Notch1-STAT3-ETBR signaling axis controls reactive astrocyte proliferation after brain injury.

PubMed

LeComte, Matthew D; Shimada, Issei S; Sherwin, Casey; Spees, Jeffrey L

2015-07-14

Defining the signaling network that controls reactive astrogliosis may provide novel treatment targets for patients with diverse CNS injuries and pathologies. We report that the radial glial cell antigen RC2 identifies the majority of proliferating glial fibrillary acidic protein-positive (GFAP(+)) reactive astrocytes after stroke. These cells highly expressed endothelin receptor type B (ETB(R)) and Jagged1, a Notch1 receptor ligand. To study signaling in adult reactive astrocytes, we developed a model based on reactive astrocyte-derived neural stem cells isolated from GFAP-CreER-Notch1 conditional knockout (cKO) mice. By loss- and gain-of-function studies and promoter activity assays, we found that Jagged1/Notch1 signaling increased ETB(R) expression indirectly by raising the level of phosphorylated signal transducer and activator of transcription 3 (STAT3), a previously unidentified EDNRB transcriptional activator. Similar to inducible transgenic GFAP-CreER-Notch1-cKO mice, GFAP-CreER-ETB(R)-cKO mice exhibited a defect in reactive astrocyte proliferation after cerebral ischemia. Our results indicate that the Notch1-STAT3-ETB(R) axis connects a signaling network that promotes reactive astrocyte proliferation after brain injury.

The glucagon-like peptide-1 receptor agonist liraglutide improves hypoxia-induced pulmonary hypertension in mice partly via normalization of reduced ET(B) receptor expression.

PubMed

Honda, J; Kimura, T; Sakai, S; Maruyama, H; Tajiri, K; Murakoshi, N; Homma, S; Miyauchi, T; Aonuma, K

2018-06-27

The glucagon-like peptide-1 receptor (GLP-1R) agonist liraglutide is an incretin hormone mimetic used in the treatment of diabetes. However, the effects of liraglutide on pulmonary hypertension (PH) and pulmonary endothelin (ET) system are unknown. Eight-week-old C57BL6/J mice were injected liraglutide or vehicle for 5 weeks. One week after injection, the mice were exposed to either room air (normoxia) or chronic hypoxia (10 % O(2)) for 4 weeks. The right ventricular systolic pressure (RVSP) was significantly higher in hypoxia + vehicle group than in normoxia + vehicle group. ET-1 mRNA expression in the lungs was comparable among all the groups. ET(B) mRNA and protein expression in the lungs was significantly lower in hypoxia + vehicle group than in normoxia + vehicle group. The above changes were normalized by liraglutide treatment. The expression of phospho-eNOS and phospho-AMPK proteins in the lungs was significantly higher in hypoxia + liraglutide group than in normoxia + vehicle group. We demonstrated for the first time that liraglutide effectively improved RVSP and RV hypertrophy in hypoxia-induced PH mice by activating eNOS through normalization of impaired ET(B) pathway and augmentation of AMPK pathway. Therefore, GLP-1R agonists can be promising therapeutic agents for PH.

Aldosterone does not alter endothelin B receptor signaling in the inner medullary collecting duct.

PubMed

Ramkumar, Nirupama; Stuart, Deborah; Yang, Tianxin; Kohan, Donald E

2017-03-01

Recent studies suggest that aldosterone-mediated sulfenic acid modification of the endothelin B receptor (ETB) promotes renal injury in an ischemia/reperfusion model through reduced ETB-stimulated nitric oxide production. Similarly, aldosterone inactivation of ETB signaling promotes pulmonary artery hypertension. Consequently, we asked whether aldosterone inhibits collecting duct ETB signaling; this could promote fluid retention since CD ETB exerts natriuretic and diuretic effects. A mouse inner medullary collecting duct cell line (IMCD3) was treated with aldosterone for 48 h followed by sarafotoxin-6c, an ETB-selective agonist, and extracellular signal-related kinase 1/2 (ERK) phosphorylation assessed. S6c increased the phospho/total-ERK ratio similarly in control and aldosterone-treated cells (aldosterone alone increased phospho/total-ERK). Since cultured IMCD cell lines lack ETB inhibited AVP signaling, the effect of S6c on AVP-stimulated cAMP in acutely isolated IMCD was assessed. Rats (have much higher CD ETB expression than mice) were exposed to 3 days of a normal or low Na + diet, or low Na + diet + desoxycorticosterone acetate. S6c inhibited AVP-stimulated cAMP in rat IMCD by the same degree in the high mineralocorticoid groups compared to controls. Finally, S6c-stimulated cGMP accumulation in cultured IMCD, or S6c-stimulated nitric oxide or cGMP in acutely isolated IMCD, was not affected by prior aldosterone exposure. These findings provide evidence that aldosterone does not modify ETB effects on ERK phosphorylation, AVP-dependent cAMP inhibition, or NO/cGMP accumulation in the IMCD Thus, while aldosterone can inhibit endothelial cell ETB activity to promote hypertension and injury, this response does not appear to occur in the IMCD. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Angiogenesis and Vascular Architecture in Pheochromocytomas

PubMed Central

Favier, Judith; Plouin, Pierre-François; Corvol, Pierre; Gasc, Jean-Marie

2002-01-01

Angiogenesis is a critical step in tumor growth and metastatic invasion. We here report the study of the vascular status of 10 benign and 9 malignant pheochromocytomas. We examined the vascular architecture after immunostaining endothelial cells (CD34) and vascular smooth muscle cells (α-actin) and identified a vascular pattern characteristic of malignant lesions. To define a gene expression profile indicative of the invasive phenotype, we studied by in situ hybridization the expression of genes encoding several pro- and anti-angiogenic factors [hypoxia-inducible factor (HIF-1α), EPAS1, vascular endothelial growth factor (VEGF), VEGF receptors, angiopoietins and their receptor Tie2, five genes of the endothelin system, and thrombospondin 1]. A semiquantitative evaluation of the labeling revealed an induction of genes encoding EPAS1, VEGF, VEGFR-1, VEGFR-2, endothelin receptor, type B (ETB) and endothelin receptor, type A (ETA) in malignant pheochromocytomas as compared to benign tumors. These differences were observed in tumor cells, in endothelial cells, or in both. Quantification by real-time reverse-transcriptase polymerase chain reaction showed an increase of EPAS1, VEGF, and ETB transcripts of 4.5-, 3.5-, and 10-fold, respectively, in malignant versus benign tumors. Furthermore, we observed a strong correlation between the expression of EPAS1 and VEGF in tumoral tissue and between EPAS1 and ETB in endothelial cells. Altogether, our observations show that analysis of angiogenesis provides promising new criteria for the diagnosis of malignant pheochromocytomas. PMID:12368197

Therapeutic potential of endothelin inhibitors in canine hemangiosarcoma.

PubMed

Fukumoto, Shinya; Saida, Kaname; Sakai, Hiroki; Ueno, Hiroshi; Iwano, Hidetomo; Uchide, Tsuyoshi

2016-08-15

Hemangiosarcoma (HSA) that originates from vascular endothelial cells is the most common splenic malignant neoplasm in dogs, as it accounts for approximately 20% of all canine soft tissue sarcomas. In this study, inhibitory effects of endothelin receptor antagonists on the growth of HSA cells were examined using cell lines established from canine HSA. The preproendothelin-1 (PPET-1), endothelin type A receptor (ETA) and endothelin type B receptor (ETB) mRNA expression levels in HSA cell lines (n=5) were analyzed quantitatively by real-time RT-PCR. These levels were compared with those in HSA tissues (n=11) and those in normal splenic tissues (n=6). ETA and ETB protein expression was examined by western blot. The production and secretion of endothelin-1 (ET-1) and big ET-1 by cell lines were analyzed by measuring the levels in the culture medium by ELISA. The inhibitory effects of endothelin receptor antagonists (ambrisentan, BQ788 and bosentan) on cell growth were evaluated by WST-8 assay. The PPET1 and ETA mRNA expression levels were elevated in HSA tissues and HSA cell lines compared with normal tissues. In cell lines, the production of ET-1 and big ET-1 peptide as well as the expression of ETA protein were detected, but the levels of ETB were not measured. Ambrisentan and bosentan inhibited growth activity in cell lines. Ambrisentan was more effective than bosentan. These findings demonstrate the importance of the ETA axis in canine HSA as well as the potential of ETA inhibitors in the treatment of canine HSA. Copyright © 2015 Elsevier Inc. All rights reserved.

Endothelin B receptor blockade attenuates pulmonary vasodilation in oxygen-ventilated fetal lambs.

PubMed

Ivy, D Dunbar; Lee, Dong-Seok; Rairigh, Robyn L; Parker, Thomas A; Abman, Steven H

2004-01-01

Endothelin-1 (ET-1) contributes to the regulation of pulmonary vascular tone in the normal ovine fetus and in models of perinatal pulmonary hypertension. In the fetal lamb lung, the effects of ET-1 depend on the balance of at least two endothelin receptor subtypes: ETA and ETB. ETA receptors are located on smooth muscle cells and mediate vasoconstriction and smooth muscle proliferation. Stimulation of endothelial ETB receptors causes vasodilation through release of nitric oxide and also functions to remove ET-1 from the circulation. However, whether activation of ETB receptors contributes to the fall in pulmonary vascular tone at birth is unknown. To determine the role of acute ETB receptor blockade in pulmonary vasodilation in response to birth-related stimuli, we studied the hemodynamic effects of selective ETB receptor blockade with BQ-788 during mechanical ventilation with low (<10%) and high FiO2 (100%) in near-term fetal sheep. Intrapulmonary infusion of BQ-788 did not change left pulmonary artery (LPA) blood flow and pulmonary vascular resistance (PVR) at baseline. In comparison with controls, BQ-788 treatment attenuated the rise in LPA flow with low and high FiO2 ventilation (p <0.001 vs. control for each FiO2 concentration). PVR progressively decreased during mechanical ventilation with low and high FiO2 in both groups, but PVR remained higher after BQ-788 treatment throughout the study period (p <0.001). We conclude that selective ETB receptor blockade attenuates pulmonary vasodilation at birth. We speculate that ETB receptor stimulation contributes to pulmonary vasodilation at birth in the ovine fetus.

Endothelin-1 signalling controls early embryonic heart rate in vitro and in vivo.

PubMed

Karppinen, S; Rapila, R; Mäkikallio, K; Hänninen, S L; Rysä, J; Vuolteenaho, O; Tavi, P

2014-02-01

Spontaneous activity of embryonic cardiomyocytes originates from sarcoplasmic reticulum (SR) Ca(2+) release during early cardiogenesis. However, the regulation of heart rate during embryonic development is still not clear. The aim of this study was to determine how endothelin-1 (ET-1) affects the heart rate of embryonic mice, as well as the pathway through which it exerts its effects. The effects of ET-1 and ET-1 receptor inhibition on cardiac contraction were studied using confocal Ca(2+) imaging of isolated mouse embryonic ventricular cardiomyocytes and ultrasonographic examination of embryonic cardiac contractions in utero. In addition, the amount of ET-1 peptide and ET receptor a (ETa) and b (ETb) mRNA levels were measured during different stages of development of the cardiac muscle. High ET-1 concentration and expression of both ETa and ETb receptors was observed in early cardiac tissue. ET-1 was found to increase the frequency of spontaneous Ca(2+) oscillations in E10.5 embryonic cardiomyocytes in vitro. Non-specific inhibition of ET receptors with tezosentan caused arrhythmia and bradycardia in isolated embryonic cardiomyocytes and in whole embryonic hearts both in vitro (E10.5) and in utero (E12.5). ET-1-mediated stimulation of early heart rate was found to occur via ETb receptors and subsequent inositol trisphosphate receptor activation and increased SR Ca(2+) leak. Endothelin-1 is required to maintain a sufficient heart rate, as well as to prevent arrhythmia during early development of the mouse heart. This is achieved through ETb receptor, which stimulates Ca(2+) leak through IP3 receptors. © 2013 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Endothelin Induces Rapid, Dynamin-mediated Budding of Endothelial Caveolae Rich in ET-B*

PubMed Central

Oh, Phil; Horner, Thierry; Witkiewicz, Halina; Schnitzer, Jan E.

2012-01-01

Clathrin-independent trafficking pathways for internalizing G protein-coupled receptors (GPCRs) remain undefined. Clathrin-mediated endocytosis of receptors including ligand-engaged GPCRs can be very rapid and comprehensive (<10 min). Caveolae-mediated endocytosis of ligands and antibodies has been reported to be much slower in cell culture (≫10 min). Little is known about the role of physiological ligands and specific GPCRs in regulating caveolae trafficking. Here, we find that one receptor for endothelin, ET-B but not ET-A, resides on endothelial cell surfaces in both tissue and cell culture primarily concentrated within caveolae. Reconstituted cell-free budding assays show that endothelins (ETs) induce the fission of caveolae from endothelial plasma membranes purified from rat lungs. Electron microcopy of lung tissue sections and tissue subcellular fractionation both show that endothelin administered intravascularly in rats also induces a significant loss of caveolae at the luminal surface of lung vascular endothelium. Endothelial cells in culture show that ET stimulates very rapid internalization of caveolae and cargo including caveolin, caveolae-targeting antibody, and itself. The ET-B inhibitor BQ788, but not the ET-A inhibitor BQ123, blocks the ET-induced budding of caveolae. Both the pharmacological inhibitor Dynasore and the genetic dominant negative K44A mutant of dynamin prevent this induced budding and internalization of caveolae. Also shRNA lentivirus knockdown of caveolin-1 expression prevents rapid internalization of ET and ET-B. It appears that endothelin can engage ET-B already highly concentrated in caveolae of endothelial cells to induce very rapid caveolae fission and endocytosis. This transport requires active dynamin function. Caveolae trafficking may occur more rapidly than previously documented when it is stimulated by a specific ligand to signaling receptors already located in caveolae before ligand engagement. PMID:22457360

Endothelin

PubMed Central

Hyndman, Kelly A.; Dhaun, Neeraj; Southan, Christopher; Kohan, Donald E.; Pollock, Jennifer S.; Pollock, David M.; Webb, David J.; Maguire, Janet J.

2016-01-01

The endothelins comprise three structurally similar 21-amino acid peptides. Endothelin-1 and -2 activate two G-protein coupled receptors, ETA and ETB, with equal affinity, whereas endothelin-3 has a lower affinity for the ETA subtype. Genes encoding the peptides are present only among vertebrates. The ligand-receptor signaling pathway is a vertebrate innovation and may reflect the evolution of endothelin-1 as the most potent vasoconstrictor in the human cardiovascular system with remarkably long lasting action. Highly selective peptide ETA and ETB antagonists and ETB agonists together with radiolabeled analogs have accurately delineated endothelin pharmacology in humans and animal models, although surprisingly no ETA agonist has been discovered. ET antagonists (bosentan, ambrisentan) have revolutionized the treatment of pulmonary arterial hypertension, with the next generation of antagonists exhibiting improved efficacy (macitentan). Clinical trials continue to explore new applications, particularly in renal failure and for reducing proteinuria in diabetic nephropathy. Translational studies suggest a potential benefit of ETB agonists in chemotherapy and neuroprotection. However, demonstrating clinical efficacy of combined inhibitors of the endothelin converting enzyme and neutral endopeptidase has proved elusive. Over 28 genetic modifications have been made to the ET system in mice through global or cell-specific knockouts, knock ins, or alterations in gene expression of endothelin ligands or their target receptors. These studies have identified key roles for the endothelin isoforms and new therapeutic targets in development, fluid-electrolyte homeostasis, and cardiovascular and neuronal function. For the future, novel pharmacological strategies are emerging via small molecule epigenetic modulators, biologicals such as ETB monoclonal antibodies and the potential of signaling pathway biased agonists and antagonists. PMID:26956245

Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension.

PubMed

Bauer, Michael; Wilkens, Heinrike; Langer, Frank; Schneider, Sven O; Lausberg, Henning; Schäfers, Hans-Joachim

2002-03-05

The pulmonary circulation is an important site for the production and clearance of endothelin (ET)-1, a potent vasoactive and mitogenic peptide. Increased plasma ET-1 levels are observed in pulmonary arterial hypertension (PHT) and may contribute to the regulation of pulmonary vascular resistance, as well as to proliferative changes in the pulmonary vascular bed. We prospectively assessed changes in plasma big ET-1 levels and changes in ET(A) and ET(B) receptor gene expression in 14 consecutive patients undergoing pulmonary thromboendarterectomy for thromboembolic PHT. Plasma big ET-1 levels were higher in patients with PHT (median, 2.2 pg/mL; 25th to 75th percentile, 1.5 to 3.0 pg/mL) compared with age-matched controls (median, 1.2 pg/mL; 25th to 75th percentile, 1.0 to 1.4 pg/mL; P=0.002). In addition to increased plasma big ET-1 levels, selective upregulation of ET(B) receptor mRNA transcripts and immunoreactive protein in the pulmonary artery was observed in the patients; however, ET(A) receptor gene expression was unaffected. These data suggest that changes in the ET signaling system in PHT caused by thromboembolic disease are not limited to an increased production of ET-1: they also affect ET receptor gene expression.

Influence of parainfluenza-1 respiratory tract viral infection on endothelin receptor-effector systems in mouse and rat tracheal smooth muscle.

PubMed Central

Knott, P. G.; Henry, P. J.; McWilliam, A. S.; Rigby, P. J.; Fernandes, L. B.; Goldie, R. G.

1996-01-01

1. In this study we have compared the effects of parainfluenza-1 respiratory tract viral infection on the density and function of ETA and ETB receptors in rat and mouse tracheal airway smooth muscle. 2. The bronchoconstrictor effect of inhaled methacholine was significantly enhanced in virus-infected rats, at both 4 and 12 days post-inoculation. That is, the concentration of methacholine causing an increase in resistance of 100% (PC100 methacholine) was significantly lower in virus-infected animals at both 4 and 12 days post-inoculation (n = 6-8; P < 0.05). 3. Total specific binding of [125I]-endothelin-1 and the relative proportions of ETA and ETB binding sites for [125I]-endothelin-1 were assessed in tracheal airway smooth muscle in parainfluenza-1-infected rats and mice at days 2, 4 and 12 post-inoculation using the ligands BQ-123 (1 microM; ETA receptor-selective) and sarafotoxin S6c (100 nM; ETB receptor-selective). Total specific binding in mice was significantly reduced at day 2 post-inoculation (n = 5; P < 0.05) but not at days 4 and 12 post-inoculation (n = 5). In control mice, the proportions of ETA and ETB binding sites were 53%:47% at day 2 and 43%:57% at day 4 and these were significantly altered by parainfluenza-1 infection such that, the ratios were 81%:19% at day 2 and 89%:11% at day 4 (P < 0.05). By day 12 post-inoculation, the proportion of ETA and ETB binding sites in tracheal smooth muscle from mice infected with parainfluenza-1 was not significantly different from control. In rat tracheal airway smooth muscle, neither total specific binding nor the ETA and ETB binding site ratio (64%:36%) were significantly altered in virus-inoculated rats at days 2, 4 or 12 post-inoculation (n = 5). 4. Parainfluenza-1 infection in mice had no effect on the sensitivity or maximal contractile effect of endothelin-1 in tracheal smooth muscle at days 2, 4 or 12 post-inoculation (n = 4). In contrast, contraction in response to the ETB receptor-selective agonist sarafotoxin S6c was attenuated by 39% at day 2 and by 93% at day 4 post-inoculation (P < 0.05). However, by day 12 post-inoculation, contractions to sarafotoxin S6c were not significantly different between control and virus-infected mice. In parainfluenza-1-infected rats, there were small but significant reductions in the sensitivity to carbachol, endothelin-1 and sarafotoxin S6c whilst the maximal responses to the highest concentrations of these agonists were not significantly altered by virus infection (n = 8). 5. BQ-123 (3 microM) had no significant effect on cumulative concentration-effect curves to endothelin-1 in tracheal preparations from control mice (n = 4) or parainfluenza-1-infected rats (n = 8). In contrast, in tissues taken from virus-infected mice at day 4 post-inoculation, BQ-123 caused a marked 9.6 fold rightward shift in the concentration-effect curve to endothelin-1 (n = 4). 6. In summary, we have demonstrated that parainfluenza-1 infection in mice transiently reduced the density of tracheal airway smooth muscle ETB receptors and this was reflected in reduced responsiveness to the ETB receptor-selective agonist sarafotoxin S6c. In contrast, whilst parainfluenza-1 infection in rats was associated with the pathological features and bronchial hyperresponsiveness common to respiratory tract viral infection, there was no selective down-regulation of ETB receptor expression or functional activity. The reasons for these species differences are not clear, but may relate to differences in the airway inflammatory response to parainfluenza-1 virus. PMID:8886411

Expression profile of endothelin receptors (ETA and ETB) and microRNAs-155 and -199 in the corpus cavernosum of rats submitted to chronic alcoholism and diabetes mellitus.

