-1 Receptors Mediate Persistent, but Not Acute, Airway Hyperreactivity to Ozone in Guinea Pigs

    PubMed Central

    Verhein, Kirsten C.; Jacoby, David B.; Fryer, Allison D.

    2008-01-01

    Ozone exposure in the lab and environment causes airway hyperreactivity lasting at least 3 days in humans and animals. In guinea pigs 1 day after ozone exposure, airway hyperreactivity is mediated by eosinophils that block neuronal M2 muscarinic receptor function, thus increasing acetylcholine release from airway parasympathetic nerves. However, mechanisms of ozone-induced airway hyperreactivity change over time, so that depleting eosinophils 3 days after ozone makes airway hyperreactivity worse rather than better. Ozone exposure increases IL-1β in bone marrow, which may contribute to acute and chronic airway hyperreactivity. To test whether IL-1β mediates ozone-induced airway hyperreactivity 1 and 3 days after ozone exposure, guinea pigs were pretreated with an IL-1 receptor antagonist (anakinra, 30 mg/kg, intraperitoneally) 30 minutes before exposure to filtered air or to ozone (2 ppm, 4 h). One or three days after exposure, airway reactivity was measured in anesthetized guinea pigs. The IL-1 receptor antagonist prevented ozone-induced airway hyperreactivity 3 days, but not 1 day, after ozone exposure. Ozone-induced airway hyperreactivity was vagally mediated, since bronchoconstriction induced by intravenous acetylcholine was not changed by ozone. The IL-1 receptor antagonist selectively prevented ozone-induced reduction of eosinophils around nerves and prevented ozone-induced deposition of extracellular eosinophil major basic protein in airways. These data demonstrate that IL-1 mediates ozone-induced airway hyperreactivity at 3 days, but not 1 day, after ozone exposure. Furthermore, preventing hyperreactivity was accompanied by decreased eosinophil major basic protein deposition within the lung, suggesting that IL-1 affects eosinophil activation 3 days after ozone exposure. PMID:18617681

  9. 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

  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. Posttranslational Modification of HOIP Blocks Toll-Like Receptor 4-Mediated Linear-Ubiquitin-Chain Formation

    PubMed Central

    Bowman, James; Rodgers, Mary A.; Shi, Mude; Amatya, Rina; Hostager, Bruce; Iwai, Kazuhiro; Gao, Shou-Jiang

    2015-01-01

    ABSTRACT Linear ubiquitination is an atypical posttranslational modification catalyzed by the linear-ubiquitin-chain assembly complex (LUBAC), containing HOIP, HOIL-1L, and Sharpin. LUBAC facilitates NF-κB activation and inflammation upon receptor stimulation by ligating linear ubiquitin chains to critical signaling molecules. Indeed, linear-ubiquitination-dependent signaling is essential to prevent pyogenic bacterial infections that can lead to death. While linear ubiquitination is essential for intracellular receptor signaling upon microbial infection, this response must be measured and stopped to avoid tissue damage and autoimmunity. While LUBAC is activated upon bacterial stimulation, the mechanisms regulating LUBAC activity in response to bacterial stimuli have remained elusive. We demonstrate that LUBAC activity itself is downregulated through ubiquitination, specifically, ubiquitination of the catalytic subunit HOIP at the carboxyl-terminal lysine 1056. Ubiquitination of Lys1056 dynamically altered HOIP conformation, resulting in the suppression of its catalytic activity. Consequently, HOIP Lys1056-to-Arg mutation led not only to persistent LUBAC activity but also to prolonged NF-κB activation induced by bacterial lipopolysaccharide-mediated Toll-like receptor 4 (TLR4) stimulation, whereas it showed no effect on NF-κB activation induced by CD40 stimulation. This study describes a novel posttranslational regulation of LUBAC-mediated linear ubiquitination that is critical for specifically directing TLR4-mediated NF-κB activation. PMID:26578682

  13. 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.

  14. Gangliosides stimulate bradykinin B2 receptors to promote calmodulin kinase II-mediated neuronal differentiation.

    PubMed

    Kanatsu, Yoshinori; Chen, Nai Hong; Mitoma, Junya; Nakagawa, Tetsuto; Hirabayashi, Yoshio; Higashi, Hideyoshi

    2012-07-01

    Gangliosides mediate neuronal differentiation and maturation and are indispensable for the maintenance of brain function and survival. As part of our ongoing efforts to understand signaling pathways related to ganglioside function, we recently demonstrated that neuronal cells react to exogenous gangliosides GT1b and GD1b. Both of these gangliosides are enriched in the synapse-forming area of the brain and induce Ca(2+) release from intracellular stores, activation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and activation of cdc42 to promote reorganization of cytoskeletal actin and dendritic differentiation. Here, we show that bradykinin B2 receptors transduce these reactions as a mediator for ganglioside glycan signals. The B2 antagonist Hoe140 inhibited ganglioside-induced CaMKII activation, actin reorganization and early development of axon- and dendrite-like processes of primary cultured hippocampal neurons. Furthermore, we confirmed by yeast reporter assay that major b-series gangliosides, GT1b, GD1b and GD3, stimulated B2 bradykinin receptors. We hypothesize that this B2 receptor-mediated ganglioside signal transduction pathway is one mechanism that modulates neuronal differentiation and maturation.

  15. 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.

  16. 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.

  17. 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.

  18. AMPA receptor involvement in 5-hydroxytryptamine2A receptor-mediated pre-frontal cortical excitatory synaptic currents and DOI-induced head shakes.

    PubMed

    Zhang, Ce; Marek, Gerard J

    2008-01-01

    Glutamate plays an important role in the psychotomimetic effects of both channel blocking N-methyl D-aspartate (NMDA) receptor antagonists and hallucinogenic drugs which activate 5-hydroxytryptamine2A (5-HT2A) receptors. Previous work suggested that activation of non-NMDA ionotropic glutamate receptors mediates the effects of 5-HT-induced excitatory post-synaptic potentials/currents (EPSPs/EPSCs) when recording from layer V pyramidal cells in the rat medial pre-frontal cortex (mPFC). However, those effects are mediated by either alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate receptors of the iGluk5 subtype. To test whether activation of AMPA receptors is sufficient to mediate 5-HT-induced EPSCs, a 2,3-benzodiazepine that selectively blocks AMPA receptors was assessed. This selective AMPA receptor antagonist potently suppressed 5-HT-induced EPSCs. Since phenethylamine hallucinogens induce head shakes by activating 5-HT2A receptors in the mPFC and this action is modulated by glutamate, we also examined whether selective blockade of AMPA receptors would suppress DOI-induced head shakes. As predicted, we found that selective blockade of AMPA receptors suppressed DOI-induced head shakes. Given evidence that activation of AMPA receptors is an important downstream effect for both channel blocking NMDA receptor antagonists and phenethylamine hallucinogens, we also tested multiple doses of DOI with a sub-anesthetic dose of MK-801. Synergistic action between these two classes of psychotomimetic drugs was demonstrated by MK-801 enhancing DOI-induced head shakes and locomotor activity. These findings expand the dependence of both channel blocking NMDA receptor antagonists and phenethylamine hallucinogens on enhancing extracellular glutamate.