PubMed

Gonçalves, F Z; Lizarte Neto, F S; Novais, P C; Gattas, D; Lourenço, L G; de Carvalho, C A M; Tirapelli, D P C; Molina, C A F; Tirapelli, L F; Tucci, S

2018-03-01

Recent evidence shows that chronic ethanol consumption increases endothelin (ET)-1 induced sustained contraction of trabecular smooth muscle cells of the corpora cavernosa in corpus cavernosum of rats by a mechanism that involves increased expression of ETA and ETB receptors. Our goal was to evaluate the effects of alcohol and diabetes and their relationship to miRNA-155, miRNA-199 and endothelin receptors in the corpus cavernosum and blood of rats submitted to the experimental model of diabetes mellitus and chronic alcoholism. Forty-eight male Wistar rats were divided into four groups: control (C), alcoholic (A), diabetic (D), and alcoholic-diabetic (AD). Samples of the corpus cavernosum were prepared to study the protein expression of endothelin receptors by immunohistochemistry and expression of miRNAs-155 and -199 in serum and the cavernous tissue. Immunostaining for endothelin receptors was markedly higher in the A, D, and AD groups than in the C group. Moreover, a significant hypoexpression of the miRNA-199 in the corpus cavernosum tissue from the AD group was observed, compared to the C group. When analyzing the microRNA profile in blood, a significant hypoexpression of miRNA-155 in the AD group was observed compared to the C group. The miRNA-199 analysis demonstrated significant hypoexpression in D and AD groups compared to the C group. Our findings in corpus cavernosum showed downregulated miRNA-155 and miRNA-199 levels associated with upregulated protein expression and unaltered mRNA expression of ET receptors suggesting decreased ET receptor turnover, which can contribute to erectile dysfunction in diabetic rats exposed to high alcohol levels.

Both endothelin-A and endothelin-B receptors are present on adult rat cardiac ventricular myocytes.

PubMed

Allen, Bruce G; Phuong, Luu Lien; Farhat, Hala; Chevalier, Dominique

2003-02-01

Endothelin-A (ET(A)) and endothelin-B (ET(B)) receptors have been demonstrated in intact heart and cardiac membranes. ET(A) receptors have been demonstrated on adult ventricular myocytes. The aim of the present study was to determine the presence of ET(B) and the relative contribution of this receptor subtype to total endothelin-1 (ET-1) binding on adult ventricular myocytes. Saturation binding experiments indicated that ET-1 bound to a single population of receptors (Kd = 0.52 +/- 0.13 nM, n = 4) with an apparent maximum binding (Bmax) of 2.10 +/- 0.25 sites (x 10(5))/cell (n = 4). Competition experiments using 40 pM [125I]ET-1 and nonradioactive ET-1 revealed a Ki of 660 +/- 71 pM (n = 10) and a Hill coefficient (nH) of 0.99 +/- 0.10 (n = 10). A selective ET(A) antagonist, BQ610, displaced 80% of the bound [125I]ET-1. No displacement was observed by concentrations of an ET(B)-selective antagonist, BQ788, up to 1.0 microM. However, in the presence of 1.0 microM BQ610, BQ788 inhibited the remaining [125I]ET-1 binding. Similarly, in the presence of 1.0 microM BQ788, BQ610 inhibited the remaining specific [125I]ET-1 binding. Binding of an ET(B1)-selective agonist, [125I]IRL-1620, confirmed the presence of ET(B). ET(B) bound to ET-1 irreversibly, whereas binding to ET(A) demonstrated both reversible and irreversible components, and BQ610 and BQ788 bound reversibly. Reducing the incubation temperature to 0 degrees C did not alter the irreversible component of ET-1 binding. Hence, both ET(A) and ET(B) receptors are present on intact adult rat ventricular myocytes, and the ratio of ET(A):ET(B) binding sites is 4:1. Both receptor subtypes bind to ET-1 by a two-step association involving the formation of a tight receptor-ligand complex; however, the kinetics of ET-1 binding to ET(A) versus ET(B) differ.

Amelioration of Cold Injury-Induced Cortical Brain Edema Formation by Selective Endothelin ETB Receptor Antagonists in Mice

PubMed Central

Michinaga, Shotaro; Nagase, Marina; Matsuyama, Emi; Yamanaka, Daisuke; Seno, Naoki; Fuka, Mayu; Yamamoto, Yui; Koyama, Yutaka

2014-01-01

Brain edema is a potentially fatal pathological condition that often occurs in stroke and head trauma. Following brain insults, endothelins (ETs) are increased and promote several pathophysiological responses. This study examined the effects of ETB antagonists on brain edema formation and disruption of the blood-brain barrier in a mouse cold injury model (Five- to six-week-old male ddY mice). Cold injury increased the water content of the injured cerebrum, and promoted extravasation of both Evans blue and endogenous albumin. In the injury area, expression of prepro-ET-1 mRNA and ET-1 peptide increased. Intracerebroventricular (ICV) administration of BQ788 (ETB antagonist), IRL-2500 (ETB antagonist), or FR139317 (ETA antagonist) prior to cold injury significantly attenuated the increase in brain water content. Bolus administration of BQ788, IRL-2500, or FR139317 also inhibited the cold injury-induced extravasation of Evans blue and albumin. Repeated administration of BQ788 and IRL-2500 beginning at 24 h after cold injury attenuated both the increase in brain water content and extravasation of markers. In contrast, FR139317 had no effect on edema formation when administrated after cold injury. Cold injury stimulated induction of glial fibrillary acidic protein-positive reactive astrocytes in the injured cerebrum. Induction of reactive astrocytes after cold injury was attenuated by ICV administration of BQ788 or IRL-2500. These results suggest that ETB receptor antagonists may be an effective approach to ameliorate brain edema formation following brain insults. PMID:25000290

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

El-Gowelli, Hanan M.; Helmy, Maged W.; Ali, Rabab M.

Endothelin (ET) signaling provokes nephrotoxicity induced by the immunosuppressant drug cyclosporine A (CSA). We tested the hypotheses that (i): celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, counterbalances renal derangements caused by CSA in rats and (ii) the COX-2/endothelin ET{sub B} receptor signaling mediates the CSA-celecoxib interaction. Ten-day treatment with CSA (20 mg/kg/day) significantly increased biochemical indices of renal function (serum urea, creatinine), inflammation (interleukin-2, IL-2) and fibrosis (transforming growth factor-β{sub 1}, TGF-β{sub 1}). Histologically, CSA caused renal tubular atrophy along with interstitial fibrosis. These detrimental renal effects of CSA were largely reduced in rats treated concurrently with celecoxib (10 mg/kg/day). Wemore » also report that cortical glomerular and medullary tubular protein expressions of COX-2 and ET{sub B} receptors were reduced by CSA and restored to near-control values in rats treated simultaneously with celecoxib. The importance of ET{sub B} receptors in renal control and in the CSA-celecoxib interaction was further verified by the findings (i) most of the adverse biochemical, inflammatory, and histopathological profiles of CSA were replicated in rats treated with the endothelin ET{sub B} receptor antagonist BQ788 (0.1 mg/kg/day, 10 days), and (ii) the BQ788 effects, like those of CSA, were alleviated in rats treated concurrently with celecoxib. Together, the data suggest that the facilitation of the interplay between the TGF-β1/IL-2/COX-2 pathway and the endothelin ET{sub B} receptors constitutes the cellular mechanism by which celecoxib ameliorates the nephrotoxic manifestations of CSA in rats. - Highlights: • Celecoxib abolishes nephrotoxic manifestations of CSA in rats. • Blockade of ETB receptors by BQ788 mimicked the nephrotoxic effects of CSA. • CSA or BQ788 reduces renal protein expression of COX-2 and endothelin ETB receptors. • Enhanced TGFβ1/IL-2/COX2/ETB signaling mediates celecoxib renoprotection.« less

Endothelin receptor-specific control of endoplasmic reticulum stress and apoptosis in the kidney

PubMed Central

De Miguel, Carmen; Hamrick, William C.; Hobbs, Janet L.; Pollock, David M.; Carmines, Pamela K.; Pollock, Jennifer S.

2017-01-01

Endothelin-1 (ET-1) promotes renal damage during cardiovascular disease; yet, the molecular mechanisms involved remain unknown. Endoplasmic reticulum (ER) stress, triggered by unfolded protein accumulation in the ER, contributes to apoptosis and organ injury. These studies aimed to determine whether the ET-1 system promotes renal ER stress development in response to tunicamycin. ETB deficient (ETB def) or transgenic control (TG-con) rats were used in the presence or absence of ETA receptor antagonism. Tunicamycin treatment similarly increased cortical ER stress markers in both rat genotypes; however, only ETB def rats showed a 14–24 fold increase from baseline for medullary GRP78, sXBP-1, and CHOP. Pre-treatment of TG-con rats with the ETA blocker ABT-627 for 1 week prior to tunicamycin injection significantly reduced the ER stress response in cortex and medulla, and also inhibited renal apoptosis. Pre-treatment with ABT-627 failed to decrease renal ER stress and apoptosis in ETB def rats. In conclusion, the ET-1 system is important for the development of tunicamycin-induced renal ER stress and apoptosis. ETA receptor activation induces renal ER stress genes and apoptosis, while functional activation of the ETB receptor has protective effects. These results highlight targeting the ETA receptor as a therapeutic approach against ER stress-induced kidney injury. PMID:28230089

Androgens influence microvascular dilation in PCOS through ET-A and ET-B receptors

PubMed Central

Wenner, Megan M.; Taylor, Hugh S.

2013-01-01

Hyperandrogenism and vascular dysfunction often coexist in women with polycystic ovary syndrome (PCOS). We hypothesized that testosterone compromises cutaneous microvascular dilation in women with PCOS via the endothelin-1 ET-B subtype receptor. To control and isolate testosterone's effects on microvascular dilation, we administered a gonadotropin-releasing hormone antagonist (GnRHant) for 11 days in obese, otherwise healthy women [controls, 22.0 (4) yr, 36.0 (3.2) kg/m2] or women with PCOS [23 (4) yr, 35.4 (1.3) kg/m2], adding testosterone (T; 2.5 mg/day) on days 8–11. Using laser Doppler flowmetry and cutaneous microdialysis, we measured changes in skin microcirculatory responsiveness (ΔCVC) to local heating while perfusing ET-A (BQ-123) and ET-B (BQ-788) receptor antagonists under three experimental conditions: baseline (BL; prehormone intervention), GnRHant (day 4 of administration), and T administration. At BL, ET-A receptor inhibition enhanced heat-induced vasodilation in both groups [ΔCVC control 2.03 (0.65), PCOS 2.10 (0.25), AU/mmHg, P < 0.05]; ET-B receptor inhibition reduced vasodilation in controls only [ΔCVC 0.98 (0.39), 1.41 (0.45) AU/mmHg for controls, PCOS] compared with saline [ΔCVC controls 1.27 (0.48), PCOS 1.31 (0.13) AU/mmHg]. GnRHant enhanced vasodilation in PCOS [saline ΔCVC 1.69 (0.23) AU/mmHg vs. BL, P < 0.05] and abolished the ET-A effect in both groups, a response reasserted with T in controls. ET-B receptor inhibition reduced heat-induced vasodilation in both groups during GnRHant and T [ΔCVC, controls: 0.95 (0.21) vs. 0.51 (13); PCOS: 1.27 (0.23) vs. 0.84 (0.27); for GnRHant vs. T, P < 0.05]. These data demonstrate that androgen suppression improves microvascular dilation in PCOS via ET-A and ET-B receptors. PMID:23921139

The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease

PubMed Central

Koehl, Bérengère; Nivoit, Pierre; El Nemer, Wassim; Lenoir, Olivia; Hermand, Patricia; Pereira, Catia; Brousse, Valentine; Guyonnet, Léa; Ghinatti, Giulia; Benkerrou, Malika; Colin, Yves; Le Van Kim, Caroline; Tharaux, Pierre-Louis

2017-01-01

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ETB receptor, strongly influences neutrophil recruitment under inflammatory conditions in sickle cell disease. We show that human neutrophils have functional ETB receptors with calcium signaling capability, leading to increased adhesion to the endothelium through effects on both endothelial cells and neutrophils. Intact ETB function was found to be required for tumor necrosis factor α-dependent upregulation of CD11b on neutrophils. Furthermore, we confirmed that human neutrophils synthesize endothelin-1, which may be involved in autocrine and paracrine pathophysiological actions. Thus, the endothelin-ETB axis should be considered as a cytokine-like potent pro-inflammatory pathway in sickle cell disease. Blockade of endothelin receptors, including ETB, may provide major benefits for preventing or treating vaso-occlusive crises in sickle cell patients. PMID:28385784

Endothelin receptor type B agonist, IRL-1620, prevents beta amyloid (Aβ) induced oxidative stress and cognitive impairment in normal and diabetic rats.

PubMed

Briyal, Seema; Shepard, Cortney; Gulati, Anil

2014-05-01

Alzheimer's disease (AD) is a progressive brain disorder leading to impairment of learning and memory. Amyloid β (Aβ) induced oxidative stress has been implicated in the initiation and progression of AD. Endothelin (ET) and its receptors have been considered as therapeutic targets for AD. Recent studies indicate that stimulation of ETB receptors may provide neuroprotection. The purpose of this study was to determine the preventative effect of selectively stimulating ETB receptors on cognitive impairment and oxidative stress in Aβ treated non-diabetic and diabetic (induced by streptozotocin) rats. Rats were concurrently treated with Aβ1-40 (day 1, 7 and 14) and either saline, IRL-1620 (an ETB agonist), and/or BQ788 (an ETB antagonist) daily for 14 days in the lateral cerebral ventricles using sterotaxically implanted cannula; experiments were performed on day 15. Aβ treatment produced a significant (p<0.0001) increase of 360% and 365% in malondialdehyde levels (a marker of lipid peroxidation) in non-diabetic and diabetic rats, respectively, compared to sham group. Antioxidants (superoxide dismutase and reduced glutathione) decreased following Aβ treatment compared to sham group. Treatment with IRL-1620 reversed these effects, indicating that ETB receptor stimulation reduces oxidative stress injury following Aβ treatment. In Morris swim task, Aβ treated rats showed impairment in spatial memory. Rats treated with IRL-1620 significantly reduced the cognitive impairment induced by Aβ. BQ788 treatment completely blocked IRL-1620 induced reduction in oxidative stress and cognitive impairment. Results of the present study demonstrate that IRL-1620 improved both acquisition (learning) and retention (memory) on water maze task and reduced oxidative stress parameters. It can be speculated that ETB receptor stimulation prevents cognitive impairment and may be useful in neurodegenerative diseases. Copyright © 2014 Elsevier Inc. All rights reserved.

Activation of either the ETA or the ETB receptors is involved in the development of electrographic seizures following intrahippocampal infusion of the endothelin-1 in immature rats.

PubMed

Tsenov, Grygoriy; Vondrakova, Katerina; Otahal, Jakub; Burchfiel, James; Kubova, Hana

2015-03-01

The period around birth is a risky time for stroke in infants, which is associated with two major acute and subacute processes: anatomical damage and seizures. It is unclear as to what extent each of these processes independently contributes to poor outcome. Furthermore, it is unclear whether there is an interaction between the two processes - does seizure activity cause additional brain damage beyond that produced by ischemia and/or does brain damage foster seizures? The model of focal cerebral ischemia induced by the intrahippocampal infusion of endothelin-1 (ET-1) in 12-day-old rat was used to examine the role of the endothelin receptors in the development of focal ischemia, symptomatic acute seizures and neurodegeneration. ET-1 (40pmol/μl) was infused either alone or co-administered with selective antagonists of ETA (BQ123; 70nmol/μl) or ETB receptors (BQ788; 70nmol/1μl). Effects of activation of ETB receptors were studied using selective agonist 4-Ala-ET-1 (40pmol/1μl). Regional cerebral blood flow (rCBF) and tissue oxygenation (pO2) were measured in anesthetized animals with a Doppler-flowmeter and a pO2-sensor, respectively. Seizure development was assessed with video-EEG in freely moving rats. Controls received the corresponding volume of the appropriate vehicle (10mM PBS or 0.01% DMSO-PBS solution; pH7.4). The extent of hippocampal lesion was determined using FluoroJade B staining performed 24h after ET-1 infusion. Infusion of ET-1 or ET-1+ETB receptor antagonist reduced rCBF to ~25% and pO2 to ~10% for about 1.5h, whereas selective ETB agonist, ET-1+ETA antagonist and the PBS vehicle had only negligible effect on the rCBF and pO2 levels. Reduction of rCBF was associated with the development of lesion in the injected hippocampus. In all groups, except sham operated and PBS controls, epileptiform activity was observed after activation of the ETA or the ETB receptors. The data revealed a positive correlation between the severity of morphological damage and all the measured seizure parameters (seizure frequency, average and total seizure duration) in the ET-1 group. In addition, the severity of morphological damage positively correlated with the average seizure duration in animals after infusion of ET-1+ETA receptor antagonist or after infusion of ET-1+ETB receptor antagonist. Our results indicate that the activation of ETA receptors is crucial for ischemia development, however either ETA or ETB receptors mediate the development of seizures following the application of ET-1 in immature rats. The dissociation between the ischemic-producing and seizure-producing processes suggests that damage is not necessary to induce seizures, although it may exacerbate them. Copyright © 2014 Elsevier Inc. All rights reserved.

Discovery of Dual ETA/ETB Receptor Antagonists from Traditional Chinese Herbs through in Silico and in Vitro Screening

PubMed Central

Wang, Xing; Zhang, Yuxin; Liu, Qing; Ai, Zhixin; Zhang, Yanling; Xiang, Yuhong; Qiao, Yanjiang

2016-01-01

Endothelin-1 receptors (ETAR and ETBR) act as a pivotal regulator in the biological effects of ET-1 and represent a potential drug target for the treatment of multiple cardiovascular diseases. The purpose of the study is to discover dual ETA/ETB receptor antagonists from traditional Chinese herbs. Ligand- and structure-based virtual screening was performed to screen an in-house database of traditional Chinese herbs, followed by a series of in vitro bioassay evaluation. Aristolochic acid A (AAA) was first confirmed to be a dual ETA/ETB receptor antagonist based intracellular calcium influx assay and impedance-based assay. Dose-response curves showed that AAA can block both ETAR and ETBR with IC50 of 7.91 and 7.40 μM, respectively. Target specificity and cytotoxicity bioassay proved that AAA is a selective dual ETA/ETB receptor antagonist and has no significant cytotoxicity on HEK293/ETAR and HEK293/ETBR cells within 24 h. It is a feasible and effective approach to discover bioactive compounds from traditional Chinese herbs using in silico screening combined with in vitro bioassay evaluation. The structural characteristic of AAA for its activity was especially interpreted, which could provide valuable reference for the further structural modification of AAA. PMID:26999111

Differential roles of endothelin-1 ETA and ETB receptors and vasoactive intestinal polypeptide in regulation of the airways and the pulmonary vasculature in isolated rat lung.

PubMed

Janosi, Tibor; Peták, Ferenc; Fontao, Fabienne; Morel, Denis R; Beghetti, Maurice; Habre, Walid

2008-11-01

The available treatment strategies against pulmonary hypertension include the administration of endothelin-1 (ET-1) receptor subtype blockers (ET(A) and ET(B) antagonists); vasoactive intestinal polypeptide (VIP) has recently been suggested as a potential new therapeutic agent. We set out to investigate the ability of these agents to protect against the vasoconstriction and impairment of lung function commonly observed in patients with pulmonary hypertension. An ET(A) blocker (BQ123), ET(B) blocker (BQ788), a combination of these selective blockers (ET(A) + ET(B) blockers) or VIP (V6130) was administered into the pulmonary circulation in four groups of perfused normal rat lungs. Pulmonary vascular resistance (PVR) and forced oscillatory lung input impedance (Z(L)) were measured in all groups under baseline conditions and at 1 min intervals following ET-1 administrations. The airway resistance, inertance, tissue damping and elastance were extracted from the Z(L) spectra. While VIP, ET(A) blocker and combined ET(A) and ET(B) blockers significantly prevented the pulmonary vasoconstriction induced by ET-1, ET(B) blockade enhanced the ET-1-induced increases in PVR. In contrast, the ET(A) and ET(B) blockers markedly elevated the ET-1-induced increases in airway resistance, while VIP blunted this constrictor response. Our results suggest that VIP potently acts against the airway and pulmonary vascular constriction mediated by endothelin-1, while the ET(A) and ET(B) blockers exert a differential effect between airway resistance and PVR.

Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

PubMed

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

1995-11-01

1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD151242 competed for the binding of [125I]-ET-1 monophasically and analysis of the competition curves indicated that a one-site fit was preferred over a two-site model, implying that the cultures express mainly ETA receptors. 6. Although messenger RNA encoding both ETA and ETB receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ETA receptor protein. Antagonism of this sub-type may be necessary to block the actions of ET-1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage.

Vascular endothelial cells mediate mechanical stimulation-induced enhancement of endothelin hyperalgesia via activation of P2X2/3 receptors on nociceptors.

PubMed

Joseph, Elizabeth K; Green, Paul G; Bogen, Oliver; Alvarez, Pedro; Levine, Jon D

2013-02-13

Endothelin-1 (ET-1) is unique among a broad range of hyperalgesic agents in that it induces hyperalgesia in rats that is markedly enhanced by repeated mechanical stimulation at the site of administration. Antagonists to the ET-1 receptors, ET(A) and ET(B), attenuated both initial as well as stimulation-induced enhancement of hyperalgesia (SIEH) by endothelin. However, administering antisense oligodeoxynucleotide to attenuate ET(A) receptor expression on nociceptors attenuated ET-1 hyperalgesia but had no effect on SIEH, suggesting that this is mediated via a non-neuronal cell. Because vascular endothelial cells are both stretch sensitive and express ET(A) and ET(B) receptors, we tested the hypothesis that SIEH is dependent on endothelial cells by impairing vascular endothelial function with octoxynol-9 administration; this procedure eliminated SIEH without attenuating ET-1 hyperalgesia. A role for protein kinase Cε (PKCε), a second messenger implicated in the induction and maintenance of chronic pain, was explored. Intrathecal antisense for PKCε did not inhibit either ET-1 hyperalgesia or SIEH, suggesting no role for neuronal PKCε; however, administration of a PKCε inhibitor at the site of testing selectively attenuated SIEH. Compatible with endothelial cells releasing ATP in response to mechanical stimulation, P2X(2/3) receptor antagonists eliminated SIEH. The endothelium also appears to contribute to hyperalgesia in two ergonomic pain models (eccentric exercise and hindlimb vibration) and in a model of endometriosis. We propose that SIEH is produced by an effect of ET-1 on vascular endothelial cells, sensitizing its release of ATP in response to mechanical stimulation; ATP in turn acts at the nociceptor P2X(2/3) receptor.

Cooperation of endothelin-1 signaling with melanosomes plays a role in developing and/or maintaining human skin hyperpigmentation

PubMed Central

Murase, Daiki; Hachiya, Akira; Kikuchi-Onoe, Mamiko; Fullenkamp, Rachel; Ohuchi, Atsushi; Kitahara, Takashi; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori

2015-01-01

ABSTRACT Skin hyperpigmentation is characterized by increased melanin synthesis and deposition that can cause significant psychosocial and psychological distress. Although several cytokine-receptor signaling cascades contribute to the formation of ultraviolet B-induced cutaneous hyperpigmentation, their possible involvement in other types of skin hyperpigmentation has never been clearly addressed. Since our continuous studies using skin specimens from more than 30 subjects with ethnic skin diversity emphasized a consistent augmentation in the expression of endothelin-1 (ET-1) and its receptor (Endothelin B receptor, ET-B) in hyperpigmented lesions, including senile lentigos (SLs), the precise function of ET-1 signaling was investigated in the present study. In line with previous studies, ET-1 significantly induced melanogenesis followed by increases in melanosome transport in melanocytes and in its transfer to keratinocytes while inhibition of ET-B function substantially depressed melanogenic ability in tissue-cultured SLs. Additionally, in agreement with a previous report that the formation of autophagosomes rather than melanosomes is stimulated according to starvation or defective melanosome production, ET-1 was found to remarkably augment the expression of components necessary for early melanosome formation, indicating its counteraction against autophagy-targeting melanosome degradation in melanocytes. Despite the lack of substantial impact of ET-1 on keratinocyte melanogenic functions, the expression of ET-1 was enhanced following melanosome uptake by keratinocytes. Taken together, our data suggest that ET-1 plays a substantial role in the development and/or maintenance of skin hyperpigmentation in reciprocal cooperation with increased melanosome incorporation. PMID:26340945

Selective endothelin A receptor antagonism with sitaxentan reduces neointimal lesion size in a mouse model of intraluminal injury

PubMed Central

Duthie, Karolina M; Hadoke, Patrick W F; Kirkby, Nicholas S; Miller, Eileen; Ivy, Jessica R; McShane, John F; Lim, Win Gel; Webb, David J

2015-01-01

Background and Purpose Endothelin (ET) receptor antagonism reduces neointimal lesion formation in animal models. This investigation addressed the hypothesis that the selective ETA receptor antagonist sitaxentan would be more effective than mixed ETA/B receptor antagonism at inhibiting neointimal proliferation in a mouse model of intraluminal injury. Experimental Approach Antagonism of ETA receptors by sitaxentan (1–100 nM) was assessed in femoral arteries isolated from adult, male C57Bl6 mice using isometric wire myography. Neointimal lesion development was induced by intraluminal injury in mice receiving sitaxentan (ETA antagonist; 15 mg·kg−1·day−1), A192621 (ETB antagonist; 30 mg·kg−1·day−1), the combination of both antagonists or vehicle. Treatment began 1 week before, and continued for 28 days after, surgery. Femoral arteries were then harvested for analysis of lesion size and composition. Key Results Sitaxentan produced a selective, concentration-dependent parallel rightward shift of ET-1-mediated contraction in isolated femoral arteries. Sitaxentan reduced neointimal lesion size, whereas ETB and combined ETA/B receptor antagonism did not. Macrophage and α-smooth muscle actin content were unaltered by ET receptor antagonism but sitaxentan reduced the amount of collagen in lesions. Conclusions and Implications These results suggest that ETA receptor antagonism would be more effective than combined ETA/ETB receptor antagonism at reducing neointimal lesion formation. PMID:25598351

Hyaluronan Production by Renomedullary Interstitial Cells: Influence of Endothelin, Angiotensin II and Vasopressin

PubMed Central

Palm, Fredrik; Takahashi, Tomoko; Ikegami-Kawai, Mayumi; Friederich-Persson, Malou; Hansell, Peter

2017-01-01

The content of hyaluronan (HA) in the interstitium of the renal medulla changes in relation to body hydration status. We investigated if hormones of central importance for body fluid homeostasis affect HA production by renomedullary interstitial cells in culture (RMICs). Simultaneous treatment with vasopressin and angiotensin II (Ang II) reduced HA by 69%. No change occurred in the mRNA expressions of hyaluronan synthase 2 (HAS2) or hyaluronidases (Hyals), while Hyal activity in the supernatant increased by 67% and CD44 expression reduced by 42%. The autocoid endothelin (ET-1) at low concentrations (10−10 and 10−8 M) increased HA 3-fold. On the contrary, at a high concentration (10−6 M) ET-1 reduced HA by 47%. The ET-A receptor antagonist BQ123 not only reversed the reducing effect of high ET-1 on HA, but elevated it to the same level as low concentration ET-1, suggesting separate regulating roles for ET-A and ET-B receptors. This was corroborated by the addition of ET-B receptor antagonist BQ788 to low concentration ET-1, which abolished the HA increase. HAS2 and Hyal2 mRNA did not alter, while Hyal1 mRNA was increased at all ET-1 concentrations tested. Hyal activity was elevated the most by high ET-1 concentration, and blockade of ET-A receptors by BQ123 prevented about 30% of this response. The present study demonstrates an important regulatory influence of hormones involved in body fluid balance on HA handling by RMICs, thereby supporting the concept of a dynamic involvement of interstitial HA in renal fluid handling. PMID:29236055

Endothelin antagonism in portal hypertensive mice: implications for endothelin receptor-specific signaling in liver disease

PubMed Central

Feng, Hong-Qiang; Weymouth, Nate D.; Rockey, Don C.

2009-01-01

Endothelin-1 (ET-1), a potent vasoactive peptide, plays an important role in the pathogenesis of liver disease and portal hypertension. Two major endothelin receptors (ET-A and ET-B) mediate biological effects, largely on the basis of their known downstream signaling pathways. We hypothesized that the different receptors are likely to mediate divergent effects in portal hypertensive mice. Liver fibrosis and cirrhosis and portal hypertension were induced in 8-wk-old male BALB/c mice by gavage with carbon tetrachloride (CCl4). Portal pressure was recorded acutely during intravenous infusion of endothelin receptor antagonists in normal or portal hypertensive mice. In vivo microscopy was used to monitor sinusoidal dynamics. Additionally, the effect of chronic exposure to endothelin antagonists was assessed in mice during induction of fibrosis and cirrhosis with CCl4 for 8 wk. Intravenous infusion of ET-A receptor antagonists into normal and cirrhotic mice reduced portal pressure whereas ET-B receptor antagonism increased portal pressure. A mixed endothelin receptor antagonist also significantly reduced portal pressure. Additionally, the ET-A receptor antagonist caused sinusoidal dilation, whereas the ET-B receptor antagonist caused sinusoidal constriction. Chronic administration of each the endothelin receptor antagonists during the induction of fibrosis and portal hypertension led to reduced fibrosis, a significant reduction in portal pressure, and altered sinusoidal dynamics relative to controls. Acute effects of endothelin receptor antagonists are likely directly on the hepatic and sinusoidal vasculature, whereas chronic endothelin receptor antagonism appears to be more complicated, likely affecting fibrogenesis and the hepatic microcirculation. The data imply a relationship between hepatic fibrogenesis or fibrosis and vasomotor responses. PMID:19299580

Tumour necrosis factor-alpha participates on the endothelin-1/nitric oxide imbalance in small arteries from obese patients: role of perivascular adipose tissue.

PubMed

Virdis, Agostino; Duranti, Emiliano; Rossi, Chiara; Dell'Agnello, Umberto; Santini, Eleonora; Anselmino, Marco; Chiarugi, Massimo; Taddei, Stefano; Solini, Anna

2015-04-01

We assessed the impact of vascular and perivascular tumour necrosis factor-alpha (TNF-α) on the endothelin (ET)-1/nitric oxide (NO) system and the molecular pathways involved in small arteries from visceral fat of obese patients (Obese) and Controls. Isolated small arteries from 16 Obese and 14 Controls were evaluated on a pressurized micromyograph. Endogenous ET-1 activity was assessed by the ETA blocker BQ-123. TNF-α and NO were tested by anti-TNF-α infliximab (IFX) and N(ω)-nitro-l-arginine methylester (L-NAME). Gene and protein expression of TNF-α, ET-1, ETA, and ETB receptors were determined by RT-PCR and IHC on arterial wall and in isolated adipocytes. Obese showed a blunted L-NAME-induced vasoconstriction, which was potentiated by IFX, and an increased relaxation to BQ-123, unaffected by L-NAME but attenuated by IFX. Perivascular adipose tissue (PVAT) removal reversed these effects. Obese showed intravascular superoxide excess, which was decreased by apocynin (NAD(P)H oxidase inhibitor), L-NAME, and BQ-123 incubations, and abolished by IFX. An increased vascular expression of ET-1, ETA, and ETB receptors, and higher vascular/perivascular TNF-α and TNF-α receptor expression were also detected. The arterial expression and phosphorylation of c-Jun N-terminal kinase (JNK) were higher in Obese vs. Controls, and downregulated by IFX. In small arteries of Obese, PVAT-derived TNF-α excess, and an increased vascular expression of ET-1 and ETA receptor, contribute to the ET-1/NO system imbalance, by impairing tonic NO release. Reactive oxygen species excess, via NAD(P)H oxidase activation, induces the endothelial nitric oxide synthase uncoupling, which in turn generates superoxide and impairs NO production. The up-regulated JNK pathway represents a crucial molecular signalling involved in this process. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.

Different actions of endothelin-1 on chemokine production in rat cultured astrocytes: reduction of CX3CL1/fractalkine and an increase in CCL2/MCP-1 and CXCL1/CINC-1

PubMed Central

2013-01-01

Background Chemokines are involved in many pathological responses of the brain. Astrocytes produce various chemokines in brain disorders, but little is known about the factors that regulate astrocytic chemokine production. Endothelins (ETs) have been shown to regulate astrocytic functions through ETB receptors. In this study, the effects of ETs on chemokine production were examined in rat cerebral cultured astrocytes. Methods Astrocytes were prepared from the cerebra of one- to two-day-old Wistar rats and cultured in serum-containing medium. After serum-starvation for 48 hours, astrocytes were treated with ETs. Total RNA was extracted using an acid-phenol method and expression of chemokine mRNAs was determined by quantitative RT-PCR. The release of chemokines was measured by ELISA. Results Treatment of cultured astrocytes with ET-1 and Ala1,3,11,15-ET-1, an ETB agonist, increased mRNA levels of CCL2/MCP1 and CXCL1/CINC-1. In contrast, CX3CL1/fractalkine mRNA expression decreased in the presence of ET-1 and Ala1,3,11,15-ET-1. The effect of ET-1 on chemokine mRNA expression was inhibited by BQ788, an ETB antagonist. ET-1 increased CCL2 and CXCL1 release from cultured astrocytes, but decreased that of CX3CL1. The increase in CCL2 and CXCL1 expression by ET-1 was inhibited by actinomycin D, pyrrolidine dithiocarbamate, SN50, mithramycin, SB203580 and SP600125. The decrease in CX3CL1 expression by ET-1 was inhibited by cycloheximide, Ca2+ chelation and staurosporine. Conclusion These findings suggest that ETs are one of the factors regulating astrocytic chemokine production. Astrocyte-derived chemokines are involved in pathophysiological responses of neurons and microglia. Therefore, the ET-induced alterations of astrocytic chemokine production are of pathophysiological significance in damaged brains. PMID:23627909

Endothelin A receptor antagonists in congestive heart failure: blocking the beast while leaving the beauty untouched?

PubMed

Spieker, L E; Noll, G; Ruschitzka, F T; Lüscher, T F

2001-12-01

Congestive heart failure (CHF) is a disease process characterized by impaired left ventricular function, increased peripheral and pulmonary vascular resistance and reduced exercise tolerance and dyspnea. Thus, mediators involved in the control of myocardial function and vascular tone may be involved in its pathophysiology. The family of endothelins (ET) consists of four closely related peptides, ET-1, ET-2, ET-3, and ET-4, which cause vasoconstriction, cell proliferation, and myocardial effects through activation of ET(A) receptors. In contrast, endothelial ET(B) receptors mediate vasodilation via release of nitric oxide and prostacyclin. In addition, ET(B) receptors in the lung are a major pathway for the clearance of ET-1 from plasma. Thus, infusion of an ET(A) receptor antagonist into the brachial artery in healthy humans leads to vasodilation whereas infusion of an ET(B) receptor antagonist causes vasoconstriction. ET-1 plasma levels are elevated in CHF and correlate both with the hemodynamic severity and with symptoms. Plasma levels of ET-1 and its precursor, big ET-1, are strong independent predictors of death in patients after myocardial infarction and with CHF. ET-1 contributes to increased systemic and pulmonary vascular resistance, vascular dysfunction, myocardial ischemia, and renal impairment in CHF. Selective ET(A) as well as combined ET(A/B) receptor antagonists have been studied in patients with CHF showing impressive hemodynamic improvements (i.e. reduced peripheral vascular and pulmonary resistance as well as increased cardiac output). These results indicate that ET receptor antagonists indeed have a potential to improve hemodynamics, symptoms, and potentially prognosis of CHF which still carries a high mortality.

Endothelin receptors and activity differ in human, dog, and rabbit lung.

PubMed

McKay, K O; Armour, C L; Black, J L

1996-01-01

In this study, we have examined dog and rabbit airways as potential models for human airways in regard to the activity of endothelin. The receptors involved in the response to endothelin-1 (ET-1) in airway tissue from human, rabbit, and dog lung were investigated, as was the mechanism responsible for the contraction to ET-1 in tissue from the three species. By using specific endothelin receptor agonists and antagonists, we have demonstrated that ETB receptors predominate in rabbit and human airways and ETA receptors in dog airways. The contraction to ET-1 is not dependent on cyclooxygenase products of arachidonic acid, as indomethacin had no effect on the response to ET-1. Extracellular calcium influx via voltage-dependent channels is necessary for contraction to ET-1 in rabbit and dog airways. These results are in contrast to our previously reported results in human airways, in which neither removal of extracellular calcium nor verapamil affected the ET-1 response. The sustained phase of the contraction to ET-1 in all three species may be mediated in part by activation of protein kinase C (PKC), as the inhibitor staurosporine significantly altered the time course of the response to endothelin. We therefore conclude that in rabbit airways ET-1 activates ETB receptors, triggers the influx of extracellular calcium through voltage-dependent channels, and induces a contractile response that is, in part, dependent upon stimulation of PKC. The same mechanism is triggered in dog bronchus; however, the receptors involved in this species are of the ETA type. Finally, in human airways, the contractile response to ET-1, while independent of extracellular calcium influx, is dependent upon PKC activation after binding of the peptide to ETB receptors.

Endothelins activate Ca(2+)-gated K(+) channels via endothelin B receptors in CD-1 mouse erythrocytes.