  19. Pharmacological and functional characterization of novel EP and DP receptor agonists: DP1 receptor mediates penile erection in multiple species.

    PubMed

    Brugger, Nadia; Kim, Noel N; Araldi, Gian Luca; Traish, Abdulmaged M; Palmer, Stephen S

    2008-02-01

    Despite the widespread use of prostaglandin E(1) as an efficacious treatment for male erectile dysfunction for more than two decades, research on prostanoid function in penile physiology has been limited. To characterize the pharmacological and physiological activity of novel subtype-selective EP and DP receptor agonists. Radioligand binding and second messenger assays were used to define receptor subtype specificity of the EP and DP agonists. Functional activity was further characterized using isolated human and rabbit penile cavernosal tissue in organ baths. In vivo activity was assessed in rabbits and rats by measuring changes in cavernous pressure after intracavernosal injection of receptor agonists. Receptor binding and signal transduction, smooth muscle contractile activity, erectile function. In organ bath preparations of human cavernosal tissue contracted with phenylephrine, EP2- and EP4-selective agonists exhibited variable potency in causing relaxation. One of the compounds caused mild contraction, and none of the compounds was as effective as PGE(1) (EC(50) = 0.23 microM). There was no consistent correlation between the pharmacological profile (receptor binding and second messenger assays) of the EP agonists and their effect on cavernosal tissue tone. In contrast, the DP1-selective agonist AS702224 (EC(50) =29 nM) was more effective in relaxing human cavernosal tissue than either PGE(1), PGD(2) (EC(50) = 58 nM), or the DP agonist BW245C (EC(50) =59 nM). In rabbit cavernosal tissue, PGE(1) and PGD(2) caused only contraction, while AS702224 and BW245C caused relaxation. Intracavernosal administration of AS702224 and BW245C also caused penile tumescence in rabbits and rats. For each compound, the erectile response improved with increasing dose and was significantly higher than vehicle alone. These data suggest that AS702224 is a potent DP1-selective agonist that causes penile erection. The DP1 receptor mediates relaxation in human cavernosal tissue, and

  20. 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

  1. The C-kit receptor-mediated signal transduction and tumor-related diseases.

    PubMed

    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.

  2. Inflammatory Mediators and Insulin Resistance in Obesity: Role of Nuclear Receptor Signaling in Macrophages

    PubMed Central

    Fuentes, Lucía; Rőszer, Tamás; Ricote, Mercedes

    2010-01-01

    Visceral obesity is coupled to a general low-grade chronic inflammatory state characterized by macrophage activation and inflammatory cytokine production, leading to insulin resistance (IR). The balance between proinflammatory M1 and antiinflammatory M2 macrophage phenotypes within visceral adipose tissue appears to be crucially involved in the development of obesity-associated IR and consequent metabolic abnormalities. The ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs) have recently been implicated in the determination of the M1/M2 phenotype. Liver X receptors (LXRs), which form another subgroup of the nuclear receptor superfamily, are also important regulators of proinflammatory cytokine production in macrophages. Disregulation of macrophage-mediated inflammation by PPARs and LXRs therefore underlies the development of IR. This review summarizes the role of PPAR and LXR signaling in macrophages and current knowledge about the impact of these actions in the manifestation of IR and obesity comorbidities such as liver steatosis and diabetic osteopenia. PMID:20508742

  3. 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.

  4. Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma.

    PubMed

    Verstraete, Kenneth; Peelman, Frank; Braun, Harald; Lopez, Juan; Van Rompaey, Dries; Dansercoer, Ann; Vandenberghe, Isabel; Pauwels, Kris; Tavernier, Jan; Lambrecht, Bart N; Hammad, Hamida; De Winter, Hans; Beyaert, Rudi; Lippens, Guy; Savvides, Savvas N

    2017-04-03

    The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency.

  5. Structure and antagonism of the receptor complex mediated by human TSLP in allergy and asthma

    PubMed Central

    Verstraete, Kenneth; Peelman, Frank; Braun, Harald; Lopez, Juan; Van Rompaey, Dries; Dansercoer, Ann; Vandenberghe, Isabel; Pauwels, Kris; Tavernier, Jan; Lambrecht, Bart N.; Hammad, Hamida; De Winter, Hans; Beyaert, Rudi; Lippens, Guy; Savvides, Savvas N.

    2017-01-01

    The pro-inflammatory cytokine thymic stromal lymphopoietin (TSLP) is pivotal to the pathophysiology of widespread allergic diseases mediated by type 2 helper T cell (Th2) responses, including asthma and atopic dermatitis. The emergence of human TSLP as a clinical target against asthma calls for maximally harnessing its therapeutic potential via structural and mechanistic considerations. Here we employ an integrative experimental approach focusing on productive and antagonized TSLP complexes and free cytokine. We reveal how cognate receptor TSLPR allosterically activates TSLP to potentiate the recruitment of the shared interleukin 7 receptor α-chain (IL-7Rα) by leveraging the flexibility, conformational heterogeneity and electrostatics of the cytokine. We further show that the monoclonal antibody Tezepelumab partly exploits these principles to neutralize TSLP activity. Finally, we introduce a fusion protein comprising a tandem of the TSLPR and IL-7Rα extracellular domains, which harnesses the mechanistic intricacies of the TSLP-driven receptor complex to manifest high antagonistic potency. PMID:28368013

  6. 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

  7. Inflammatory mediators and insulin resistance in obesity: role of nuclear receptor signaling in macrophages.

    PubMed

    Fuentes, Lucía; Roszer, Tamás; Ricote, Mercedes

    2010-01-01

    Visceral obesity is coupled to a general low-grade chronic inflammatory state characterized by macrophage activation and inflammatory cytokine production, leading to insulin resistance (IR). The balance between proinflammatory M1 and antiinflammatory M2 macrophage phenotypes within visceral adipose tissue appears to be crucially involved in the development of obesity-associated IR and consequent metabolic abnormalities. The ligand-dependent transcription factors peroxisome proliferator activated receptors (PPARs) have recently been implicated in the determination of the M1/M2 phenotype. Liver X receptors (LXRs), which form another subgroup of the nuclear receptor superfamily, are also important regulators of proinflammatory cytokine production in macrophages. Disregulation of macrophage-mediated inflammation by PPARs and LXRs therefore underlies the development of IR. This review summarizes the role of PPAR and LXR signaling in macrophages and current knowledge about the impact of these actions in the manifestation of IR and obesity comorbidities such as liver steatosis and diabetic osteopenia.

  8. 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

  9. 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

  10. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    PubMed Central

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A.; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell differentiation, as measured by the formation of cornified envelopes. We show that this acceleration is mediated by the AHR; also, that dioxin increases the expression of several genes known to be regulated by ARNT, which have critical roles in the cornification and epidermal barrier function of the skin. Importantly, we demonstrate that all of these responses are opposed by ligand-activation of the EGF receptor (R), an important regulator of keratinocyte cell fate. In the CYP1A1 enhancer, EGFR activation prevents recruitment of the p300 coactivator, although not affecting the binding of the AHR or ARNT. The total cellular level of p300 protein does not decrease, and overexpression of p300 relieves EGFR-mediated repression of transcription, indicating that p300 is a critical target for the repression of the AHR complex by EGFR signaling. These results provide a mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin is able to disrupt epidermal homeostasis and identify EGFR signaling as a regulator of the AHR. This signaling may modulate the incidence and severity of chloracne and be of therapeutic relevance to human poisonings by dioxin. PMID:19255421

  11. 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.