PubMed

Rivera, A; Rotter, M A; Brugnara, C

1999-10-01

Cell dehydration mediated by Ca(2+)-activated K(+) channels plays an important role in the pathogenesis of sickle cell disease. CD-1 mouse erythrocytes possess a Ca(2+)-activated K(+) channel (Gardos channel) with maximal velocity (V(max)) of 0.154 +/- 0.02 mmol. l cells(-1). min(-1) and an affinity constant (K(0.5)) for Ca(2+) of 286 +/- 83 nM in the presence of A-23187. Cells pretreated with 500 nM endothelin-1 (ET-1) increased their V(max) by 88 +/- 9% (n = 8) and decreased their K(0.5) for Ca(2+) to 139 +/- 63 nM (P < 0.05; n = 4). Activation of the Gardos channel resulted in an EC(50) of 75 +/- 20 nM for ET-1 and 374 +/- 97 nM for ET-3. Analysis of the affinity of unlabeled ET-1 for its receptor showed two classes of binding sites with apparent dissociation constants of 167 +/- 51 and 785 +/- 143 nM and with capacity of binding sites of 298 +/- 38 and 1,568 +/- 211 sites/cell, respectively. The Gardos channel was activated by the endothelin B (ET(B)) receptor agonist IRL 1620 and inhibited by BQ-788, demonstrating the involvement of ET(B) receptors. Calphostin C inhibited 73% of ET-1-induced Gardos activation and 84% of the ET-1-induced membrane protein kinase C activity. Thus endothelins regulate erythrocyte Gardos channels via ET(B) receptors and a calphostin-sensitive mechanism.

ENOS, ET-1 and ETB-R immunoreactivities in the porcine mesometrial lymphatics during the estrous cycle.

PubMed

Doboszyńska, Teresa; Andronowska, Aneta

2002-01-01

Abstract: Immunohistochemical localization and distribution of nitric oxide synthase (eNOS), endothelin (ET-1) and endothelin beta receptor (ETB-R) were investigated in precollector and collector lymph vessels in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and ETB-R and monoclonal antibody for eNOS isoform were used to perform observations on the light microscopic level. Immunoreactivities to ET-1, ETB-R and eNOS were observed in the endothelium of precollector and collector lymphangions but not in smooth muscle cells of the lymphatics examined. The staining for eNOS in the endothelial cells of all studied lymphatic vessels was stronger comparing to ET-1 and ETB-R. During the estrous cycle, only eNOS showed the correlation with the particular phases of the estrous cycle. The differences between ET-1 and ETB-R immunoreactivities were very slight and rather independent of the size or type of the lymphatic lymphangions and estrous cycle. The highest immunoreactivity level for eNOS was displayed by collector lymphangions with widened lumen in the follicular phase comparing to the precollector ones. During the luteal phase, a slight decrease in the reaction intensity was observed. The immunoreactivities for ET-1 in the endothelium of the studied vessels was not comparable with the presence or with the reactivity level of ETB-R. Optically stronger immunoreaction for ETB-R was observed in the cytoplasm of collector lymphangions in the follicular phase. eNOS, ET-1 and ETB-R were also present in the cytoplasm of the lymphatic valves. These results suggest that ET-1 and eNOS can play a role in the mechanisms regulating the vascular contractile activity, promoting lymph flow during the estrous cycle in the porcine broad ligament.

Polymorphism in endothelin-related genes limits exercise-induced decreases in arterial stiffness in older subjects.

PubMed

Iemitsu, Motoyuki; Maeda, Seiji; Otsuki, Takeshi; Sugawara, Jun; Tanabe, Takumi; Jesmin, Subrina; Kuno, Shinya; Ajisaka, Ryuichi; Miyauchi, Takashi; Matsuda, Mitsuo

2006-05-01

Increase in arterial stiffness is associated with aging, which is improved by regular exercise. Endothelin (ET) system has crucial roles in regulating vascular tone and in the progression of atherosclerosis. We hypothesized that molecular variations (ie, gene polymorphisms) in ET-related gene might affect exercise-induced improvement in arterial stiffness with age in human subjects. The present study provides a cross-sectional investigation of 191 healthy middle-aged and older (65+/-1 years) human subjects to clarify the relationship between the regular exercise-induced improvement of arterial stiffness and the gene polymorphisms of ET converting enzyme (ECE)-1, ECE-2, ET-A receptor (ET-A), and ET-B receptor (ET-B). The study subjects were divided into active and inactive groups based on the median value (186 kcal/d) of energy expenditure. Brachial-ankle arterial pulse wave velocity (baPWV) was used to evaluate arterial stiffness. All individuals were genotyped for 4 different polymorphisms of the ET system: 2013(+289)A/G in intron 17 of ECE-1, 669(+17)T/C in intron 5 of ECE-2, 958A/G in exon 6 of ET-A, and 831A/G in exon 4 of ET-B. The baseline baPWV was significantly lower in the active group without any change in blood pressure. Polymorphisms in ECE-1 influenced basal blood pressure. Polymorphisms in ECE-1 and ECE-2 had no effect on baPWV between active and inactive groups. However, polymorphisms in both ET-A and ET-B affected baPWV in the 2 groups. The present results suggest that differences in ET-A and ET-B polymorphisms may influence the response of the vascular wall to exercise whereas ECE-1 polymorphisms may affect basal blood pressure.

Dual ETA/ETB blockade with macitentan improves both vascular remodeling and angiogenesis in pulmonary arterial hypertension

PubMed Central

Nadeau, Valerie; Potus, Francois; Boucherat, Olivier; Paradis, Renee; Tremblay, Eve; Iglarz, Marc; Paulin, Roxane; Bonnet, Sebastien

2017-01-01

Dysregulated metabolism and rarefaction of the capillary network play a critical role in pulmonary arterial hypertension (PAH) etiology. They are associated with a decrease in perfusion of the lungs, skeletal muscles, and right ventricle (RV). Previous studies suggested that endothelin-1 (ET-1) modulates both metabolism and angiogenesis. We hypothesized that dual ETA/ETB receptors blockade improves PAH by improving cell metabolism and promoting angiogenesis. Five weeks after disease induction, Sugen/hypoxic rats presented severe PAH with pulmonary artery (PA) remodeling, RV hypertrophy and capillary rarefaction in the lungs, RV, and skeletal muscles (microCT angiogram, lectin perfusion, CD31 staining). Two-week treatment with dual ETA/ETB receptors antagonist macitentan (30 mg/kg/d) significantly improved pulmonary hemodynamics, PA vascular remodeling, and RV function and hypertrophy compared to vehicle-treated animals (all P = 0.05). Moreover, macitentan markedly increased lung, RV and quadriceps perfusion, and microvascular density (all P = 0.05). In vitro, these effects were associated with increases in oxidative phosphorylation (oxPhox) and markedly reduced cell proliferation of PAH-PA smooth muscle cells (PASMCs) treated with macitentan without affecting apoptosis. While macitentan did not affect oxPhox, proliferation, and apoptosis of PAH–PA endothelial cells (PAECs), it significantly improved their angiogenic capacity (tube formation assay). Exposure of control PASMC and PAEC to ET-1 fully mimicked the PAH cells phenotype, thus confirming that ET-1 is implicated in both metabolism and angiogenesis abnormalities in PAH. Dual ETA/ETB receptor blockade improved the metabolic changes involved in PAH-PASMCs’ proliferation and the angiogenic capacity of PAH-PAEC leading to an increased capillary density in lungs, RV, and skeletal muscles. PMID:29064353

Aldh2 knockout mice were more sensitive to DNA damage in leukocytes due to ethyl tertiary butyl ether exposure.

PubMed

Weng, Zuquan; Suda, Megumi; Ohtani, Katsumi; Mei, Nan; Kawamoto, Toshihiro; Nakajima, Tamie; Wang, Rui-Sheng

2011-01-01

To clarify the genotoxicity of ethyl tertiary butyl ether (ETBE), a gasoline additive, male and female C57BL/6 mice of Aldh2+/+ and Aldh2-/- genotypes, aged 8 wk, were exposed to 0, 500, 1,750, or 5,000 ppm ETBE for 6 h/day, 5 d per week for 13 wk. DNA damage in leukocytes was measured by the alkaline comet assay and expressed quantitatively as Tail Intensity (TI). For male mice, TI was significantly higher in all three groups exposed to ETBE than in those without exposure within Aldh2-/- mice, whereas within Aldh2+/+ mice, TI increased only in those exposed to 5,000 ppm of ETBE as compared with mice without exposure. For female mice, a significant increase in TI values was observed in the group exposed to 5,000 ppm of ETBE as compared with those without exposure within Aldh2-/- mice; TI in Aldh2-/- mice exposed to 1,750 and 5,000 ppm was significantly higher than in Aldh2+/+ mice without exposure. TI did not significantly increase in any of the groups exposed to ETBE within female Aldh2+/+ mice. Based on the results we suggest that Aldh2-/- mice are more sensitive to DNA damage caused by ETBE than Aldh2+/+ mice and that males seem more susceptible to this effect than females.

TRPC3- and ETB receptor-mediated PI3K/AKT activation induces vasogenic edema formation following status epilepticus.

PubMed

Kim, Ji-Eun; Kang, Tae-Cheon

2017-10-01

Status epilepticus (SE, a prolonged seizure activity) is a high risk factor of developing vasogenic edema, which leads to secondary complications following SE. In the present study, we investigated whether transient receptor potential canonical channel-3 (TRPC3) may link vascular endothelial growth factor (VEGF) pathway to NFκB/ET B receptor axis in the rat piriform cortex during vasogenic edema formation. Following SE, TRPC3 and ET B receptor independently activated phosphatidylinositol 3 kinase (PI3K)/AKT/eNOS signaling pathway. SN50 (a NFκB inhibitor) attenuated the up-regulations of eNOS, TRPC3 and ET B receptor expressions following SE, accompanied by reductions in PI3K/AKT phosphorylations. Inhibition of SE-induced VEGF over-expression by leptomycin B also abrogated PI3K and AKT phosphorylations, but not TRPC3 expression. Wortmannin (a PI3K inhibitor) and 3CAI (an AKT inhibitor) effectively inhibited up-regulation of eNOS expressions and vasogenic edema lesion following SE. These findings indicate that PI3K/AKT may be common down-stream molecules for TRPC3- and ET B receptor signaling pathways during vasogenic edema formation. In addition, the present data demonstrate for the first time that TRPC3 may integrate VEGF- and NFκB-mediated vasogenic edema formation following SE. Thus, we suggest that PI3K/AKT signaling pathway may be one of considerable therapeutic targets for vasogenic edema. Copyright © 2017 Elsevier B.V. All rights reserved.

Expression profiling of G-protein-coupled receptors in human urothelium and related cell lines.

PubMed

Ochodnický, Peter; Humphreys, Sian; Eccles, Rachel; Poljakovic, Mirjana; Wiklund, Peter; Michel, Martin C

2012-09-01

What's known on the subject? and What does the study add? Urothelium emerged as a crucial integrator of sensory inputs and outputs in the bladder wall, and urothelial G-protein-coupled receptors (GPCRs) may represent plausible targets for treatment of various bladder pathologies. Urothelial cell lines provide a useful tool to study urothelial receptor function, but their validity as models for native human urothelium remains unclear. We characterize the mRNA expression of genes coding for GPCRs in human freshly isolated urothelium and compare the expression pattern with those in human urothelial cell lines. To characterize the mRNA expression pattern of genes coding for G-protein-coupled receptors (GPCRs) in human freshly isolated urothelium. To compare GPCR expression in human urothelium-derived cell lines to explore the suitability of these cell lines as model systems to study urothelial function. Native human urothelium (commercially sourced) and human urothelium-derived non-cancer (UROtsa and TERT-NHUC) and cancer (J82) cell lines were used. For mRNA expression profiling we used custom-designed real-time polymerase chain reaction array for 40 receptors and several related genes. Native urothelium expressed a wide variety of GPCRs, including α(1A), α(1D) and all subtypes of α(2) and β adrenoceptors. In addition, M(2) and M(3) cholinergic muscarinic receptors, angiotensin II AT(1) receptor, serotonin 5-HT(2A) receptor and all subtypes of bradykinin, endothelin, cannabinoid, tachykinin and sphingosine-1-phosphate receptors were detected. Nerve growth factor and both its low- and high-affinity receptors were also expressed in urothelium. In all cell lines expression of most GPCRs was markedly downregulated, with few exceptions. In UROtsa cells, but much less in other cell lines, the expression of β(2) adrenoceptors, M(3) muscarinic receptors, B(1) and B(2) bradykinin receptors, ET(B) endothelin receptors and several subtypes of sphingosine-1-phosphate receptors was largely retained. Human urothelium expresses a wide range of receptors which enables sensing and integration of various extracellular signals. Human urothelium-derived cell lines, especially UROtsa cells, show comparable mRNA expression to native tissue for several physiologically relevant GPCRs, but lose expression of many other receptors. The use of cell lines as model systems of human urothelium requires careful validation of suitability for the genes of interest. © 2012 BJU INTERNATIONAL.

Disparate effects of single endothelin A and B receptor blocker therapy on the progression of renal injury in advanced renovascular disease

PubMed Central

Chade, Alejandro R.; Stewart, Nicholas J.; Peavy, Patrick R.

2013-01-01

We hypothesized that chronic specific endothelin (ET)-A receptor blockade therapy would reverse renal dysfunction and injury in advanced experimental renovascular disease. To test this, unilateral renovascular disease was induced in 19 pigs and after 6 weeks, single-kidney hemodynamics and function was quantified in vivo using computed-tomography. All pigs with renovascular disease were divided such that 7 were untreated, 7 were treated with ET-A blockers, and 5 were treated with ET-B blockers. Four weeks later, all pigs were re-studied in vivo, then euthanized and ex vivo studies performed on the stenotic kidney to quantify microvascular density, remodeling, renal oxidative stress, inflammation, and fibrosis. RBF, GFR, and redox status were significantly improved in the stenotic kidney after ET-A but not ET-B blockade. Furthermore, only ET-A blockade therapy reversed renal microvascular rarefaction and diminished remodeling, which was accompanied by a marked decreased in renal inflammatory and fibrogenic activity. Thus, ET-A but not ET-B blockade ameliorated renal injury in pigs with advanced renovascular disease by stimulating microvascular proliferation and decreasing the progression of microvascular remodeling, renal inflammation and fibrosis in the stenotic kidney. These effects were functionally consequential since ET-A blockade improved single kidney microvascular endothelial function, RBF, and GFR, and decreased albuminuria. PMID:24352153

Human cytochrome P450 isozymes in metabolism and health effects of gasoline ethers.

PubMed

Hong, J Y; Wang, Y Y; Mohr, S N; Bondoc, F Y; Deng, C

2001-05-01

To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE*), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Among them, MTBE is the most widely used. The possible adverse effect of MTBE in humans is a public concern, but the human enzymes responsible for metabolism of these gasoline ethers and the causes or factors for increased sensitivity to MTBE in certain individuals are totally unknown. This information is important to understanding the health effects of MTBE in humans and to assessing the human relevance of pharmacokinetics and toxicity data obtained from animals. In the present study, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and an exposure marker of MTBE. The activity is localized in the microsomal fraction but not in the cytosol. Formation of TBA in human liver microsomes is NADPH-dependent and is significantly inhibited by carbon monoxide, which inhibits cytochrome P450 (CYP) enzymes. These results provide strong evidence that CYP enzymes play a critical role in the metabolism of MTBE in human livers. Human liver is also active in the oxidative metabolism of 2 other gasoline ethers, ETBE and TAME. We observed a large interindividual variation in metabolizing these gasoline ethers in 15 microsomal samples prepared from normal human livers. The activity level (pmol metabolite/min/mg) ranged from 204 to 2,890 for MTBE; 179 to 3,134 for ETBE; and 271 to 8,532 for TAME. The microsomal activities in metabolizing MTBE, ETBE, and TAME correlated highly with each other (r = 0.91 to 0.96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the human liver microsomes showed that the highest degree of correlation was with CYP isoform 2A6 (CYP2A6)+ (r = 0.94 for MTBE, 0.95 for ETBE, and 0.90 for TAME), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. CYP2A6 coexpressed with human CYP reductase by a baculovirus expression system was also more active than CYP isoform 2E1 (CYP2E1) in the metabolism of MTBE, ETBE, and TAME. Kinetic studies on MTBE metabolism with human liver microsomes (n = 3) exhibited an apparent Michaelis constant (Km) of 28 to 89 microM and a maximum rate of metabolism (Vmax) of 215 to 783 pmol/min/mg. Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that, in human liver, CYP2A6 is a major enzyme responsible for metabolism of MTBE, ETBE, and TAME. Although CYP2E1 metabolizes diethyl ether and was previously suggested to be involved

Pharmacological characteristics of endothelin receptors on sheep rectal blood vessels.

PubMed

Lohsiriwat, Varut; Scholefield, John H; Dashwood, Michael R; Wilson, Vincent G

2011-06-01

Haemorrhoids is associated with high blood flow of the anorectal region. The question of whether pharmacological manipulation of vascular supply can relieve the symptoms of haemorrhoids has been raised. In order to undertake this type of clinical investigation, it is first essential to gain a better understanding of the properties of vascular receptors that may regulate blood flow into anal cushions and haemorrhoids. Due to the limited availability of human anorectal specimens and the good reliability of sheep tissue as an experimental model of human anorectal diseases, we studied the properties of endothelin receptors in sheep rectal artery (SRA) and vein (SRV), the vessels contributing to the blood flow of haemorrhoidal plexus, using isometric tension recordings. We found that endothelin-1 and sarafotoxin 6a were very potent constrictor agents in both SRA and SRV. The selective ET(A) receptor antagonist PD156707 (100 nM) produced a parallel rightward displacement of ET-1-induced contractions in both vessels and abolished sarafotoxin 6a-induced contractions in the SRA. PD156707 (3 μM) practically abolished contractions to ET-1 in the SRA, suggesting that the response is entirely mediated by ET(A) receptors. While, the selective ET(B) receptor antagonist BQ788 (100 nM) caused no significant change in ET-1-induced contractions in both vessels, a minor role for ET(B) receptor subtype to responses to sarafotoxin 6a in the artery was suggested. Copyright © 2011 Elsevier Ltd. All rights reserved.

Effects of endothelin receptor antagonists on renal hemodynamics in angiotensin II-infused rats on high NaCl intake.

PubMed

Saeed, Aso; Dibona, Gerald F; Guron, Gregor

2012-01-01

The aim was to investigate effects of selective endothelin (ET) receptor antagonists on renal hemodynamics and dynamic renal blood flow autoregulation (RBFA) in angiotensin II (Ang II)-infused rats on a high NaCl intake. Sprague-Dawley rats received Ang II (250 ng/kg/min, s.c.) and an 8% NaCl diet for 14 days after which renal clearance experiments were performed. After baseline measurements animals were administered either: (a) saline vehicle; (b) ETA receptor antagonist BQ-123 (30 nmol/kg/min); (c) ETB receptor antagonist BQ-788 (30 nmol/kg/min); or (d) BQ-123 + BQ-788, for six consecutive 20-minute clearance periods. BQ-123 reduced arterial pressure (AP) and selectively increased outer medullary perfusion versus vehicle (p<0.05). These effects were attenuated or abolished by combined BQ-123 and BQ-788. BQ-788 reduced renal blood flow and increased renovascular resistance (p<0.05). Ang II-infused rats on high NaCl intake showed abnormalities in dynamic RBFA characterized by an impaired myogenic response that were not significantly affected by ET receptor antagonists. In hypertensive Ang II-infused rats on a high-NaCl intake selective ETA antagonism with BQ-123 reduced AP and specifically increased OM perfusion and these effects were dependent on intact ETB receptor stimulation. Furthermore, ET receptor antagonists did not attenuate abnormalities in dynamic RBFA. Copyright © 2012 S. Karger AG, Basel.