  12. Direct involvement of σ-1 receptors in the dopamine D1 receptor-mediated effects of cocaine

    PubMed Central

    Navarro, Gemma; Moreno, Estefanía; Aymerich, Marisol; Marcellino, Daniel; McCormick, Peter J.; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Canela, Enric I.; Ortiz, Jordi; Fuxe, Kjell; Lluís, Carmen; Ferré, Sergi; Franco, Rafael

    2010-01-01

    It is well known that cocaine blocks the dopamine transporter. This mechanism should lead to a general increase in dopaminergic neurotransmission, and yet dopamine D1 receptors (D1Rs) play a more significant role in the behavioral effects of cocaine than the other dopamine receptor subtypes. Cocaine also binds to σ-1 receptors, the physiological role of which is largely unknown. In the present study, D1R and σ1R were found to heteromerize in transfected cells, where cocaine robustly potentiated D1R-mediated adenylyl cyclase activation, induced MAPK activation per se and counteracted MAPK activation induced by D1R stimulation in a dopamine transporter-independent and σ1R-dependent manner. Some of these effects were also demonstrated in murine striatal slices and were absent in σ1R KO mice, providing evidence for the existence of σ1R-D1R heteromers in the brain. Therefore, these results provide a molecular explanation for which D1R plays a more significant role in the behavioral effects of cocaine, through σ1R-D1R heteromerization, and provide a unique perspective toward understanding the molecular basis of cocaine addiction. PMID:20956312

  13. 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.

  14. Folate receptor mediated intracellular protein delivery using PLL-PEG-FOL conjugate.

    PubMed

    Hwa Kim, Sun; Hoon Jeong, Ji; Joe, Cheol O; Gwan Park, Tae

    2005-04-18

    To develop a receptor-mediated intracellular delivery system that can transport therapeutic proteins or other bioactive macromolecules into a specific cell, a di-block copolymer conjugate, poly(L-lysine)-poly(ethylene glycol)-folate (PLL-PEG-FOL), was synthesized. The PLL-PEG-FOL conjugate was physically complexed with fluorescein isothiocyanate conjugated bovine serum albumin (FITC-BSA) in an aqueous phase by ionic interactions. Cellular uptake of PLL-PEG-FOL/FITC-BSA complexes was greatly enhanced against a folate receptor over-expressing cell line (KB cells) compared to a folate receptor deficient cell line (A549 cells). The presence of an excess amount of free folate (1 mM) in the medium inhibited the intracellular delivery of PLL-PEG-FOL/FITC-BSA complexes. This suggests that the enhanced cellular uptake of FITC-BSA by KB cells in a specific manner was attributed to folate receptor-mediated endocytosis of the complexes having folate moieties on the surface. The PLL-PEG-FOL di-block copolymer could be potentially applied for intracellular delivery of a wide range of other biological active agents that have negative charges on the surface.

  15. 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.

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. Cannabinoid receptor 2 and its agonists mediate hematopoiesis and hematopoietic stem and progenitor cell mobilization

    PubMed Central

    Jiang, Shuxian; Alberich-Jorda, Meritxell; Zagozdzon, Radoslaw; Parmar, Kalindi; Fu, Yigong; Mauch, Peter; Banu, Naheed; Makriyannis, Alexandros; Tenen, Daniel G.; Avraham, Shalom; Groopman, Jerome E.

    2011-01-01

    Endocannabinoids are arachidonic acid derivatives and part of a novel bioactive lipid signaling system, along with their G-coupled cannabinoid receptors (CB1 and CB2) and the enzymes involved in their biosynthesis and degradation. However, their roles in hematopoiesis and hematopoietic stem and progenitor cell (HSPC) functions are not well characterized. Here, we show that bone marrow stromal cells express endocannabinoids (anandamide and 2-arachidonylglycerol), whereas CB2 receptors are expressed in human and murine HSPCs. On ligand stimulation with CB2 agonists, CB2 receptors induced chemotaxis, migration, and enhanced colony formation of bone marrow cells, which were mediated via ERK, PI3-kinase, and Gαi-Rac1 pathways. In vivo, the CB2 agonist AM1241 induced mobilization of murine HSPCs with short- and long-term repopulating abilities. In addition, granulocyte colony-stimulating factor -induced mobilization of HSPCs was significantly decreased by specific CB2 antagonists and was impaired in Cnr2−/− cannabinoid type 2 receptor knockout mice. Taken together, these results demonstrate that the endocannabinoid system is involved in hematopoiesis and that CB2/CB2 agonist axis mediates repopulation of hematopoiesis and mobilization of HSPCs. Thus, CB2 agonists may be therapeutically applied in clinical conditions, such as bone marrow transplantation. PMID:21063029

  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. 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.

  4. Thrombin functions as an inflammatory mediator through activation of its receptor

    PubMed Central

    1996-01-01

    A rat model of inflammation was used to investigate the biological effects of thrombin. The thrombin-specific inhibitor Hirulog markedly attentuated the carrageenin-induced edema of the paw of the rat. Injection of thrombin into the paw also produced edema. The effect of thrombin was due to activation of its receptor; a thrombin receptor activating peptide (TRAP) reproduced the effects of thrombin in causing edema. TRAP also increased vascular permeability as demonstrated by extravasation of Evans blue and 125I-labeled serum albumin. The release of bioactive amines played an important role in mediating the TRAP- induced edema; the serotonin/histamine antagonist cryproheptadine and the histamine H2 receptor antagonist cimetidine reduced significantly the edema caused by TRAP. Treatment of rats with the mast cell degranulator 48/80 to deplete these cells of their stores of histamine and serotonin abolished completely the ability of TRAP to produce edema. Histochemical examination confirmed that TRAP treatment led to mast cell degranulation. Thus, it has been possible to demonstrate that thrombin acts as an inflammatory mediator in vivo by activating its receptor, which in turn leads to release of vasoactive amines from mast cells. PMID:8642286

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

    PubMed

    Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

    2015-12-01

    Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

  10. 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.

  11. Functional evaluation of the receptors mediating vasoconstriction of rat aorta by trace amines and amphetamines.

    PubMed

    Broadley, Kenneth J; Fehler, Martina; Ford, William R; Kidd, Emma J

    2013-09-05

    Trace amines including β-phenylethylamine (β-PEA) and amphetamines classically exert pharmacological actions via indirect sympathomimetic mechanisms. However, there is evidence for other mechanisms and this study explores the receptors mediating vasoconstriction in rat aorta. β-PEA, d-amphetamine, MDMA, cathinone and methylphenidate caused concentration-dependent contractions of rat isolated aortic rings which were unaffected by prazosin (1 μM), ICI-118,551 (1 μM), cocaine (10 μM) and pargyline (10 μM), to inhibit α1- and β2-adrenoceptors, neuronal transport and monoamine oxidase (MAO), respectively. Octopamine concentration-response curves, however, were shifted to the right. In the presence of the inhibitors, the rate of onset of octopamine contractions was slowed. Lineweaver-Burk analysis of the kinetics of the response generated different KM values for octopamine in the absence (2.35 × 10(-6)M) and presence (6.09 × 10(-5)M) of inhibitors, indicating mediation by different receptors. Tryptamine-induced vasoconstriction also resisted blockade by adrenergic inhibitors and the 5-HT1A, 1B, 1D and 5-HT2A receptor antagonists, methiothepin (50 nM) and ketanserin (30 nM), respectively. Trace amines and amphetamines therefore exert vasoconstriction independently of adrenoceptors, neuronal transport and 5-HT receptor activation. There was no evidence of tachyphylaxis or cross-tachyphylaxis of the vasoconstriction to these amines. Tyramine was a partial agonist and in its presence, β-PEA, d-amphetamine and octopamine were antagonised indicating that they all act through a common receptor for which tyramine serves as an antagonist. We conclude that the vasoconstriction is via TAAR-1, because of structural similarities between amines, ability to stimulate recombinant trace amine-associated receptor 1 (TAAR-1) and the presence of TAAR-1 in rat aorta. © 2013 Elsevier B.V. All rights reserved.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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

  18. Methamphetamine Inhibits Toll-Like Receptor 9-Mediated Anti-HIV Activity in Macrophages

    PubMed Central

    Cen, Ping; Ye, Li; Su, Qi-Jian; Wang, Xu; Li, Jie-Liang; Lin, Xin-Qin

    2013-01-01

    Abstract Toll-like receptor 9 (TLR9) is one of the key sensors that recognize viral infection/replication in the host cells. Studies have demonstrated that methamphetamine (METH) dysregulated host cell innate immunity and facilitated HIV infection of macrophages. In this study, we present new evidence that METH suppressed TLR9-mediated anti-HIV activity in macrophages. Activation of TLR9 by its agonist CpG-ODN 2216 inhibits HIV replication, which was demonstrated by increased expression of TLR9, interferon (IFN)-α, IFN regulatory factor-7 (IRF-7), myeloid differentiation factor 88 (MyD88), and myxovirus resistance gene A (MxA) in macrophages. However, METH treatment of macrophages greatly compromised the TLR9 signaling-mediated anti-HIV effect and inhibited the expression of TLR9 downstream signaling factors. Dopamine D1 receptor (D1R) antagonists (SCH23390) could block METH-mediated inhibition of anti-HIV activity of TLR9 signaling. Investigation of the underlying mechanisms of the METH action showed that METH treatment selectively down-regulated the expression of TLR9 on macrophages, whereas it had little effect on the expression of other TLRs. Collectively, our results provide further evidence that METH suppresses host cell innate immunity against HIV infection by down-regulating TLR9 expression and its signaling-mediated antiviral effect in macrophages. PMID:23751096

  19. 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.

  20. Analgesic effects mediated by neuronal nicotinic acetylcholine receptor agonists: correlation with desensitization of α4β2* receptors.

    PubMed

    Zhang, Jiahui; Xiao, Yun-De; Jordan, Kristen G; Hammond, Phil S; Van Dyke, Katherine M; Mazurov, Anatoly A; Speake, Jason D; Lippiello, Patrick M; James, John W; Letchworth, Sharon R; Bencherif, Merouane; Hauser, Terry A

    2012-12-18

    which are more potent at desensitization of α4β2* receptors display better analgesia scores in the formalin test. Consideration of desensitization propertiesat α4β2* receptors, especially at α4β2α5, in multiple linear regression analyses significantly improves correlations with efficacies of analgesia. Thus, α4β2* nicotinic acetylcholine receptor desensitization may contribute to efficacy in the mediation of pain, and represent a mechanism for analgesic effects mediated by nicotinic agonists.

  1. The type I interferon receptor mediates tyrosine phosphorylation of insulin receptor substrate 2.

    PubMed

    Platanias, L C; Uddin, S; Yetter, A; Sun, X J; White, M F

    1996-01-05

    Binding of interferon alpha (IFN alpha) to its receptor induces activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine phosphorylation of multiple downstream signaling elements, including the Stat components of the interferon-stimulated gene factor 3 (ISGF-3). IFN alpha also induces tyrosine phosphorylation of IRS-1, the principle substrate of the insulin receptor. In this study we demonstrate that various Type I IFNs rapidly stimulate tyrosine phosphorylation of IRS-2. This is significant since IRS-2 is the major IRS protein found in hematopoietic cells. The IFN alpha-induced phosphorylated form of IRS-2 associates with the p85 regulatory subunit of the phosphatidylinositol 3'-kinase, suggesting that this kinase participates in an IFN alpha-signaling cascade downstream of IRS-2. We also provide evidence for an interaction of IRS-2 with Tyk-2, suggesting that Tyk-2 is the kinase that phosphorylates this protein during IFN alpha stimulation. A conserved region in the pleckstrin homology domain of IRS-2 may be required for the interaction of IRS-2 with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the IRS-2-IH1PH or IRS-1-IH1PH domains to Tyk-2 but not other Janus kinases in vitro.

  2. Ionotropic Receptors Mediate Drosophila Oviposition Preference through Sour Gustatory Receptor Neurons.

    PubMed

    Chen, Yan; Amrein, Hubert

    2017-09-25

    Carboxylic acids are present in many foods, being especially abundant in fruits. Yet, relatively little is known about how acids are detected by gustatory systems and whether they have a potential role in nutrition or provide other health benefits. Here we identify sour gustatory receptor neurons (GRNs) in tarsal taste sensilla of Drosophila melanogaster. We find that most tarsal sensilla harbor a sour GRN that is specifically activated by carboxylic and mineral acids but does not respond to sweet- and bitter-tasting chemicals or salt. One pair of taste sensilla features two GRNs that respond only to a subset of carboxylic acids and high concentrations of salt. All sour GRNs prominently express two Ionotropic Receptor (IR) genes, IR76b and IR25a, and we show that both these genes are necessary for the detection of acids. Furthermore, we establish that IR25a and IR76b are essential in sour GRNs of females for oviposition preference on acid-containing food. Our investigations reveal that acids activate a unique set of taste cells largely dedicated to sour taste, and they indicate that both pH/proton concentration and the structure of carboxylic acids contribute to sour GRN activation. Together, our studies provide new insights into the cellular and molecular basis of sour taste. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. 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

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules.

    PubMed

    Bush, Kristin A; Krukowski, Karen; Eddy, Justin L; Janusek, Linda Witek; Mathews, Herbert L

    2012-01-01

    Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. 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.

  10. 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.

  11. Multivalent ligand-receptor-mediated interaction of small filled vesicles with a cellular membrane

    NASA Astrophysics Data System (ADS)

    Zhdanov, Vladimir P.

    2017-07-01

    The ligand-receptor-mediated contacts of small sub-100-nm-sized lipid vesicles (or nanoparticles) with the cellular membrane are of interest in the contexts of cell-to-cell communication, endocytosis of membrane-coated virions, and drug (RNA) delivery. In all these cases, the interior of vesicles is filled by biologically relevant content. Despite the diversity of such systems, the corresponding ligand-receptor interaction possesses universal features. One of them is that the vesicle-membrane contacts can be accompanied by the redistribution of ligands and receptors between the contact and contact-free regions. In particular, the concentrations of ligands and receptors may become appreciably higher in the contact regions and their composition may there be different compared to that in the suspended state in the solution. A statistical model presented herein describes the corresponding distribution of various ligands and receptors and allows one to calculate the related change of the free energy with variation of the vesicle-engulfment extent. The results obtained are used to clarify the necessary conditions for the vesicle-assisted pathway of drug delivery.