Tight-binding analysis of Si and GaAs ultrathin bodies with subatomic wave-function resolution

NASA Astrophysics Data System (ADS)

Tan, Yaohua P.; Povolotskyi, Michael; Kubis, Tillmann; Boykin, Timothy B.; Klimeck, Gerhard

2015-08-01

Empirical tight-binding (ETB) methods are widely used in atomistic device simulations. Traditional ways of generating the ETB parameters rely on direct fitting to bulk experiments or theoretical electronic bands. However, ETB calculations based on existing parameters lead to unphysical results in ultrasmall structures like the As-terminated GaAs ultrathin bodies (UTBs). In this work, it is shown that more transferable ETB parameters with a short interaction range can be obtained by a process of mapping ab initio bands and wave functions to ETB models. This process enables the calibration of not only the ETB energy bands but also the ETB wave functions with corresponding ab initio calculations. Based on the mapping process, ETB models of Si and GaAs are parameterized with respect to hybrid functional calculations. Highly localized ETB basis functions are obtained. Both the ETB energy bands and wave functions with subatomic resolution of UTBs show good agreement with the corresponding hybrid functional calculations. The ETB methods can then be used to explain realistically extended devices in nonequilibrium that cannot be tackled with ab initio methods.

Evaporation characteristics of ETBE-blended gasoline.

PubMed

Okamoto, Katsuhiro; Hiramatsu, Muneyuki; Hino, Tomonori; Otake, Takuma; Okamoto, Takashi; Miyamoto, Hiroki; Honma, Masakatsu; Watanabe, Norimichi

2015-04-28

To reduce greenhouse gas emissions, which contribute to global warming, production of gasoline blended with ethyl tert-buthyl ether (ETBE) is increasing annually. The flash point of ETBE is higher than that of gasoline, and blending ETBE into gasoline will change the flash point and the vapor pressure. Therefore, it is expected that the fire hazard caused by ETBE-blended gasoline would differ from that caused by normal gasoline. The aim of this study was to acquire the knowledge required for estimating the fire hazard of ETBE-blended gasoline. Supposing that ETBE-blended gasoline was a two-component mixture of gasoline and ETBE, we developed a prediction model that describes the vapor pressure and flash point of ETBE-blended gasoline in an arbitrary ETBE blending ratio. We chose 8-component hydrocarbon mixture as a model gasoline, and defined the relation between molar mass of gasoline and mass loss fraction. We measured the changes in the vapor pressure and flash point of gasoline by blending ETBE and evaporation, and compared the predicted values with the measured values in order to verify the prediction model. The calculated values of vapor pressures and flash points corresponded well to the measured values. Thus, we confirmed that the change in the evaporation characteristics of ETBE-blended gasoline by evaporation could be predicted by the proposed model. Furthermore, the vapor pressure constants of ETBE-blended gasoline were obtained by the model, and then the distillation curves were developed. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation of endothelin receptors in the failing right ventricle of the heart and vasculature of the lung in human pulmonary arterial hypertension.

PubMed

Kuc, Rhoda E; Carlebur, Myrna; Maguire, Janet J; Yang, Peiran; Long, Lu; Toshner, Mark; Morrell, Nicholas W; Davenport, Anthony P

2014-11-24

In pulmonary arterial hypertension (PAH), increases in endothelin-1 (ET-1) contribute to elevated pulmonary vascular resistance which ultimately causes death by right ventricular (RV) heart failure. ET antagonists are effective in treating PAH but lack efficacy in treating left ventricular (LV) heart failure, where ETA receptors are significantly increased. The aim was to quantify the density of ETA and ETB receptors in cardiopulmonary tissue from PAH patients and the monocrotaline (MCT) rat, which recapitulates some of the pathophysiological features, including increased RV pressure. Radioligand binding assays were used to quantify affinity, density and ratio of ET receptors. In RV from human PAH hearts, there was a significant increase in the ratio of ETA to ETB receptors compared with normal hearts. In the RV of the MCT rat, the ratio also changed but was reversed. In both human and rat, there was no change in LV. In human PAH lungs, ETA receptors were significantly increased in the medial layer of small pulmonary arteries with no change detectable in MCT rat vessels. Current treatments for PAH focus mainly on pulmonary vasodilatation. The increase in ETA receptors in arteries provides a mechanism for the beneficial vasodilator actions of ET antagonists. The increase in the ratio of ETA in RV also implicates changes to ET signalling although it is unclear if ET antagonism is beneficial but the results emphasise the unexploited potential for therapies that target the RV, to improve survival in patients with PAH. Copyright © 2014. Published by Elsevier Inc.

Pancreatic acini possess endothelin receptors whose internalization is regulated by PLC-activating agents.

PubMed

Hildebrand, P; Mrozinski, J E; Mantey, S A; Patto, R J; Jensen, R T

1993-05-01

Endothelin-1 (ET-1) and ET-3 mRNA have been found in the pancreas. We investigated the ability of ET-1, ET-2, and ET-3 to interact with and alter dispersed rat pancreatic acinar cell function. Radiolabeled ETs bound in a time- and temperature-dependent fashion, which was specific and saturable. Analysis demonstrated two classes of receptors, one class (ETA receptor) had a high affinity for ET-1 but a low affinity for ET-3, and the other class (ETB receptor) had equally high affinities for ET-1 and ET-3. No specific receptor for ET-2 was identified. Pancreatic secretagogues that activate phospholipase C (PLC) inhibited binding of 125I-labeled ET-1 (125I-ET-1) or 125I-ET-3, whereas agents that act through adenosine 3',5'-cyclic monophosphate (cAMP) did not. A23187 had no effect on 125I-ET-1 or 125I-ET-3 binding, whereas the phorbol ester 12-O-tetradecanoylphorbol 13-acetate reduced binding. The effect of cholecystokinin octapeptide (CCK-8) was mediated through its own receptor. Stripping of surface bound ligand studies demonstrated that both 125I-labeled ET-1 and 125I-labeled ET-3 were rapidly internalized. CCK-8 decreased the internalization but did not change the amount of surface bound ligand. Endothelins neither stimulate nor alter changes in enzyme secretion, intracellular calcium, cAMP, or [3H]inositol trisphosphate (IP3). This study demonstrates the presence of ETA and ETB receptors on rat pancreatic acini; occupation of both receptors resulted in rapid internalization, which is regulated by PLC-activating secretagogues. Occupation of either ET receptor did not alter intracellular calcium, cAMP, IP3, or stimulate amylase release.

Pharmacological endothelin receptor interaction does not occur in veins from ET(B) receptor deficient rats.

PubMed

Thakali, Keshari; Galligan, James J; Fink, Gregory D; Gariepy, Cheryl E; Watts, Stephanie W

2008-07-01

Heterodimerization of G-protein coupled receptors can alter receptor pharmacology. ET A and ET B receptors heterodimerize when co-expressed in heterologous expression lines. We hypothesized that ET A and ET B receptors heterodimerize and pharmacologically interact in vena cava from wild-type (WT) but not ET B receptor deficient (sl/sl) rats. Pharmacological endothelin receptor interaction was assessed by comparing ET-1-induced contraction in rings of rat thoracic aorta and thoracic vena cava from male Sprague Dawley rats under control conditions, ET A receptor blockade (atrasentan, 10 nM), ET B receptor blockade (BQ-788, 100 nM) or ET B receptor desensitization (Sarafotoxin 6c, 100 nM) and ET A plus ET B receptor blockade or ET A receptor blockade plus ET B receptor desensitization. In addition, similar pharmacological ET receptor antagonism experiments were performed in rat thoracic aorta and vena cava from WT and sl/sl rats. ET A but not ET B receptor blockade or ET B receptor desensitization inhibited aortic and venous ET-1-induced contraction. In vena cava but not aorta, when ET B receptors were blocked (BQ-788, 100 nM) or desensitized (S6c, 100 nM), atrasentan caused a greater inhibition of ET-1-induced contraction. Vena cava from WT but not sl/sl rats exhibited similar pharmacological ET receptor interaction. Immunocytochemistry was performed on freshly dissociated aortic and venous vascular smooth muscle cells to determine localization of ET A and ET B receptors. ET A and ET B receptors qualitatively co-localized more strongly to the plasma membrane of aortic compared to venous vascular smooth muscle cells. Our data suggest that pharmacological ET A and ET B receptor interaction may be dependent on the presence of functional ET B receptors and independent of receptor location.

Evaluation of ethyl tert-butyl ether biodegradation in a contaminated aquifer by compound-specific isotope analysis and in situ microcosms.

PubMed

Bombach, Petra; Nägele, Norbert; Rosell, Mònica; Richnow, Hans H; Fischer, Anko

2015-04-09

Ethyl tert-butyl ether (ETBE) is an upcoming groundwater pollutant in Europe whose environmental fate has been less investigated, thus far. In the present study, we investigated the in situ biodegradation of ETBE in a fuel-contaminated aquifer using compound-specific stable isotope analysis (CSIA), and in situ microcosms in combination with total lipid fatty acid (TLFA)-stable isotope probing (SIP). In a first field investigation, CSIA revealed insignificant carbon isotope fractionation, but low hydrogen isotope fractionation of up to +14‰ along the prevailing anoxic ETBE plume suggesting biodegradation of ETBE. Ten months later, oxygen injection was conducted to enhance the biodegradation of petroleum hydrocarbons (PH) at the field site. Within the framework of this remediation measure, in situ microcosms loaded with [(13)C6]-ETBE (BACTRAP(®)s) were exposed for 119 days in selected groundwater wells to assess the biodegradation of ETBE by TLFA-SIP under the following conditions: (i) ETBE as main contaminant; (ii) ETBE as main contaminant subjected to oxygen injection; (iii) ETBE plus other PH; (iv) ETBE plus other PH subjected to oxygen injection. Under all conditions investigated, significant (13)C-incorporation into microbial total lipid fatty acids extracted from the in situ microcosms was found, providing clear evidence of ETBE biodegradation. Copyright © 2014 Elsevier B.V. All rights reserved.

Reduced sickle erythrocyte dehydration in vivo by endothelin-1 receptor antagonists.

PubMed

Rivera, Alicia

2007-09-01

Elevated plasma levels of cytokines such as endothelin-1 (ET-1) have been shown to be associated with sickle cell disease (SCD). However, the role of ET-1 in the pathophysiology of SCD is not entirely clear. I now show that treatment of SAD mice, a transgenic mouse model of SCD, with BQ-788 (0.33 mg.kg(-1).day(-1) intraperitoneally for 14 days), an ET-1 receptor B (ET(B)) antagonist, induced a significant decrease in Gardos channel activity (1.7 +/- 0.1 to 1.0 +/- 0.4 mmol.10(13) cell(-1).h(-1), n = 3, P = 0.019) and reduced the erythrocyte density profile by decreasing the mean density (D(50); n = 4, P = 0.012). These effects were not observed in mice treated with BQ-123, an ET-1 receptor A (ET(A)) antagonist. A mixture of both antagonists induced a similar change in density profile as with BQ-788 alone that was associated with an increase in mean cellular volume and a decrease in corpuscular hemoglobin concentration mean. I also observed in vitro effects of ET-1 on human sickle erythrocyte dehydration that was blocked by BQ-788 and a mixture of ET(B)/ET(A) antagonists but not by ET(A) antagonist alone. These results show that erythrocyte hydration status in vivo is mediated via activation of the ET(B) receptor, leading to Gardos channel modulation in SCD.

Lack of micronucleus induction activity of ethyl tertiary-butyl ether in the bone marrow of F344 rats by sub-chronic drinking-water treatment, inhalation exposure, or acute intraperitoneal injection.

PubMed

Noguchi, Tadashi; Kamigaito, Tomoyuki; Katagiri, Taku; Kondou, Hitomi; Yamazaki, Kazunori; Aiso, Shigetoshi; Nishizawa, Tomoshi; Nagano, Kasuke; Fukushima, Shoji

2013-01-01

Ethyl tertiary-butyl ether (ETBE) is an oxygenated gasoline additive synthesized from ethanol and isobutene that is used to reduce CO2 emissions. To support the Kyoto Protocol, the production of ETBE has undergone a marked increase. Previous reports have indicated that exposure to ETBE or methyl tertiary-butyl ether resulted in liver and kidney tumors in rats and/or mice. These reports raise concern about the effects of human exposure being brought about by the increased use of ETBE. The present study was conducted to evaluate the genotoxicity of ETBE using micronucleus induction of polychromatic erythrocytes in the bone marrow of male and female rats treated with ETBE in the drinking-water at concentrations of 0, 1,600, 4,000 or 10,000 ppm or exposed to ETBE vapor at 0, 500, 1,500 or 5,000 ppm for 13 weeks. There were no significant increases in micronucleus induction in either the drinking water-administered or inhalation-administered groups at any concentration of ETBE; although, in both groups red blood cells and hemoglobin concentration were slightly reduced in the peripheral blood in rats administered the highest concentration of ETBE. In addition, two consecutive daily intraperitoneal injections of ETBE at doses of 0, 250, 500 or 1,000 mg/kg did not increase the frequency of micronucleated bone marrow cells in either sex; all rats receiving intraperitoneal injections of ETBE at a dose of 2,000 mg/kg died after treatment day 1. These data suggest that ETBE is not genotoxic in vivo.

Transcriptomic Assessment of Isozymes in the Biphenyl Pathway of Rhodococcus sp. Strain RHA1†

PubMed Central

Gonçalves, Edmilson R.; Hara, Hirofumi; Miyazawa, Daisuke; Davies, Julian E.; Eltis, Lindsay D.; Mohn, William W.

2006-01-01

Rhodococcus sp. RHA1 grows on a broad range of aromatic compounds and vigorously degrades polychlorinated biphenyls (PCBs). Previous work identified RHA1 genes encoding multiple isozymes for most of the seven steps of the biphenyl (BPH) pathway, provided evidence for coexpression of some of these isozymes, and indicated the involvement of some of these enzymes in the degradation of BPH, ethylbenzene (ETB), and PCBs. To investigate the expression of these isozymes and better understand how they contribute to the robust degradative capacity of RHA1, we comprehensively analyzed the 9.7-Mb genome of RHA1 for BPH pathway genes and characterized the transcriptome of RHA1 growing on benzoate (BEN), BPH, and ETB. Sequence analyses revealed 54 potential BPH pathway genes, including 28 not previously reported. Transcriptomic analysis with a DNA microarray containing 70-mer probes for 8,213 RHA1 genes revealed a suite of 320 genes of diverse functions that were upregulated during growth both on BPH and on ETB, relative to growth on the control substrate, pyruvate. By contrast, only 65 genes were upregulated during growth on BEN. Quantitative PCR assays confirmed microarray results for selected genes and indicated that some of the catabolic genes were upregulated over 10,000-fold. Our analysis suggests that up to 22 enzymes, including 8 newly identified ones, may function in the BPH pathway of RHA1. The relative expression levels of catabolic genes did not differ for BPH and ETB, suggesting a common regulatory mechanism. This study delineated a suite of catabolic enzymes for biphenyl and alkyl-benzenes in RHA1, which is larger than previously recognized and which may serve as a model for catabolism in other environmentally important bacteria having large genomes. PMID:16957245

Transferable tight binding model for strained group IV and III-V heterostructures

NASA Astrophysics Data System (ADS)

Tan, Yaohua; Povolotskyi, Micheal; Kubis, Tillmann; Boykin, Timothy; Klimeck, Gerhard

Modern semiconductor devices have reached critical device dimensions in the range of several nanometers. For reliable prediction of device performance, it is critical to have a numerical efficient model that are transferable to material interfaces. In this work, we present an empirical tight binding (ETB) model with transferable parameters for strained IV and III-V group semiconductors. The ETB model is numerically highly efficient as it make use of an orthogonal sp3d5s* basis set with nearest neighbor inter-atomic interactions. The ETB parameters are generated from HSE06 hybrid functional calculations. Band structures of strained group IV and III-V materials by ETB model are in good agreement with corresponding HSE06 calculations. Furthermore, the ETB model is applied to strained superlattices which consist of group IV and III-V elements. The ETB model turns out to be transferable to nano-scale hetero-structure. The ETB band structures agree with the corresponding HSE06 results in the whole Brillouin zone. The ETB band gaps of superlattices with common cations or common anions have discrepancies within 0.05eV.

Endothelin-like action of Pausinystalia yohimbe aqueous extract on vascular and renal regional hemodynamics in Sprague Dawley rats.

PubMed

Ajayi, A A; Newaz, M; Hercule, H; Saleh, M; Bode, C O; Oyekan, A O

2003-12-01

The bark of the African tree Pausinystalia yohimbe has been used as a food additive with aphrodisiac and penile erection enhancing properties. The effect of an aqueous extract of P. yohimbe (CCD-X) on renal circulation was assessed in order to test the hypothesis that it possesses additional effects on nitric oxide production and/or endothelin-1 (ET-1)-like actions. In vivo studies with CCD-X in Sprague Dawley rats demonstrated a dose-dependent (1-1000 ng/kg) increase in mean blood pressure (p < 0.001) and an increase in medullary blood flow (MBF) (p < 0.001). Both the pressor action and renal medullary vasodilation were blocked by endothelinA (ETA) receptor antagonist BMS182874 and endothelinB (ETB) receptor antagonist BQ788 in combination. L-Nomega-nitro-l-arginine methyl ester (L-NAME; 10 mg/kg) also inhibited the increase in MBF induced by CCD-X. In vitro studies in isolated perfused kidney and in pressurized renal microvessels confirmed the dose-dependent vasoconstrictor action of this extract. ETA receptor antagonist BQ610 and ETB receptor antagonist BQ788 separately and significantly attenuated the renal vasoconstrictor actions of the extract (p < 0.001 ANOVA). These preliminary observations indicate that, in addition to the alpha-adrenergic antagonist actions that characterize yohimbine, CCD-X possesses endothelin-like actions and affects nitric oxide (NO) production in renal circulation. These findings suggest a strong possibility of post-receptor cross-talk between alpha2-adrenoceptors and endothelin, as well as a direct effect of alpha2-adrenoceptors on renal NO production. (c) 2003 Prous Science

Comparison of pharmacological activity of macitentan and bosentan in preclinical models of systemic and pulmonary hypertension.

PubMed

Iglarz, Marc; Bossu, Alexandre; Wanner, Daniel; Bortolamiol, Céline; Rey, Markus; Hess, Patrick; Clozel, Martine

2014-11-24

The endothelin (ET) system is a tissular system, as the production of ET isoforms is mostly autocrine or paracrine. Macitentan is a novel dual ETA/ETB receptor antagonist with enhanced tissue distribution and sustained receptor binding properties designed to achieve a more efficacious ET receptor blockade. To determine if these features translate into improved efficacy in vivo, a study was designed in which rats with either systemic or pulmonary hypertension and equipped with telemetry were given macitentan on top of maximally effective doses of another dual ETA/ETB receptor antagonist, bosentan, which does not display sustained receptor occupancy and shows less tissue distribution. After establishing dose-response curves of both compounds in conscious, hypertensive Dahl salt-sensitive and pulmonary hypertensive bleomycin-treated rats, macitentan was administered on top of the maximal effective dose of bosentan. In hypertensive rats, macitentan 30 mg/kg further decreased mean arterial blood pressure (MAP) by 19 mm Hg when given on top of bosentan 100 mg/kg (n=9, p<0.01 vs. vehicle). Conversely, bosentan given on top of macitentan failed to induce an additional MAP decrease. In pulmonary hypertensive rats, macitentan 30 mg/kg further decreased mean pulmonary artery pressure (MPAP) by 4 mm Hg on top of bosentan (n=8, p<0.01 vs. vehicle), whereas a maximal effective dose of bosentan given on top of macitentan did not cause any additional MPAP decrease. The add-on effect of macitentan on top of bosentan in two pathological models confirms that this novel compound can achieve a superior blockade of ET receptors and provides evidence for greater maximal efficacy. Copyright © 2014. Published by Elsevier Inc.

Exposure assessment of ETBE in gas station workers and gasoline tanker truck drivers.

PubMed

Eitaki, Yoko; Kawai, Toshio; Omae, Kazuyuki

2011-01-01

In order to measure occupational exposure concentrations of ethyl tertiary-butyl ether (ETBE), we developed a diffusive sampling method for monitoring ETBE and performed an ETBE exposure assessment. The applicability of diffusive samplers was examined by exposing the samplers to ETBE vapor in test chambers. The personal exposure levels of workers and airborne concentrations were measured at 4 gas stations. The ETBE sampling rate for the diffusive samplers (VOC-SD, Sigma-Aldrich Japan) was 25.04 ml/min (25°C). Compared with the active sampling method, the diffusive samplers could be used for short-term measurements and in environments containing a mixture of organic solvents. The geometric mean (GM) of TWA-8h ETBE was 0.08 ppm (0.02-0.28 ppm) in 28 gas station workers and 0.04 ppm (0.01-0.21 ppm) in 2 gasoline tanker truck drivers. With regard to ETBE airborne concentrations, the GM was 4.12 ppm (0.93-8.71 ppm) at the handles of hanging pumps but dropped to less than 0.01 ppm (less than 0.01-0.01 ppm) at the side of a public road. The diffusive sampling method can be used for the measurement of occupational ETBE exposure. The threshold limit of TLV-TWA 5 ppm recommended by the ACGIH was not exceeded in any of the workers in this study.

Effect of endothelin-1 on the serotonin-induced contraction of smooth muscle in the guinea pig trachea.

PubMed

Yoshida, M; Aizawa, H; Hara, N

1999-01-01

Endothelin (ET), a potent constrictor of smooth muscle including that of the airways, may contribute to the development of airway hyperresponsiveness. To investigate the role of ET-1 on the airway smooth muscle, we examined the effects of ET-1 on the serotonin-induced contraction of guinea pig tracheal smooth muscle. The changes in isometric tension evoked by serotonin were measured before and after the application of a subthreshold dose (a dose which did not induce smooth muscle contraction by itself) of ET-1. Serotonin caused smooth muscle contraction in a dose-dependent manner. The subthreshold doses of ET-1 (1 pM) and sarafotoxin 6c (1 pM), a selective ETB receptor agonist, were found to potentiate significantly the contraction induced by serotonin. A potentiating effect of ET-1 was not altered by indomethacin or calphostin C, a protein kinase C inhibitor. These results suggest that a subthreshold concentration of ET-1 can potentiate serotonin-induced contraction of smooth muscle through the activation of ETB receptor, while in contrast cyclooxygenase and protein kinase C were found not to be involved in this mechanism.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (Etbe) ...

EPA Pesticide Factsheets

The IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) was released for external peer review in June 2017. EPA’s Science Advisory Board’s (SAB) Chemical Assessment Advisory Committee (CAAC) will conduct a peer review of the scientific basis supporting the ETBE assessment and release a final report of their review. Information regarding the peer review can be found on the SAB website. EPA is conducting an Integrated Risk Information System (IRIS) health assessment for Ethyl Tertiary Butyl Ether (ETBE). The outcome of this project is a Toxicological Review and IRIS Summary for ETBE that will be entered into the IRIS database.

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-α in the induction of endothelin system genes.

PubMed

Donate, Paula B; Cunha, Thiago M; Verri, Waldiceu A; Junta, Cristina M; Lima, Flavia O; Vieira, Silvio M; Peres, Rafael S; Bombonato-Prado, Karina F; Louzada, Paulo; Ferreira, Sergio H; Donadi, Eduardo A; Passos, Geraldo A S; Cunha, Fernando Q

2012-04-01

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1β, TNFα and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNFα upregulated ET system genes. These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNFα.

Activation of particulate guanylyl cyclase by endothelins in cultured SV-40 transformed cat iris sphincter smooth muscle cells.

PubMed

Ding, K H; Latimer, A J; Abdel-Latif, A A

1999-01-01

We investigated the effects of endothelins (ETs) on cGMP production in cultured SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. ET-3 increased cGMP formation in a concentration-dependent manner (EC50 = 98nM), which was 2.5 times higher than that of ET-1. The ET(B)receptor agonists sarafotoxin-S6c and IRL 1620 also increased cGMP production, mimicking the effects of the ETs. The ET(B) receptor antagonist BQ 788, but not the ET(A) receptor antagonist BQ610, dose-dependently blocked ET-3-stimulated cGMP formation (IC50=10nM). The phorbol ester, Phorbol 12, 13-dibutyrate (PDBu), which inhibits particulate guanylyl cyclase in smooth muscle, dose-dependently inhibited ET-3-stimulated cGMP accumulation (IC50=66nM). LY83583 and ODQ, inhibitors of soluble guanylyl cyclases, as well as inhibitors of the nitric oxide cascade and of intracellular Ca2+ elevation had no appreciable effect on ET-3-induced cGMP production. ET-3 markedly inhibited carbachol-induced intracellular Ca2+ mobilization. We conclude that ET-3 increases intracellular cGMP levels in SV-CISM-2 cells through activation of the ET(B) receptor subtype and subsequent stimulation of the membrane-bound guanylyl cyclase. Elevation of cGMP by ET and the subsequent inhibition of muscarinic stimulation of intracellular Ca2+ mobilization by the cyclic nucleotide could serve to modulate the contractile effects of Ca2+-mobilizing agonists in the iris sphincter smooth muscle.

Physiologically based pharmacokinetic model for ethyl tertiary‐butyl ether and tertiary‐butyl alcohol in rats: Contribution of binding to α2u–globulin in male rats and high‐exposure nonlinear kinetics to toxicity and cancer outcomes

PubMed Central

Ring, Caroline; Banton, Marcy I.; Leavens, Teresa L.

2016-01-01

Abstract In cancer bioassays, inhalation, but not drinking water exposure to ethyl tertiary‐butyl ether (ETBE), caused liver tumors in male rats, while tertiary‐butyl alcohol (TBA), an ETBE metabolite, caused kidney tumors in male rats following exposure via drinking water. To understand the contribution of ETBE and TBA kinetics under varying exposure scenarios to these tumor responses, a physiologically based pharmacokinetic model was developed based on a previously published model for methyl tertiary‐butyl ether, a structurally similar chemical, and verified against the literature and study report data. The model included ETBE and TBA binding to the male rat‐specific protein α2u–globulin, which plays a role in the ETBE and TBA kidney response observed in male rats. Metabolism of ETBE and TBA was described as a single, saturable pathway in the liver. The model predicted similar kidney AUC0–∞ for TBA for various exposure scenarios from ETBE and TBA cancer bioassays, supporting a male‐rat‐specific mode of action for TBA‐induced kidney tumors. The model also predicted nonlinear kinetics at ETBE inhalation exposure concentrations above ~2000 ppm, based on blood AUC0–∞ for ETBE and TBA. The shift from linear to nonlinear kinetics at exposure concentrations below the concentration associated with liver tumors in rats (5000 ppm) suggests the mode of action for liver tumors operates under nonlinear kinetics following chronic exposure and is not relevant for assessing human risk. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd PMID:27885692

Physiologically based pharmacokinetic model for ethyl tertiary-butyl ether and tertiary-butyl alcohol in rats: Contribution of binding to α2u-globulin in male rats and high-exposure nonlinear kinetics to toxicity and cancer outcomes.

PubMed

Borghoff, Susan J; Ring, Caroline; Banton, Marcy I; Leavens, Teresa L

2017-05-01

In cancer bioassays, inhalation, but not drinking water exposure to ethyl tertiary-butyl ether (ETBE), caused liver tumors in male rats, while tertiary-butyl alcohol (TBA), an ETBE metabolite, caused kidney tumors in male rats following exposure via drinking water. To understand the contribution of ETBE and TBA kinetics under varying exposure scenarios to these tumor responses, a physiologically based pharmacokinetic model was developed based on a previously published model for methyl tertiary-butyl ether, a structurally similar chemical, and verified against the literature and study report data. The model included ETBE and TBA binding to the male rat-specific protein α2u-globulin, which plays a role in the ETBE and TBA kidney response observed in male rats. Metabolism of ETBE and TBA was described as a single, saturable pathway in the liver. The model predicted similar kidney AUC 0-∞ for TBA for various exposure scenarios from ETBE and TBA cancer bioassays, supporting a male-rat-specific mode of action for TBA-induced kidney tumors. The model also predicted nonlinear kinetics at ETBE inhalation exposure concentrations above ~2000 ppm, based on blood AUC 0-∞ for ETBE and TBA. The shift from linear to nonlinear kinetics at exposure concentrations below the concentration associated with liver tumors in rats (5000 ppm) suggests the mode of action for liver tumors operates under nonlinear kinetics following chronic exposure and is not relevant for assessing human risk. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.

Ether oxygenate additives in gasoline reduce toxicity of exhausts.

PubMed

Westphal, G A; Krahl, J; Brüning, T; Hallier, E; Bünger, J

2010-02-09

Fuel additives can improve combustion and knock resistance of gasoline engines. Common additives in commercial fuels are "short-chain, oxygen containing hydrocarbons" such as methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE). Since these additives change the combustion characteristics, this may as well influence toxic effects of the resulting emissions. Therefore we compared toxicity and BTEX emissions of gasoline engine exhaust regarding addition of MTBE or ETBE. Non-reformulated gasoline served as basic fuel. This fuel was supplemented with 10%, 20%, 25% and 30% ETBE or 15% MTBE. The fuels were combusted in a gasoline engine at idling, part load and rated power. Condensates and particulate matter (PM) were collected and PM samples extracted with dichloromethane. Cytotoxic effects were investigated in murine fibroblasts (L929) using the neutral red uptake assay and mutagenicity using the bacterial reverse mutation assay. BTEX emissions were analyzed by gas chromatography. PM-extracts showed mutagenicity with and without metabolic activation. Mutagenicity was reduced by the addition of MTBE and ETBE, 10% ETBE being most effective. The condensates produced no significant mutagenic response. The cytotoxicity of the condensates from ETBE- and MTBE-reformulated fuels was reduced as well. The BTEX content in the exhaust was lowered by the addition of MTBE and ETBE. This effect was significantly related to the ETBE content at rated power and part load. Addition of MTBE and ETBE to fuels can improve combustion and leads to decreased toxicity and BTEX content of the exhaust. Reduction of mutagenicity in the PM-extracts is most probably caused by a lower content of polycyclic aromatic hydrocarbons. (c) 2009 Elsevier Ireland Ltd. All rights reserved.

Ethyl tertiary-butyl ether: a toxicological review.

PubMed

McGregor, Douglas

2007-05-01

A number of oxygenated compounds (oxygenates) are available for use in gasoline to reduce vehicle exhaust emissions, reduce the aromatic compound content, and avoid the use of organo-lead compounds, while maintaining high octane numbers. Ethyl tertiary-butyl ether (ETBE) is one such compound. The current use of ETBE in gasoline or petrol is modest but increasing, with consequently similar trends in the potential for human exposure. Inhalation is the most likely mode of exposure, with about 30% of inhaled ETBE being retained by the lungs and distributed around the body. Following cessation of exposure, the blood concentration of ETBE falls rapidly, largely as a result of its metabolism to tertiary-butyl alcohol (TBA) and acetaldehyde. TBA may be further metabolized, first to 2-methyl-1,2-propanediol and then to 2-hydroxyisobutyrate, the two dominant metabolites found in urine of volunteers and rats. The rapid oxidation of acetaldehyde suggests that its blood concentration is unlikely to rise above normal as a result of human exposure to sources of ETBE. Single-dose toxicity tests show that ETBE has low toxicity and is essentially nonirritant to eyes and skin; it did not cause sensitization in a maximization test in guinea pigs. Neurological effects have been observed only at very high exposure concentrations. There is evidence for an effect of ETBE on the kidney of rats. Increases in kidney weight were seen in both sexes, but protein droplet accumulation (with alpha(2u)-globulin involvement) and sustained increases in cell proliferation occurred only in males. In liver, centrilobular necrosis was induced in mice, but not rats, after exposure by inhalation, although this lesion was reported in some rats exposed to very high oral doses of ETBE. The proportion of liver cells engaged in S-phase DNA synthesis was increased in mice of both sexes exposed by inhalation. ETBE has no specific effects on reproduction, development, or genetic material. Carcinogenicity studies have been conducted with ETBE, TBA, and ethanol (included in this review as an endogenous precursor of acetaldehyde in the absence of TBA). A single experiment with ETBE in rats and several experiments with ethanol in rats and mice were not considered adequate for an evaluation of ETBE carcinogenicity. In male rats only, TBA induced alpha(2u)-globulin nephropathy-related renal tubule adenomas. These are generally considered to have no human relevance. In addition, increases in thyroid follicular cell adenoma incidence were associated with TBA treatment in female mice. This result lacks independent confirmation and is not supported by experiments in which similar or higher internal doses of TBA were delivered.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) (External Review Draft)

EPA Science Inventory

The IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) was released for external peer review in June 2017. EPA’s Science Advisory Board’s (SAB) Chemical Assessment Advisory Committee (CAAC) will conduct a peer review of the scientific basis supporting the ETB...

Endothelial nitric-oxide synthase (eNOS) is activated through G-protein-coupled receptor kinase-interacting protein 1 (GIT1) tyrosine phosphorylation and Src protein.

PubMed

Liu, Songling; Premont, Richard T; Rockey, Don C

2014-06-27

Nitric oxide (NO) is a critical regulator of vascular tone and plays an especially prominent role in liver by controlling portal blood flow and pressure within liver sinusoids. Synthesis of NO in sinusoidal endothelial cells by endothelial nitric-oxide synthase (eNOS) is regulated in response to activation of endothelial cells by vasoactive signals such as endothelins. The endothelin B (ETB) receptor is a G-protein-coupled receptor, but the mechanisms by which it regulates eNOS activity in sinusoidal endothelial cells are not well understood. In this study, we built on two previous strands of work, the first showing that G-protein βγ subunits mediated activation of phosphatidylinositol 3-kinase and Akt to regulate eNOS and the second showing that eNOS directly bound to the G-protein-coupled receptor kinase-interacting protein 1 (GIT1) scaffold protein, and this association stimulated NO production. Here we investigated the mechanisms by which the GIT1-eNOS complex is formed and regulated. GIT1 was phosphorylated on tyrosine by Src, and Y293F and Y554F mutations reduced GIT1 phosphorylation as well as the ability of GIT1 to bind to and activate eNOS. Akt phosphorylation activated eNOS (at Ser(1177)), and Akt also regulated the ability of Src to phosphorylate GIT1 as well as GIT1-eNOS association. These pathways were activated by endothelin-1 through the ETB receptor; inhibiting receptor-activated G-protein βγ subunits blocked activation of Akt, GIT1 tyrosine phosphorylation, and ET-1-stimulated GIT1-eNOS association but did not affect Src activation. These data suggest a model in which Src and Akt cooperate to regulate association of eNOS with the GIT1 scaffold to facilitate NO production. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Endothelin@25 - new agonists, antagonists, inhibitors and emerging research frontiers: IUPHAR Review 12.

PubMed

Maguire, J J; Davenport, A P

2014-12-01

Since the discovery of endothelin (ET)-1 in 1988, the main components of the signalling pathway have become established, comprising three structurally similar endogenous 21-amino acid peptides, ET-1, ET-2 and ET-3, that activate two GPCRs, ETA and ETB . Our aim in this review is to highlight the recent progress in ET research. The ET-like domain peptide, corresponding to prepro-ET-193-166 , has been proposed to be co-synthesized and released with ET-1, to modulate the actions of the peptide. ET-1 remains the most potent vasoconstrictor in the human cardiovascular system with a particularly long-lasting action. To date, the major therapeutic strategy to block the unwanted actions of ET in disease, principally in pulmonary arterial hypertension, has been to use antagonists that are selective for the ETA receptor (ambrisentan) or that block both receptor subtypes (bosentan). Macitentan represents the next generation of antagonists, being more potent than bosentan, with longer receptor occupancy and it is converted to an active metabolite; properties contributing to greater pharmacodynamic and pharmacokinetic efficacy. A second strategy is now being more widely tested in clinical trials and uses combined inhibitors of ET-converting enzyme and neutral endopeptidase such as SLV306 (daglutril). A third strategy based on activating the ETB receptor, has led to the renaissance of the modified peptide agonist IRL1620 as a clinical candidate in delivering anti-tumour drugs and as a pharmacological tool to investigate experimental pathophysiological conditions. Finally, we discuss biased signalling, epigenetic regulation and targeting with monoclonal antibodies as prospective new areas for ET research. © 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.

No Promoting Effect of Ethyl Tertiary-butyl Ether (ETBE) on Rat Urinary Bladder Carcinogenesis Initiated with N-Butyl-N-(4-hydroxybutyl)nitrosamine

PubMed Central

Hagiwara, Akihiro; Imai, Norio; Doi, Yuko; Suguro, Mayuko; Kawabe, Mayumi; Furukawa, Fumio; Nagano, Kasuke; Fukushima, Shoji

2013-01-01

The effects of ethyl tertiary-butyl ether (ETBE) on two-stage urinary bladder carcinogenesis in male F344 rats initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) were investigated at various dose levels with regard to possible promoting activity. Groups of 30 rats were given drinking water containing 500 ppm BBN, as an initiator, for 4 weeks and starting one week thereafter received ETBE by gavage (daily, 7 days/week) at dose levels of 0 (control), 100, 300, 500 or 1000 mg/kg/day until experimental week 36. No statistically significant differences in incidences of preneoplastic lesions, papillomas, and carcinomas of the urinary bladder were evident in rats treated with 100–1000 mg/kg/day ETBE as compared with control values. Furthermore, the average numbers of preneoplastic or neoplastic lesions per unit length of basement membrane in rats given 100–1000 mg/kg/day ETBE were also comparable to control values. However, papillomatosis of the urinary bladder was found in 4 out of 30 rats (13%) in the group given 1000 mg/kg/day ETBE, and soft stones in the urinary bladder were found in 3 out of these 4 rats. The results thus demonstrated that ETBE did not exert promotional activity on urinary bladder carcinogenesis. However, papillomatosis of the urinary bladder developed in small numbers of the rats given ETBE at 1000 mg/kg/day but not in rats given 500 mg/kg/day or lower doses. PMID:24526807

Development and application of a rat PBPK model to elucidate kidney and liver effects induced by ETBE and tert-butanol.