  12. Motivational properties of opioids: evidence that an activation of delta-receptors mediates reinforcement processes.

    PubMed

    Shippenberg, T S; Bals-Kubik, R; Herz, A

    1987-12-15

    The role of central delta-opioid receptors in the mediation of opioid reinforcement and endogenous reward processes was examined using a non-biased place-preference conditioning procedure. Intracerebroventricular (i.c.v.) administration of the selective delta-receptor agonist, [D-Pen2, D-Pen5]-enkephalin (DPDPE, 10.0-25.0 micrograms) produced a significant preference for the drug-associated place and a similar effect was observed following i.c.v. injections of morphine (10.0 micrograms). Administration of the delta-receptor antagonist, ICI 174,864, at doses (1.0-5.0 micrograms, i.c.v.) which had no aversive effects when tested alone, abolished the reinforcing properties of DPDPE. Such treatment did not, however, modify the effect of morphine. These findings demonstrate the involvement delta- as well as mu-receptors in the motivational properties of opioids and suggest that the activation of either receptor type is sufficient for the elicitation of appetitively reinforcing effects.

  13. 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.

  14. Mediation of ACTH and prolactin responses to 5-HTP by 5-HT2 receptors.

    PubMed

    Gartside, S E; Cowen, P J

    1990-04-10

    Serotonin has a facilitatory role in the role of prolactin and adrenocorticotropin (ACTH) secretion. The serotonin precursor 5-hydroxy-L-tryptophan (5-HTP) dose dependently (30-100 mg/kg i.p.) increased plasma prolactin and ACTH in the male rat. Prolactin and ACTH responses to 5-HTP (100 mg/kg) were attenuated by pretreatment with the non-selective 5-HT receptor antagonist, metergoline (0.5 mg/kg), and by the selective 5-HT2 receptor antagonists, ritanserin (0.4 mg/kg), ketanserin (2.5 mg/kg), ICI (5.0 mg/kg) and spiperone (1.0 mg/kg). The 5-HT1 receptor antagonists, propranolol (40 mg/kg) and pindolol (4.0 mg/kg), failed to antagonize the prolactin and ACTH responses to 5-HTP (100 mg/kg), as did the selective 5-HT3 receptor antagonist, BRL 43694 (1.0 mg/kg). The results suggest that the prolactin and ACTH responses to 5-HTP in the male rat are mediated by 5-HT2 receptors.

  15. Activation by SLAM Family Receptors Contributes to NK Cell Mediated “Missing-Self” Recognition

    PubMed Central

    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. PMID:27054584

  16. Pharmacological characterization of cholecystokinin receptors mediating contraction of human gallbladder and ascending colon.

    PubMed

    Morton, M F; Welsh, N J; Tavares, I A; Shankley, N P

    2002-04-15

    Cholecystokinin (CCK) produces contractions of gallbladder and colon in a number of different species. Although the effects of CCK on the human gallbladder are relatively well documented, the CCK receptors in the human colon have not been clearly characterised. Therefore, in this study, the CCK receptors in the human gallbladder and colon were compared using pharmacological techniques. Contraction of specimens of the human tissue was measured using in vitro organ bath bioassay. The effect of selective concentrations of CCK(1) and CCK(2) receptor antagonists (L-364,718 and JB93182, respectively) was determined on agonist concentration-effect (E/[A]) curves obtained by cumulative dosing with sulphated CCK. The CCK(1) antagonist L-364,718 produced a rightward shift of the CCK-8S [E/[A] curve in the human gallbladder (pA(2)=9.15 +/- 0.26) and ascending colon (pA(2)=9.20 +/- .33). In both tissues, the CCK(2) receptor antagonist, JB93182, had no effect on the CCK E/[A] curves. In addition, in the colon, pentagastrin responses were inhibited by L-364,718 but unaffected by JB93182. These data indicate that the CCK-induced contraction of the human colon and gallbladder smooth muscle is mediated solely through the CCK(1) receptor subtype, and the antagonist affinity estimates are consistent with those previously obtained in experiments on animal tissue.

  17. Elastase-mediated phosphatidylserine receptor cleavage impairs apoptotic cell clearance in cystic fibrosis and bronchiectasis

    PubMed Central

    Vandivier, R. William; Fadok, Valerie A.; Hoffmann, Peter R.; Bratton, Donna L.; Penvari, Churee; Brown, Kevin K.; Brain, Joseph D.; Accurso, Frank J.; Henson, Peter M.

    2002-01-01

    Cystic fibrosis is characterized by an early and sustained influx of inflammatory cells into the airways and by release of proteases. Resolution of inflammation is normally associated with the orderly removal of dying apoptotic inflammatory cells through cell recognition receptors, such as the phosphatidylserine receptor, CD36, and αv integrins. Accordingly, removal of apoptotic inflammatory cells may be impaired in persistent inflammatory responses such as that seen in cystic fibrosis airways. Examination of sputa from cystic fibrosis and non–cystic fibrosis bronchiectasis patients demonstrated an abundance of apoptotic cells, in excess of that seen in patients with chronic bronchitis. In vitro, cystic fibrosis and bronchiectasis airway fluid directly inhibited apoptotic cell removal by alveolar macrophages in a neutrophil elastase-dependent manner, suggesting that elastase may impair apoptotic cell clearance in vivo. Flow cytometry demonstrated that neutrophil elastase cleaved the phosphatidylserine receptor, but not CD36 or CD32 (FcγRII). Cleavage of the phosphatidylserine receptor by neutrophil elastase specifically disrupted phagocytosis of apoptotic cells, implying a potential mechanism for delayed apoptotic cell clearance in vivo. Therefore, defective airway clearance of apoptotic cells in cystic fibrosis and bronchiectasis may be due to elastase-mediated cleavage of phosphatidylserine receptor on phagocytes and may contribute to ongoing airway inflammation. PMID:11877474

  18. FLIP ing the coin? Death receptor-mediated signals during skin tumorigenesis.

    PubMed

    Leverkus, Martin; Diessenbacher, Philip; Geserick, Peter

    2008-07-01

    Keratinocyte skin cancer is a multi-step process, during which a number of obstacles have to be overcome by the tumor cell to allow the development of a manifest tumor. Beside proliferation and immortality, apoptosis resistance is one additional and critical step during skin carcinogenesis. Over the past two decades, much has been learned about the prototypical membrane-bound inducers of apoptosis, namely the death receptors and their ligands, and the apoptosis signalling pathways activated by death receptors have been elucidated in great detail. In contrast, much less is known about the tissue-specific role of the death receptor/ligands systems during the development of skin cancer. Here, we summarize and discuss the role of this intriguing receptor family and the potential mechanistical impact of the intracellular caspase-8 inhibitor cFLIP for keratinocyte skin cancer. Given more recent data about cFLIP and its isoforms, a more complex regulatory role of cFLIP can be suspected. Indeed, cFLIP may not solely interfere with death receptor-mediated apoptosis signalling pathways, but may positively or negatively influence other, potential harmful signalling pathways such as the production of inflammatory cytokines, tumor cell migration or the activation of transcription factors such as NF-kappaB, considered crucial during skin tumorigenesis. In this respect, cFLIP may act to 'FLIP the coin' during the development of keratinocyte skin cancer.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. Interleukin-1β Mediates Virus-Induced M2 Muscarinic Receptor Dysfunction and Airway Hyperreactivity

    PubMed Central

    Rynko, Abby E.; Fryer, Allison D.

    2014-01-01

    Respiratory viral infections are associated with the majority of asthma attacks. Inhibitory M2 receptors on parasympathetic nerves, which normally limit acetylcholine (ACh) release, are dysfunctional after respiratory viral infection. Because IL-1β is up-regulated during respiratory viral infections, we investigated whether IL-1β mediates M2 receptor dysfunction during parainfluenza virus infection. Virus-infected guinea pigs were pretreated with the IL-1β antagonist anakinra. In the absence of anakinra, viral infection increased bronchoconstriction in response to vagal stimulation but not to intravenous ACh, and neuronal M2 muscarinic receptors were dysfunctional. Pretreatment with anakinra prevented virus-induced increased bronchoconstriction and M2 receptor dysfunction. Anakinra did not change smooth muscle M3 muscarinic receptor response to ACh, lung viral loads, or blood and bronchoalveolar lavage leukocyte populations. Respiratory virus infection decreased M2 receptor mRNA expression in parasympathetic ganglia extracted from infected animals, and this was prevented by blocking IL-1β or TNF-α. Treatment of SK-N-SH neuroblastoma cells or primary cultures of guinea pig parasympathetic neurons with IL-1β directly decreased M2 receptor mRNA, and this was not synergistic with TNF-α treatment. Treating guinea pig trachea segment with TNF-α or IL-1β in vitro increased tracheal contractions in response to activation of airway nerves by electrical field stimulation. Blocking IL-1β during TNF-α treatment prevented this hyperresponsiveness. These data show that virus-induced hyperreactivity and M2 dysfunction involves IL-1β and TNF-α, likely in sequence with TNF-α causing production of IL-1β. PMID:24735073

  9. Receptor-Mediated Enhancement of Beta Adrenergic Drug Activity by Ascorbate In Vitro and In Vivo

    PubMed Central

    Dillon, Patrick F.; Root-Bernstein, Robert; Robinson, N. Edward; Abraham, William M.; Berney, Catherine

    2010-01-01

    Rationale Previous in vitro research demonstrated that ascorbate enhances potency and duration of activity of agonists binding to alpha 1 adrenergic and histamine receptors. Objectives Extending this work to beta 2 adrenergic systems in vitro and in vivo. Methods Ultraviolet spectroscopy was used to study ascorbate binding to adrenergic receptor preparations and peptides. Force transduction studies on acetylcholine-contracted trachealis preparations from pigs and guinea pigs measured the effect of ascorbate on relaxation due to submaximal doses of beta adrenergic agonists. The effect of inhaled albuterol with and without ascorbate was tested on horses with heaves and sheep with carbachol-induced bronchoconstriction. Measurements Binding constants for ascorbate binding to beta adrenergic receptor were derived from concentration-dependent spectral shifts. Dose- dependence curves were obtained for the relaxation of pre-contracted trachealis preparations due to beta agonists in the presence and absence of varied ascorbate. Tachyphylaxis and fade were also measured. Dose response curves were determined for the effect of albuterol plus-and-minus ascorbate on airway resistance in horses and sheep. Main Results Ascorbate binds to the beta 2 adrenergic receptor at physiological concentrations. The receptor recycles dehydroascorbate. Physiological and supra-physiological concentrations of ascorbate enhance submaximal epinephrine and isoproterenol relaxation of trachealis, producing a 3–10-fold increase in sensitivity, preventing tachyphylaxis, and reversing fade. In vivo, ascorbate improves albuterol's effect on heaves and produces a 10-fold enhancement of albuterol activity in “asthmatic” sheep. Conclusions Ascorbate enhances beta-adrenergic activity via a novel receptor-mediated mechanism; increases potency and duration of beta adrenergic agonists effective in asthma and COPD; prevents tachyphylaxis; and reverses fade. These novel effects are probably caused by a

  10. Evidence for the putative cannabinoid receptor (GPR55)-mediated inhibitory effects on intestinal contractility in mice.

    PubMed

    Ross, Gracious R; Lichtman, Aron; Dewey, William L; Akbarali, Hamid I

    2012-01-01

    Cannabinoids inhibit intestinal motility via presynaptic cannabinoid receptor type I (CB1) in enteric neurons while cannabinoid receptor type II (CB2) receptors are located mainly in immune cells. The recently de-orphanized G-protein-coupled receptor, GPR55, has been proposed to be the 'third' cannabinoid receptor. Although gene expression of GPR55 is evident in the gut, functional evidence for GPR55 in the gut is unknown. In this study, we tested the hypothesis that GPR55 activation inhibits neurogenic contractions in the gut. We assessed the inhibitory effect of the atypical cannabinoid O-1602, a GPR55 agonist, in mouse colon. Isometric tension recordings in colonic tissue strips were used from either wild-type, GPR55(-/-) or CB1(-/-)/CB2(-/-) knockout mice. O-1602 inhibited the electrical field- induced contractions in the colon strips from wild-type and CB1(-/-)/CB2(-/-) in a concentration-dependent manner, suggesting a non-CB1/CB2 receptor-mediated prejunctional effect. The concentration-dependent response of O-1602 was significantly inhibited in GPR55(-/-) mice. O-1602 did not relax colonic strips precontracted with high K(+) (80 mmol/l), indicating no involvement of Ca(2+) channel blockade in O-1602-induced relaxation. However, 10 µmol/l O-1602 partially inhibited the exogenous acetylcholine (10 µmol/l)-induced contractions. Moreover, we also assessed the inhibitory effects of JWH015, a CB2/GPR55 agonist on neurogenic contractions of mouse ileum. Surprisingly, the effects of JWH015 were independent of the known cannabinoid receptors. Taken together, these findings suggest that activation of GPR55 leads to inhibition of neurogenic contractions in the gut and are predominantly prejunctional. Copyright © 2012 S. Karger AG, Basel.

  11. Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle

    PubMed Central

    Ayman, Sinem; Wallace, Pat; Wayman, Chris P; Gibson, Alan; McFadzean, Ian

    2003-01-01

    The aim of this work was to determine whether Rho-kinase-mediated calcium sensitisation contributes to contractions of the mouse anococcygeus smooth muscle and, if so, whether the process was activated by receptor-dependent or receptor-independent mechanisms.The Rho-kinase inhibitor Y27632 produced concentration-dependent decreases in tone raised by either the muscarinic receptor agonist carbachol (CCh), or the sarco-endoplasmic reticulum calcium ATPase inhibitor thapsigargin (Tg) (EC50 values against CCh and Tg of 8.4±3.3 (n=6) and 6.1±2.1 (n=7) μM, respectively). Pretreatment of tissues with Y27632 also inhibited contractions produced by 65 mM external potassium (69±7% (n=4) inhibition using 10 μM Y27632). Y27632 had no effect on contractions produced by the inhibitor of smooth muscle myosin light-chain phosphatase, calyculin-A.In β-escin-permeabilised preparations, both CCh and Tg produced significant increases in tone over-and-above that produced by a combination of calcium (1 μM) and GTP (100 μM). These responses to CCh and Tg were inhibited by Y27632 (10 μM).Western blot analysis of fractionated tissue samples probed for RhoA immunoreactivity, indicated that both CCh and Tg were able to induce translocation of RhoA from the cytosol to the membrane.These findings suggest that Rho-kinase-mediated calcium sensitisation is activated by both receptor-dependent and receptor-independent mechanisms in the mouse anococcygeus. PMID:12922941

  12. 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).

  13. 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.

  14. 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

  15. A critical role for Syk in signal transduction and phagocytosis mediated by Fcgamma receptors on macrophages.

    PubMed

    Crowley, M T; Costello, P S; Fitzer-Attas, C J; Turner, M; Meng, F; Lowell, C; Tybulewicz, V L; DeFranco, A L

    1997-10-06

    Receptors on macrophages for the Fc region of IgG (FcgammaR) mediate a number of responses important for host immunity. Signaling events necessary for these responses are likely initiated by the activation of Src-family and Syk-family tyrosine kinases after FcgammaR cross-linking. Macrophages derived from Syk-deficient (Syk-) mice were defective in phagocytosis of particles bound by FcgammaRs, as well as in many FcgammaR-induced signaling events, including tyrosine phosphorylation of a number of cellular substrates and activation of MAP kinases. In contrast, Syk- macrophages exhibited normal responses to another potent macrophage stimulus, lipopolysaccharide. Phagocytosis of latex beads and Escherichia coli bacteria was also not affected. Syk- macrophages exhibited formation of polymerized actin structures opposing particles bound to the cells by FcgammaRs (actin cups), but failed to proceed to internalization. Interestingly, inhibitors of phosphatidylinositol 3-kinase also blocked FcgammaR-mediated phagocytosis at this stage. Thus, PI 3-kinase may participate in a Syk-dependent signaling pathway critical for FcgammaR-mediated phagocytosis. Macrophages derived from mice deficient for the three members of the Src-family of kinases expressed in these cells, Hck, Fgr, and Lyn, exhibited poor Syk activation upon FcgammaR engagement, accompanied by a delay in FcgammaR-mediated phagocytosis. These observations demonstrate that Syk is critical for FcgammaR-mediated phagocytosis, as well as for signal transduction in macrophages. Additionally, our findings provide evidence to support a model of sequential tyrosine kinase activation by FcgammaR's analogous to models of signaling by the B and T cell antigen receptors.

  16. High molecular weight kininogen binds phosphatidylserine and opsonizes urokinase plasminogen activator receptor-mediated efferocytosis

    PubMed Central

    Yang, Aizhen; Dai, Jihong; Xie, Zhanli; Colman, Robert W.; Wu, Qingyu; Birge, Raymond B.; Wu, Yi

    2014-01-01

    Summary Phagocytosis of apoptotic cells (efferocytosis) is essential for regulation of immune responses and tissue homeostasis, and is mediated by phagocytic receptors. In this study we found that urokinase plasminogen activator receptor (uPAR) plays an important role in internalization of apoptotic cells, and also characterized the underlying mechanisms. In a flow cytometry-based phagocytic assay, uPAR-deficient (uPAR−/−) macrophages displayed significant defect in internalization but not tethering of apoptotic cells. When uPAR−/− mice were challenged with apoptotic cells, they exhibited pronounced splenomegaly resulting from accumulation of abundant apoptotic cells in spleen. Overexpression of uPAR in HEK-293 cells enhanced efferocytosis, which was inhibited by annexin V and phosphatidylserine (PS) liposome, suggesting that uPAR-mediated efferocytosis is dependent on PS. In serum lacking high-molecular-weight kininogen (HK), a uPAR ligand, uPAR-mediated efferocytosis was significantly attenuated, which was rescued by replenishment of HK. As detected by flow cytometry, HK selectively bound to apoptotic cells, but not viable cells. In purified systems, HK was specifically associated with PS liposome. HK binding to apoptotic cells induced its rapid cleavage to two-chain HKa and bradykinin. Both heavy chain and light chain of HKa were associated with PS liposome and apoptotic cells. HKa has higher binding affinity than HK to uPAR. Overexpression of Rac1/N17 cDNA inhibited uPAR-mediated efferocytosis. HK plus PS liposome stimulated a complex formation of CrkII with p130Cas and Dock-180, and Rac1 activation in uPAR-293 cells, but not in control HEK-293 cells. Thus, uPAR mediates efferocytosis through HK interaction with PS on apoptotic cells and activation of Rac1 pathway. PMID:24688027

  17. Lysosomal trafficking regulator Lyst links membrane trafficking to toll-like receptor-mediated inflammatory responses.

    PubMed

    Westphal, Andreas; Cheng, Weijia; Yu, Jinbo; Grassl, Guntram; Krautkrämer, Martina; Holst, Otto; Föger, Niko; Lee, Kyeong-Hee

    2017-01-01

    Subcellular compartmentalization of receptor signaling is an emerging principle in innate immunity. However, the functional integration of receptor signaling pathways into membrane trafficking routes and its physiological relevance for immune responses is still largely unclear. In this study, using Lyst-mutant beige mice, we show that lysosomal trafficking regulator Lyst links endolysosomal organization to the selective control of toll-like receptor 3 (TLR3)- and TLR4-mediated proinflammatory responses. Consequently, Lyst-mutant mice showed increased susceptibility to bacterial infection and were largely resistant to endotoxin-induced septic shock. Mechanistic analysis revealed that Lyst specifically controls TLR3- and TLR4-induced endosomal TRIF (TIR domain-containing adapter-inducing interferon β) signaling pathways. Loss of functional Lyst leads to dysregulated phagosomal maturation, resulting in a failure to form an activation-induced Rab7(+) endosomal/phagosomal compartment. This specific Rab7(+) compartment was further demonstrated to serve as a major site for active TRIF signaling events, thus linking phagosomal maturation to specific TLR signaling pathways. The immunoregulatory role of Lyst on TLR signaling pathways was confirmed in human cells by CRISPR/Cas9-mediated gene inactivation. As mutations in LYST cause human Chédiak-Higashi syndrome, a severe immunodeficiency, our findings also contribute to a better understanding of human disease mechanisms. © 2016 Westphal et al.

  18. 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

  19. Regulation of ENaC-Mediated Sodium Reabsorption by Peroxisome Proliferator-Activated Receptors

    PubMed Central

    Pavlov, Tengis S.; Imig, John D.; Staruschenko, Alexander

    2010-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of a steroid hormone receptor superfamily that responds to changes in lipid and glucose homeostasis. Peroxisomal proliferator-activated receptor subtype γ (PPARγ) has received much attention as the target for antidiabetic drugs, as well as its role in responding to endogenous compounds such as prostaglandin J2. However, thiazolidinediones (TZDs), the synthetic agonists of the PPARγ are tightly associated with fluid retention and edema, as potentially serious side effects. The epithelial sodium channel (ENaC) represents the rate limiting step for sodium absorption in the renal collecting duct. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. The role of PPARγ agonists on ENaC activity remains controversial. While PPARγ agonists were shown to stimulate ENaC-mediated renal salt absorption, probably via Serum- and Glucocorticoid-Regulated Kinase 1 (SGK1), other studies reported that PPARγ agonist-induced fluid retention is independent of ENaC activity. The current paper provides new insights into the control and function of ENaC and ENaC-mediated sodium transport as well as several other epithelial channels/transporters by PPARs and particularly PPARγ. The potential contribution of arachidonic acid (AA) metabolites in PPAR-dependent mechanisms is also discussed. PMID:20613963

  20. 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.

  1. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1.

    PubMed

    Li, Li; Fang, Celia J; Ryan, James C; Niemi, Eréne C; Lebrón, José A; Björkman, Pamela J; Arase, Hisashi; Torti, Frank M; Torti, Suzy V; Nakamura, Mary C; Seaman, William E

    2010-02-23

    Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules.

  2. Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

    PubMed Central

    Li, Li; Fang, Celia J.; Ryan, James C.; Niemi, Eréne C.; Lebrón, José A.; Björkman, Pamela J.; Arase, Hisashi; Torti, Frank M.; Torti, Suzy V.; Nakamura, Mary C.; Seaman, William E.

    2010-01-01

    Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules. PMID:20133674

  3. 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.

  4. 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.

  5. 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

  6. The Amyloid Precursor Protein Is a Conserved Receptor for Slit to Mediate Axon Guidance

    PubMed Central

    Li, Hongmei; Mutlu, Sena A.; Bowser, Devon A.; Wang, Meng C.

    2017-01-01

    Abstract The amyloid precursor protein (APP) is a receptor-like membrane protein. Although APP processing and β-amyloid production play a central role in Alzheimer’s disease (AD) pathogenesis, the physiological function of APP remains elusive. Here, we identify APP as a novel receptor for Slit that mediates axon guidance and neural circuit formation. APP deficiency abolishes the Slit repulsive effect in a 3D olfactory explant culture, consistent with its callosal projection deficit in vivo and reminiscent of Slit loss. Inactivation of APP ortholog APL-1 in Caenorhabditis elegans results in pioneer axon mistargeting and genetic analysis places APL-1 in the SLT-1 (Slit)/SAX-3 (Robo) repulsive pathway. Slit binds to APP through the E1 domain, which triggers APP ectodomain shedding and recruitment of the intracellular FE65 and Pak1 complex and associated Rac1 GTPase activation. Our study establishes APP as a novel receptor for Slit ligand mediating axon guidance and neural circuit formation. PMID:28785723

  7. Mannose Receptor Mediates the Immune Response to Ganoderma atrum Polysaccharides in Macrophages.

    PubMed

    Li, Wen-Juan; Tang, Xiao-Fang; Shuai, Xiao-Xue; Jiang, Cheng-Jia; Liu, Xiang; Wang, Le-Feng; Yao, Yu-Fei; Nie, Shao-Ping; Xie, Ming-Yong

    2017-01-18

    The ability of mannose receptor (MR) to recognize the carbohydrate structures is well-established. Here, we reported that MR was crucial for the immune response to a Ganoderma atrum polysaccharide (PSG-1), as evidenced by elevation of MR in association with increase of phagocytosis and concentrations of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in normal macrophages. Elevation of MR triggered by PSG-1 also led to control lipopolysaccharide (LPS)-triggered inflammatory response via the increase of interleukin-10 (IL-10) and inhibition of phagocytosis and IL-1β. Anti-MR antibody partly attenuated PSG-1-mediated anti-inflammatory responses, while it could not affect TNF-α secretion, suggesting that another receptor was involved in PSG-1-triggered immunomodulatory effects. MR and toll-like receptor (TLR)4 coordinated the influences on the TLR4-mediated signaling cascade by the nuclear factor-κB (NF-κB) pathway in LPS-stimulated macrophages subjected to PSG-1. Collectively, immune response to PSG-1 required recognition by MR in macrophages. The NF-κB pathway served as a central role for the coordination of MR and TLR4 to elicit immune response to PSG-1.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

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

    PubMed

    Marques, Maud; Laflamme, Liette; Gaudreau, Luc

    2013-09-01

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

  13. IgG-mediated anaphylaxis via Fcγ receptor in CD40-deficient mice

    PubMed Central

    Wakayama, H; Hasegawa, Y; Kawabe, T; Saito, H; Kikutani, H; Shimokata, K

    1998-01-01

    Anaphylaxis denotes an immediate hypersensitivity reaction to allergen, exclusively mediated by IgE antibodies. However, IgE antibodies do not explain all the syndromes that are encountered. We investigated potent IgG-mediated anaphylaxis in CD40-deficient mice that lack the immunoglobulin class switching for T cell-dependent antigens. Immunization with ovalbumin did not induce either humoral responses of IgG, IgA, and IgE, or systemic anaphylaxis in CD40-deficient mice. Although systemic anaphylaxis by active immunization was not observed in CD40-deficient mice, both passive cutaneous anaphylaxis (PCA) and passive systemic anaphylaxis assessed by mouse blood pressure monitoring with cervical artery catheterization did take place when antigen-specific IgG was transferred and then antigen challenge given. Further, to investigate the inflammatory pathway of IgG-mediated immediate hypersensitivity reactions, we focused on the Fcγ receptor (FcγR) function. Pretreatment of the mice with the anti-FcγRII/FcγRIII MoAb clearly blocked the response of PCA and passive systemic anaphylaxis, suggesting that they were initiated through FcγR. In conclusion, we directly demonstrate the IgG-mediated anaphylaxis and its triggering mechanism through FcγR in in vivo conditions. In addition to IgE-mediated anaphylaxis, IgG-mediated anaphylaxis should be considered and the blocking of FcγR would provide one of the therapeutic targets for the control of IgG-mediated hypersensitivity diseases. PMID:9822270

  14. 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

  15. 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.

  16. The Orphan Tyrosine Kinase Receptor, ROR2, Mediates Wnt5A Signaling in Metastatic Melanoma

    PubMed Central

    O’Connell, Michael P.; Fiori, Jennifer L.; Xu, Mai; Carter, Arnell D.; Frank, Brittany P.; Camilli, Tura C.; French, Amanda D.; Dissanayake, Samudra K.; Indig, Fred E.; Bernier, Michel; Taub, Dennis D.; Hewitt, Stephen M.; Weeraratna, Ashani T.

    2009-01-01

    Tyrosine kinase receptors represent targets of great interest for cancer therapy. Here we demonstrate, for the first time, the importance of the orphan tyrosine kinase receptor, ROR2, in melanoma progression. Using melanoma tissue microarrays we show that ROR2 is expressed predominantly in metastatic melanoma. Because ROR2 has been shown to specifically interact with the non-canonical Wnt ligand, Wnt5A, this corroborates our previous data implicating Wnt5A as a mediator of melanoma metastasis. We show here that increases in Wnt5A cause increases in ROR2 expression, as well as the PKC-dependent, clathrin-mediated internalization of ROR2. WNT5A knockdown by siRNA decreases ROR2 expression, but silencing of ROR2 has no effect on WNT5A levels. ROR2 knockdown does, however, result in a decrease in signaling downstream of Wnt5A. Using in vitro and in vivo metastasis