PubMed

Salazar, Keith D; Brinkerhoff, Christopher J; Lee, Janice S; Chiu, Weihsueh A

2015-11-01

Subchronic and chronic studies in rats of the gasoline oxygenates ethyl tert-butyl ether (ETBE) and tert-butanol (TBA) report similar noncancer kidney and liver effects but differing results with respect to kidney and liver tumors. Because TBA is a major metabolite of ETBE, it is possible that TBA is the active toxic moiety in all these studies, with reported differences due simply to differences in the internal dose. To test this hypothesis, a physiologically-based pharmacokinetic (PBPK) model was developed for ETBE and TBA to calculate internal dosimetrics of TBA following either TBA or ETBE exposure. This model, based on earlier PBPK models of methyl tert-butyl ether (MTBE), was used to evaluate whether kidney and liver effects are consistent across routes of exposure, as well as between ETBE and TBA studies, on the basis of estimated internal dose. The results demonstrate that noncancer kidney effects, including kidney weight changes, urothelial hyperplasia, and chronic progressive nephropathy (CPN), yielded consistent dose-response relationships across routes of exposure and across ETBE and TBA studies using TBA blood concentration as the dose metric. Relative liver weights were also consistent across studies on the basis of TBA metabolism, which is proportional to TBA liver concentrations. However, kidney and liver tumors were not consistent using any dose metric. These results support the hypothesis that TBA mediates the noncancer kidney and liver effects following ETBE administration; however, additional factors besides internal dose are necessary to explain the induction of liver and kidney tumors. Published by Elsevier Inc.

Endothelinergic Contractile Hyperreactivity in Rat Contralateral Carotid to Balloon Injury: Integrated Role for ETB Receptors and Superoxide Anion

PubMed Central

Gimenes, Lilian R.; Gomes, Mayara S.; do Vale, Bruno N.; Cardoso, Cristina R. B.; de Oliveira, Ana M.; Moreira, Josimar D.

2017-01-01

Temporal consequences of neurocompensation to balloon injury on endothelinergic functionality in rat contralateral carotid were evaluated. Rats underwent balloon injury in left carotid and were treated with CP-96345 (NK1 antagonist). Concentration-response curves for endothelin-1 were obtained in contralateral (right) carotid at 2, 8, 16, 30, or 45 days after surgery in the absence or presence of BQ-123 (ETA antagonist), BQ-788 (ETB antagonist), or Tempol (superoxide-dismutase mimic). Endothelin-1-induced calcium mobilization was evaluated in functional assays carried out with BQ-123, BQ-788, or Tempol. Endothelin-1-induced NADPH oxidase-driven superoxide generation was measured by lucigenin chemiluminescence assays performed with BQ-123 or BQ-788. Endothelin-1-induced contraction was increased in contralateral carotid from the sixteenth day after surgery. This response was restored in CP-96345-treated rats. Endothelium removal or BQ-123 did not change endothelin-1-induced contraction in contralateral carotid. This response was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced calcium mobilization, which was restored by BQ-788 or Tempol. Contralateral carotid exhibited an increased endothelin-1-induced lucigenin chemiluminescence, which was restored by BQ-788. We conclude that the NK1-mediated neurocompensatory response to balloon injury elicits a contractile hyperreactivity to endothelin-1 in rat contralateral carotid by enhancing the muscular ETB-mediated NADPH oxidase-driven generation of superoxide, which activates calcium channels. PMID:29062837

Inhibition of IgE-induced mast cell activation by ethyl tertiary-butyl ether, a bioethanol-derived fuel oxygenate.

PubMed

Yamaki, Kouya; Yoshino, Shin

2009-09-01

The effect of ethyl tertiary-butyl ether (ETBE), which is widely used as a fuel oxygenate commonly produced from bioethanol, on immunoglobulin (Ig)E-dependent mast cell activation was investigated. The rat mast cell line RBL2H3 sensitised with monoclonal anti-ovalbumin IgE was challenged with ovalbumin in the presence or absence of ETBE, tert-butanol (TBA), which is the main metabolite of ETBE in humans, and ethanol. Degranulation of RBL2H3 was examined by the release of beta-hexosaminidase. To understand the mechanisms responsible for regulating mast cell function, the effects of ETBE, TBA and ethanol on the levels of intracellular calcium, phosphorylation of Akt (as a marker of phosphatidylinositol 3-kinase) and global tyrosine phosphorylation were also measured as indicators of mast cell activation. In the presence of ETBE, TBA or ethanol, IgE-induced release of beta-hexosaminidase was decreased. These compounds also attenuated the IgE-mediated increase in the levels of intracellular Ca(2+), phosphorylation of Akt and global tyrosine phosphorylation in RBL2H3 cells. ETBE, TBA and ethanol inhibited mast cell degranulation by inhibiting the increase in intracellular calcium ion concentration and activation of phosphatidylinositol 3-kinase and protein tyrosine kinase activation, suggesting that exposure to ETBE might affect immune responses, particularly in allergic diseases.

Endothelin-1 (ET-1) induces resistance to bortezomib in human multiple myeloma cells via a pathway involving the ETB receptor and upregulation of proteasomal activity.

PubMed

Vaiou, Maria; Pangou, Evanthia; Liakos, Panagiotis; Sakellaridis, Nikos; Vassilopoulos, George; Dimas, Konstantinos; Papandreou, Christos

2016-10-01

Bortezomib (BTZ) is used for the treatment of multiple myeloma (MM). However, a significant proportion of patients may be refractory to the drug. This study aimed to investigate whether the endothelin (ET-1) axis may act as an escape mechanism to treatment with bortezomib in MM cells. NCI-H929 and RPMI-8226 (human MM cell lines) were cultured with or without ET-1, BTZ, and inhibitors of the endothelin receptors. ET-1 levels were determined by ELISA, while the protein levels of its receptors and of the PI3K and MAPK pathways' components by western blot. Effects of ET-1 on cell proliferation were studied by MTT and on the ubiquitin proteasome pathway by assessing the chymotryptic activity of the 20S proteasome in cell lysates. Endothelin receptors A and B (ETAR and ETBR, respectively) were found to be expressed in both cell lines, with the RPMI-8226 cells that are considered resistant to BTZ, expressing higher levels of ETBR and in addition secreting ET-1. Treatment of the NCI-H929 cells with ET-1 increased proliferation, while co-incubation of these cells with ET-1 and BTZ decreased BTZ efficacy with concomitant upregulation of 20S proteasomal activity. Si-RNA silencing or chemical blockade of ETBR abrogated the protective effects of ET-1. Finally, data suggest that the predominant signaling pathway involved in ET-1/ETBR-induced BTZ resistance in MM cells may be the MAPK pathway. Our data suggest a possible role of the ET-1/ETBR axis in regulating the sensitivity of MM cells to BTZ. Thus, combining bortezomib with strategies to target the ET-1 axis could prove to be a novel promising therapeutic approach in MM.

Enhanced expression of Gqα and PLC-β1 proteins contributes to vascular smooth muscle cell hypertrophy in SHR: role of endogenous angiotensin II and endothelin-1.

PubMed

Atef, Mohammed Emehdi; Anand-Srivastava, Madhu B

2014-07-01

Vascular Gqα signaling has been shown to contribute to cardiac hypertrophy. In addition, angiotensin II (ANG II) was shown to induce vascular smooth muscle cell (VSMC) hypertrophy through Gqα signaling; however, the studies on the role of Gqα and PLC-β1 proteins in VSMC hypertrophy in animal model are lacking. The present study was therefore undertaken to examine the role of Gqα/PLC-β1 proteins and the signaling pathways in VSMC hypertrophy using spontaneously hypertensive rats (SHR). VSMC from 16-wk-old SHR and not from 12-wk-old SHR exhibited enhanced levels of Gqα/PLC-β1 proteins compared with age-matched Wistar-Kyoto (WKY) rats as determined by Western blotting. However, protein synthesis as determined by [(3)H]leucine incorporation was significantly enhanced in VSMC from both 12- and 16-wk-old SHR compared with VSMC from age-matched WKY rats. Furthermore, the knockdown of Gqα/PLC-β1 in VSMC from 16-wk-old SHR by antisense and small interfering RNA resulted in attenuation of protein synthesis. In addition, the enhanced expression of Gqα/PLC-β1 proteins, enhanced phosphorylation of ERK1/2, and enhanced protein synthesis in VSMC from SHR were attenuated by the ANG II AT1 and endothelin-1 (ET-1) ETA receptor antagonists losartan and BQ123, respectively, but not by the ETB receptor antagonist BQ788. In addition, PD98059 decreased the enhanced expression of Gqα/PLC-β1 and protein synthesis in VSMC from SHR. These results suggest that the enhanced levels of endogenous ANG II and ET-1 through the activation of AT1 and ETA receptors, respectively, and MAP kinase signaling, enhanced the expression of Gqα/PLC-β1 proteins in VSMC from 16-wk-old SHR and result in VSMC hypertrophy. Copyright © 2014 the American Physiological Society.

Development and application of a rat PBPK model to elucidate kidney and liver effects induced by ETBE and tert-butanol

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

Salazar, Keith D., E-mail: Salazar.keith@epa.gov; Brinkerhoff, Christopher J., E-mail: Brinkerhoff.Chris@epa.gov; Lee, Janice S., E-mail: Lee.JaniceS@epa.gov

Subchronic and chronic studies in rats of the gasoline oxygenates ethyl tert-butyl ether (ETBE) and tert-butanol (TBA) report similar noncancer kidney and liver effects but differing results with respect to kidney and liver tumors. Because TBA is a major metabolite of ETBE, it is possible that TBA is the active toxic moiety in all these studies, with reported differences due simply to differences in the internal dose. To test this hypothesis, a physiologically-based pharmacokinetic (PBPK) model was developed for ETBE and TBA to calculate internal dosimetrics of TBA following either TBA or ETBE exposure. This model, based on earlier PBPKmore » models of methyl tert-butyl ether (MTBE), was used to evaluate whether kidney and liver effects are consistent across routes of exposure, as well as between ETBE and TBA studies, on the basis of estimated internal dose. The results demonstrate that noncancer kidney effects, including kidney weight changes, urothelial hyperplasia, and chronic progressive nephropathy (CPN), yielded consistent dose–response relationships across routes of exposure and across ETBE and TBA studies using TBA blood concentration as the dose metric. Relative liver weights were also consistent across studies on the basis of TBA metabolism, which is proportional to TBA liver concentrations. However, kidney and liver tumors were not consistent using any dose metric. These results support the hypothesis that TBA mediates the noncancer kidney and liver effects following ETBE administration; however, additional factors besides internal dose are necessary to explain the induction of liver and kidney tumors. - Highlights: • We model two metabolically-related fuel oxygenates to address toxicity data gaps. • Kidney and liver effects are compared on an internal dose basis. • Noncancer kidney effects are consistent using TBA blood concentration. • Liver weight changes are consistent using TBA metabolic rate. • Kidney and liver tumors are not consistent using any internal dose metric.« less

Effects of a thirteen-week inhalation exposure to ethyl tertiary butyl ether on fischer-344 rats and CD-1 mice.

PubMed

Medinsky, M A; Wolf, D C; Cattley, R C; Wong, B; Janszen, D B; Farris, G M; Wright, G A; Bond, J A

1999-09-01

The 1990 Clean Air Act Amendments require that oxygenates be added to automotive fuels to reduce emissions of carbon monoxide and hydrocarbons. One potential oxygenate is the aliphatic ether ethyl tertiary butyl ether (ETBE). Our objective was to provide data on the potential toxic effects of ETBE. Male and female Fisher 344 rats and CD-1 mice were exposed to 0 (control), 500, 1750, or 5000 ppm of ETBE for 6 h/day and 5 days/wk over a 13-week period. ETBE exposure had no effect on mortality and body weight with the exception of an increase in body weights of the female rats in the 5000-ppm group. No major changes in clinical pathology parameters were noted for either rats or mice exposed to ETBE for 6 (rats only) or 13 weeks. Liver weights increased with increasing ETBE-exposure concentration for both sexes of rats and mice. Increases in kidney, adrenal, and heart (females only) weights were noted in rats. Degenerative changes in testicular seminiferous tubules were observed in male rats exposed to 1750 and 5000 ppm but were not seen in mice. This testicular lesion has not been reported previously for aliphatic ethers. Increases in the incidence of regenerative foci, rates of renal cell proliferation, and alpha2u-globulin containing protein droplets were noted in the kidneys of all treated male rats. These lesions are associated with the male rat-specific syndrome of alpha2u-globulin nephropathy. Increases in the incidence of centrilobular hepatocyte hypertrophy and rates of hepatocyte cell proliferation were seen in the livers of male and female mice in the 5000-ppm group, consistent with a mitogenic response to ETBE. These two target organs for ETBE toxicity, mouse liver and male rat kidney, have also been reported for methyl tertiary butyl ether and unleaded gasoline.

Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic.

PubMed

Sauer, P; Síla, J; Stosová, T; Vecerová, R; Hejnar, P; Vágnerová, I; Kolár, M; Raclavsky, V; Petrzelová, J; Lovecková, Y; Koukalová, D

2008-04-01

A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages.

PubMed

Soldano, Stefano; Pizzorni, Carmen; Paolino, Sabrina; Trombetta, Amelia Chiara; Montagna, Paola; Brizzolara, Renata; Ruaro, Barbara; Sulli, Alberto; Cutolo, Maurizio

2016-01-01

Alternatively activated (M2) macrophages are phenotypically characterized by the expression of specific markers, mainly macrophage scavenger receptors (CD204 and CD163) and mannose receptor-1 (CD206), and participate in the fibrotic process by over-producing pro-fibrotic molecules, such as transforming growth factor-beta1 (TGFbeta1) and metalloproteinase (MMP)-9. Endothelin-1 (ET-1) is implicated in the fibrotic process, exerting its pro-fibrotic effects through the interaction with its receptors (ETA and ETB). The study investigated the possible role of ET-1 in inducing the transition from cultured human macrophages into M2 cells. Cultured human monocytes (THP-1 cell line) were activated into macrophages (M0 macrophages) with phorbol myristate acetate and subsequently maintained in growth medium (M0-controls) or treated with either ET-1 (100nM) or interleukin-4 (IL-4, 10ng/mL, M2 inducer) for 72 hours. Similarly, primary cultures of human peripheral blood monocyte (PBM)-derived macrophages obtained from healthy subjects, were maintained in growth medium (untreated cells) or treated with ET-1 or IL-4 for 6 days. Both M0 and PBM-derived macrophages were pre-treated with ET receptor antagonist (ETA/BRA, bosentan 10-5M) for 1 hour before ET-1 stimulation. Protein and gene expression of CD204, CD206, CD163, TGFbeta1 were analysed by immunocytochemistry, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Gene expression of interleukin(IL)-10 and macrophage derived chemokine (CCL-22) was evaluated by qRT-PCR. MMP-9 production was investigated by gel zymography. ET-1 significantly increased the expression of M2 phenotype markers CD204, CD206, CD163, IL-10 and CCL-22, and the production of MMP-9 in both cultures of M0 and PBM-derived macrophages compared to M0-controls and untreated cells. In cultured PBM-derived macrophages, ET-1 increased TGFbeta1 protein and gene expression compared to untreated cells. The ET-1-mediated effects were contrasted by ETA/BRA treatment in both cultured cell types. ET-1 seems to induce the M2 phenotype in cultured human macrophages, a process apparently contrasted by the action of the ETA/BRA, suggesting possible clinical implications in those fibrotic diseases characterized by increased ET-1 concentrations, such as systemic sclerosis but also type 2 diabetes.

Endothelin type B (ETB) receptors: friend or foe in the pathogenesis of pre-eclampsia and future cardiovascular disease (CVD) risk?

PubMed

Mirabito Colafella, Katrina M

2018-01-16

In a recent issue of Clinical Science, Stanhewicz et al. investigated persistent microvascular dysfunction in women up to 16 months postpartum. The authors found sensitivity to the pressor effects of endothelin-1 (ET-1) was enhanced when compared with women who had a normotensive pregnancy. Importantly, the authors demonstrated that this effect was mediated via the endothelin type B (ET B ) receptors. Therefore, the present study highlights the possibility that alterations in the localization of the ET B receptor contributes to the pathogenesis of pre-eclampsia and future cardiovascular disease (CVD) risk. Currently, there is great interest in the role of the endothelin system in pre-eclampsia. Targetting the endothelin system, potentially by modulating upstream pathways to prevent ET B receptor dysfunction, may improve health outcomes for women and their offspring during pre-eclampsia and later life. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Homology modeling, active site prediction, and targeting the anti hypertension activity through molecular docking on endothelin – B receptor domain

PubMed Central

Rayalu, Daddam Jayasimha; Selvaraj, Chandrabose; Singh, Sanjeev Kumar; Ganeshan, Ramakrishan; Kumar, Nagapatla Udaya; Seshapani, Panthangi

2012-01-01

In cardiovascular system, activation of Endothelin receptors causes vasoconstriction which leads to Pulmonary Arterial Hypertension (PAH). Endothelin receptor antagonism has emerged as an important therapeutic strategy in pulmonary arterial hypertension. Bosentan is intended to affect vasoconstriction, hypertrophic and fibrotic effects by blocking the actions of receptors ETA and ETB. In this study we identified the action of Bosentan on endothelin B receptor using docking studies with homology modeled endothelin B receptor. Through the modeled protein, the flexible Docking study was performed with Bosentan and its derivatives with theoretically predicted active sites. The results indicated that amino acid ARG82, ARG84 and HIS197 present in endothelin B receptor are core important for binding activities and these residues are having strong hydrogen bond interactions with Bosentan. We have investigated the Bosentan and its derivatives interactions and scoring parameters using gold docking package. Among the docked compounds, one of the Bosentan derivatives BD6 shows better interaction than Bosentan with endothelin B receptor. Our results may be helpful for further investigations in both in vivo and in vitro conditions. PMID:22359440

Binding and activation of major histocompatibility complex class II-deficient macrophages by staphylococcal exotoxins

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Iandolo, J. J.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1994-01-01

Macrophages from C2D transgenic mice deficient in the expression of major histocompatibility complex (MHC) class II proteins were used to identify binding sites for superantigens distinct from the MHC class II molecule. Iodinated staphylococcal enterotoxins A and B (SEA and SEB) and exfoliative toxins A and B (ETA and ETB) bound to C2D macrophages in a concentration-dependent and competitive manner. All four toxins increased F-actin concentration within 30 s of their addition to C2D macrophages, indicating that signal transduction occurred in response to toxin in the absence of class II MHC. Furthermore, ETA, ETB, SEA, and, to a lesser extent, SEB induced C2D macrophages to produce interleukin 6. Several molecular species on C2D macrophages with molecular masses of 140, 97, 61, 52, 43, and 37 kDa bound SEA in immunoprecipitation experiments. These data indicate the presence of novel, functionally active toxin binding sites on murine macrophages distinct from MHC class II molecules.

Perturbations in Endothelial Dysfunction-Associated Pathways in the Nitrofen-Induced Congenital Diaphragmatic Hernia Model.

PubMed

Zhaorigetu, Siqin; Bair, Henry; Lu, Jonathan; Jin, Di; Olson, Scott D; Harting, Matthew T

2018-01-01

Although it is well known that nitrofen induces congenital diaphragmatic hernia (CDH), including CDH-associated lung hypoplasia and pulmonary hypertension (PH) in rodents, the mechanism of pathogenesis remains largely unclear. It has been reported that pulmonary artery (PA) endothelial cell (EC) dysfunction contributes to the development of PH in CDH. Thus, we hypothesized that there is significant alteration of endothelial dysfunction-associated proteins in nitrofen-induced CDH PAs. Pregnant SD rats received either nitrofen or olive oil on gestational day 9.5. The newborn rats were sacrificed and divided into a CDH (n = 81) and a control (n = 23) group. After PA isolation, the expression of PA endothelial dysfunction-associated proteins was assessed on Western blot and immunostaining. We demonstrate that the expression of C-reactive protein and endothelin-1 and its receptors, ETA and ETB, were significantly increased in the CDH PAs. Levels of phosphorylated myosin light chain were significantly elevated, but those of phosphorylated endothelial nitric oxide synthase, caveolin-1, and mechanistic target of rapamycin were significantly decreased in the CDH PAs. In this work, we elucidate alterations in the expression of endothelial dysfunction-associated proteins specific to nitrofen-induced CDH rodent PAs, thereby advancing our understanding of the critical role of endothelial dysfunction-associated pathways in the pathogenesis of nitrofen-induced CDH. © 2017 S. Karger AG, Basel.

Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

PubMed

Jung, Christian; Lichtenauer, Michael; Wernly, Bernhard; Franz, Marcus; Goebel, Bjoern; Rafnsson, Arnar; Figulla, Hans-Reiner; Pernow, John

2016-08-01

Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms. © 2016 Stichting European Society for Clinical Investigation Journal Foundation.

Fundus findings in a series of patients with extrapulmonary tuberculosis in Thailand.

PubMed

Jirawison, Choeng; Liu, Yingna; Surasit, Karjbundid; Maningding, Ernest; Kamphaengkham, Siripim; Ausayakhun, Somsanguan; Heiden, David; Margolis, Todd P; Gonzales, John A; Acharya, Nisha R; Keenan, Jeremy David

2017-06-01

The aim of this study was to determine the frequency of fundus abnormalities among patients who are undergoing or have recently completed treatment for extrapulmonary tuberculosis (eTB). This is a prospective cross-sectional study conducted in a TB clinic of a tertiary hospital in northern Thailand. All patients who had eTB between January 2014 and August 2015 were invited by telephone to return to the clinic for fundus photography. Three uveitis specialists reviewed all photographs to identify posterior segment lesions that were consistent with ocular TB. A total of 265 patients were diagnosed with eTB during the specified period, of which 118 (44.5%) were reached by telephone and 60 (50.8%) participated in the study. A total of 7 eyes from six patients (10.0% of participants, 95% CI 2.2% to 17.8%) had lesions consistent with ocular TB. The group with possible ocular TB lesions was on average 16.8 years older than those without ocular lesions (p=0.01), but the two groups were otherwise not significantly different. Ocular lesions consistent with TB were not rare in a group of patients who were undergoing or had recently completed treatment for eTB. Fundus examination may provide diagnostic information that could influence a clinician's beliefs when diagnosing eTB. Given the low costs and immediate results of eye examination, this diagnostic test should be considered in patients suspected for eTB, especially when other tests are negative. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Roles of Ring-Hydroxylating Dioxygenases in Styrene and Benzene Catabolism in Rhodococcus jostii RHA1▿ †

PubMed Central

Patrauchan, Marianna A.; Florizone, Christine; Eapen, Shawn; Gómez-Gil, Leticia; Sethuraman, Bhanu; Fukuda, Masao; Davies, Julian; Mohn, William W.; Eltis, Lindsay D.

2008-01-01

Proteomics and targeted gene disruption were used to investigate the catabolism of benzene, styrene, biphenyl, and ethylbenzene in Rhodococcus jostii RHA1, a well-studied soil bacterium whose potent polychlorinated biphenyl (PCB)-transforming properties are partly due to the presence of the related Bph and Etb pathways. Of 151 identified proteins, 22 Bph/Etb proteins were among the most abundant in biphenyl-, ethylbenzene-, benzene-, and styrene-grown cells. Cells grown on biphenyl, ethylbenzene, or benzene contained both Bph and Etb enzymes and at least two sets of lower Bph pathway enzymes. By contrast, styrene-grown cells contained no Etb enzymes and only one set of lower Bph pathway enzymes. Gene disruption established that biphenyl dioxygenase (BPDO) was essential for growth of RHA1 on benzene or styrene but that ethylbenzene dioxygenase (EBDO) was not required for growth on any of the tested substrates. Moreover, whole-cell assays of the ΔbphAa and etbAa1::cmrA etbAa2::aphII mutants demonstrated that while both dioxygenases preferentially transformed biphenyl, only BPDO transformed styrene. Deletion of pcaL of the β-ketoadipate pathway disrupted growth on benzene but not other substrates. Thus, styrene and benzene are degraded via meta- and ortho-cleavage, respectively. Finally, catalases were more abundant during growth on nonpolar aromatic compounds than on aromatic acids. This suggests that the relaxed specificities of BPDO and EBDO that enable RHA1 to grow on a range of compounds come at the cost of increased uncoupling during the latter's initial transformation. The stress response may augment RHA1's ability to degrade PCBs and other pollutants that induce similar uncoupling. PMID:17965160

Endothelin Blockade in Diabetic Kidney Disease.

PubMed

Anguiano, Lidia; Riera, Marta; Pascual, Julio; Soler, María José

2015-05-25

Diabetic kidney disease (DKD) remains the most common cause of chronic kidney disease and multiple therapeutic agents, primarily targeted at the renin-angiotensin system, have been assessed. Their only partial effectiveness in slowing down progression to end-stage renal disease, points out an evident need for additional effective therapies. In the context of diabetes, endothelin-1 (ET-1) has been implicated in vasoconstriction, renal injury, mesangial proliferation, glomerulosclerosis, fibrosis and inflammation, largely through activation of its endothelin A (ETA) receptor. Therefore, endothelin receptor antagonists have been proposed as potential drug targets. In experimental models of DKD, endothelin receptor antagonists have been described to improve renal injury and fibrosis, whereas clinical trials in DKD patients have shown an antiproteinuric effect. Currently, its renoprotective effect in a long-time clinical trial is being tested. This review focuses on the localization of endothelin receptors (ETA and ETB) within the kidney, as well as the ET-1 functions through them. In addition, we summarize the therapeutic benefit of endothelin receptor antagonists in experimental and human studies and the adverse effects that have been described.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) (Public Comment Draft)

EPA Science Inventory

In September 2016, the U.S. Environmental Protection Agency's (USEPA) released the draft Integrated Risk Information System (IRIS) Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE). Consistent with the 2013 IRIS Enhancements, draft IRIS assessments are released prior to e...

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) (Preliminary Assessment Materials)

EPA Science Inventory

In August 2013, EPA released the draft literature searches and associated search strategies, evidence tables, and exposure response arrays for ETBE to obtain input from stakeholders and the public prior to developing the draft IRIS assessment. Specifically, EPA was interested in ...

Effects of age and caloric restriction in the vascular response of renal arteries to endothelin-1 in rats.

PubMed

Amor, Sara; García-Villalón, Angel Luis; Rubio, Carmen; Carrascosa, Jose Ma; Monge, Luis; Fernández, Nuria; Martín-Carro, Beatriz; Granado, Miriam

2017-02-01

Cardiovascular alterations are the most prevalent cause of impaired physiological function in aged individuals with kidney being one the most affected organs. Aging-induced alterations in renal circulation are associated with a decrease in endothelium-derived relaxing factors such as nitric oxide (NO) and with an increase in contracting factors such as endothelin-1(ET-1). As caloric restriction (CR) exerts beneficial effects preventing some of the aging-induced alterations in cardiovascular system, the aim of this study was to analyze the effects of age and caloric restriction in the vascular response of renal arteries to ET-1 in aged rats. Vascular function was studied in renal arteries from 3-month-old Wistar rats fed ad libitum (3m) and in renal arteries from 8-and 24-month-old Wistar rats fed ad libitum (8m and 24m), or subjected to 20% caloric restriction during their three last months of life (8m-CR and 24m-CR). The contractile response to ET-1 was increased in renal arteries from 8m and 24m compared to 3m rats. ET-1-induced contraction was mediated by ET-A receptors in all experimental groups and also by ET-B receptors in 24m rats. Caloric restriction attenuated the increased contraction to ET-1 in renal arteries from 8m but not from 24m rats possibly through NO release proceeding from ET-B endothelial receptors. In 24m rats, CR did not attenuate the aging-increased response of renal arteries to ET-1, but it prevented the aging-induced increase in iNOS mRNA levels and the aging-induced decrease in eNOS mRNA levels in arterial tissue. In conclusion, aging is associated with an increased response to ET-1 in renal arteries that is prevented by CR in 8m but not in 24m rats. Copyright © 2016 Elsevier Inc. All rights reserved.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) (Interagency Science Consultation Draft)

EPA Science Inventory

In September 2016, EPA released the draft IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) for public comment and discussion. The draft assessment was reviewed internally by EPA and by other federal agencies and White House Offices before public release. Consistent ...

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) (External Review Draft, 2009)

EPA Science Inventory

EPA is conducting a peer review and public comment of the scientific basis supporting the human health hazard and dose-response assessment of ethyl tertiary butyl ether (ETBE) that when finalized will appear on the Integrated Risk Information System (IRIS) database.

Victims of Educator-Targeted Bullying: A Qualitative Study

ERIC Educational Resources Information Center

de Wet, Corene

2010-01-01

I report on findings emanating from in-depth personal interviews with victims of educator-targeted bullying (ETB). Qualitative content analysis was used to analyse the narratives. The findings indicate that the victims of ETB were exposed repeatedly over time to verbal, non-verbal, psychological, and physical abuse during and after school hours.…

Anaerobic degradation of a mixture of MtBE, EtBE, TBA, and benzene under different redox conditions.

PubMed

van der Waals, Marcelle J; Pijls, Charles; Sinke, Anja J C; Langenhoff, Alette A M; Smidt, Hauke; Gerritse, Jan

2018-04-01

The increasing use of biobased fuels and fuel additives can potentially change the typical fuel-related contamination in soil and groundwater. Anaerobic biotransformation of the biofuel additive ethyl tert-butyl ether (EtBE), as well as of methyl tert-butyl ether (MtBE), benzene, and tert-butyl alcohol (TBA, a possible oxygenate metabolite), was studied at an industrially contaminated site and in the laboratory. Analysis of groundwater samples indicated that in the field MtBE was degraded, yielding TBA as major product. In batch microcosms, MtBE was degraded under different conditions: unamended control, with medium without added electron acceptors, or with ferrihydrite or sulfate (with or without medium) as electron acceptor, respectively. Degradation of EtBE was not observed under any of these conditions tested. TBA was partially depleted in parallel with MtBE. Results of microcosm experiments with MtBE substrate analogues, i.e., syringate, vanillate, or ferulate, were in line with the hypothesis that the observed TBA degradation is a cometabolic process. Microcosms with ferulate, syringate, isopropanol, or diethyl ether showed EtBE depletion up to 86.5% of the initial concentration after 83 days. Benzene was degraded in the unamended controls, with medium without added electron acceptors and with ferrihydrite, sulfate, or chlorate as electron acceptor, respectively. In the presence of nitrate, benzene was only degraded after addition of an anaerobic benzene-degrading community. Nitrate and chlorate hindered MtBE, EtBE, and TBA degradation.

Design of ET(B) receptor agonists: NMR spectroscopic and conformational studies of ET7-21[Leu7, Aib11, Cys(Acm)15].

PubMed

Hewage, Chandralal M; Jiang, Lu; Parkinson, John A; Ramage, Robert; Sadler, Ian H

2002-03-01

In a previous report we have shown that the endothelin-B receptor-selective linear endothelin peptide, ET-1[Cys (Acm)1,15, Ala3, Leu7, Aib11], folds into an alpha-helical conformation in a methanol-d3/water co-solvent [Hewage et al. (1998) FEBS Lett., 425, 234-238]. To study the requirements for the structure-activity relationships, truncated analogues of this peptide were subjected to further studies. Here we report the solution conformation of ET7-21[Leu7, Aib11, Cys(Acm)15], in a methanol-d3/water co-solvent at pH 3.6, by NMR spectroscopic and molecular modelling studies. Further truncation of this short peptide results in it displaying poor agonist activity. The modelled structure shows that the peptide folds into an alpha-helical conformation between residues Lys9-His16, whereas the C-terminus prefers no fixed conformation. This truncated linear endothelin analogue is pivotal for designing endothelin-B receptor agonists.

Endothelin-1 (ET-1) stimulates carboxy terminal Smad2 phosphorylation in vascular endothelial cells by a mechanism dependent on ET receptors and de novo protein synthesis.

PubMed

Sharifat, Narges; Mohammad Zadeh, Ghorban; Ghaffari, Mohammad-Ali; Dayati, Parisa; Kamato, Danielle; Little, Peter J; Babaahmadi-Rezaei, Hossein

2017-01-01

G protein-coupled receptor (GPCR) agonists through their receptors can transactivate protein tyrosine kinase receptors such as epidermal growth factor receptor and serine/threonine kinase receptors most notably transforming growth factor (TGF)-β receptor (TβRI). This signalling mechanism represents a major expansion in the cellular outcomes attributable to GPCR signalling. This study addressed the role and mechanisms involved in GPCR agonist, endothelin-1 (ET-1)-mediated transactivation of the TβRI in bovine aortic endothelial cells (BAECs). The in-vitro model used BAECs. Signalling intermediate phospho-Smad2 in the carboxy terminal was detected and quantified by Western blotting. ET-1 treatment of BAECs resulted in a time and concentration-dependent increase in pSmad2C. Peak phosphorylation was evident with 100 nm treatment of ET-1 at 4-6 h. TβRI antagonist, SB431542 inhibited ET-1-mediated pSmad2C. In the presence of bosentan, a mixed ET A and ET B receptor antagonist ET-1-mediated pSmad2C levels were inhibited. The ET-mediated pSmad2C was blocked by the protein synthesis inhibitor, cycloheximide. In BAECs, ET-1 via the ETB receptor is involved in transactivation of the TβRI. The transactivation-dependent response is dependent upon de novo protein synthesis. © 2016 Royal Pharmaceutical Society.

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

PubMed

Tiwari, Jagesh Kumar; Devi, Sapna; Sundaresha, S; Chandel, Poonam; Ali, Nilofer; Singh, Brajesh; Bhardwaj, Vinay; Singh, Bir Pal

2015-06-01

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

Stochastic bio-economic modeling of mastitis in Ethiopian dairy farms.

PubMed

Getaneh, Abraham Mekibeb; Mekonnen, Sefinew Alemu; Hogeveen, Henk

2017-03-01

Mastitis is an inflammation of the mammary gland that is considered to be one of the most frequent and costly diseases in the dairy industry. Also in Ethiopia, bovine mastitis is one of the most frequently encountered diseases of dairy cows. However, there was no study, so far, regarding the costs of clinical mastitis and only two studies were reported on costs of subclinical mastitis. Presenting an appropriate and complete study of the costs of mastitis will help farmers in making management decisions for mastitis control. The objective of this study was to estimate the economic effects of mastitis on Ethiopian market-oriented dairy farms. Market-oriented dairy farming is driven by making profits through selling milk in the market on a regular basis. A dynamic stochastic Monte-Carlo simulation model (bio-economic model) was developed taking into account both clinical and subclinical mastitis. Production losses, culling, veterinarian costs, treatment, discarded milk, and labour were the main cost factors which were modeled in this study. The annual incidence of clinical mastitis varied from 0 to 50% with a mean annual incidence of 21.6%, whereas the mean annual incidence of subclinical mastitis was 36.2% which varied between 0 and 75%. The total costs due to mastitis for a default farm size of 8 lactating cows were 6,709 ETB per year (838 ETB per cow per year). The costs varied considerably, with 5th and 95th percentiles of 109 ETB and 22,009 ETB, respectively. The factor most contributing to the total annual cost of mastitis was culling. On average a clinical case costs 3,631 ETB, varying from 0 to 12,401, whereas a sub clinical case costs 147 ETB, varying from 0 to 412. The sensitivity analysis showed that the total costs at the farm level were most sensitive for variation in the probability of occurrence of clinical mastitis and the probability of culling. This study helps farmers to raise awareness about the actual costs of mastitis and motivate them to timely treat and/or take preventive measures. As a results, the dairy industry will improve. Copyright © 2017 Elsevier B.V. All rights reserved.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (Etbe) ...

EPA Pesticide Factsheets

In September 2016, EPA released the draft IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE) for public comment and discussion. The draft assessment was reviewed internally by EPA and by other federal agencies and White House Offices before public release. Consistent with the May 2009 IRIS assessment development process, all written comments on IRIS assessments submitted by other federal agencies and White House Offices are made publicly available. Accordingly, interagency comments and the interagency science consultation materials provided to other agencies, including interagency review drafts of the IRIS Toxicological Review of Ethyl Tertiary Butyl Ether are posted on this site. EPA is undertaking an new health assessment for ethyl tertiary butyl ether (ETBE) for the Integrated Risk Information System (IRIS). The outcome of this project will be a Toxicological Review and IRIS Summary of ETBE that will be entered on the IRIS database. IRIS is an EPA database containing Agency scientific positions on potential adverse human health effects that may result from chronic (or lifetime) exposure to chemicals in the environment. IRIS contains chemical-specific summaries of qualitative and quantitative health information in support of two steps of the risk assessment process, i.e., hazard identification and dose-response evaluation. IRIS assessments are used nationally and internationally in combination with specific situational exposure assessment infor

Enthalpy relaxation studies of two structurally related amorphous drugs and their binary dispersions.

PubMed

Bansal, Shyam Sunder; Kaushal, Aditya Mohan; Bansal, Arvind Kumar

2010-11-01

The purpose of the current study was to evaluate the enthalpy relaxation behavior of valdecoxib (VLB) and etoricoxib (ETB) and their binary dispersions to derive relaxation constants and to understand their molecular mobilities. Solid dispersions of VLB and ETB were prepared with 1%, 2%, 5%, 10%, 15%, and 20% (w/w) concentrations of polyvinylpyrrolidone (PVP) in situ using differential scanning calorimetry (DSC). Enthalpy relaxation studies were carried out with isothermal storage periods of 1, 2, 4, 6, 16, and 24 hours at 40°C and 0% relative humidity (RH). PVP increased the glass transition temperature (T(g)) and decreased the enthalpy relaxation. Significant differences between two drugs were observed with respect to their relaxation behavior which may be due to differences in intermolecular interactions as predicted by Couchman-Karasz equation and molecular mobility. Kohlrausch-Williams-Watts equation was found to be inadequate in describing complex molecular relaxations in binary dispersions. The enthalpy relaxation behavior of VLB and ETB was found to be significantly different. PVP stabilized VLB significantly; however, its effect on ETB was negligible. The extent of enthalpy relaxation was found to correlate with hydrogen bonding tendency of the drug molecules. The outcome can help in rational designing of amorphous systems with optimal performance.

Flux-driven simulations of turbulence collapse

DOE PAGES

Park, G. Y.; Kim, S. S.; Jhang, Hogun; ...

2015-03-12

In this study, using self-consistent three-dimensional nonlinear simulations of tokamak turbulence, we show that an edge transport barrier (ETB) forms naturally due to mean E x B shear feedback through evolving pressure gradient once input power exceeds a threshold value. The temporal evolution and development of the transition are elucidated. Profiles, turbulence-driven flows and neoclassical coefficients are evolved self-consistently. A slow power ramp-up simulation shows that ETB transition is triggered by the turbulence-driven flows via an intermediate phase which involves coherent oscillation of turbulence intensity and E x B flow shear. A novel observation of the evolution is that themore » turbulence collapses and the ETB transition begins when R T > 1 at t = t R (R T : normalized Reynolds power), while the conventional transition criterion (ω E x B > γlin) is satisfied only after t = t C (> t R), when the mean ow shear grows due to positive feedback.« less

Ideal MHD stability of double transport barrier plasmas in DIII-D

NASA Astrophysics Data System (ADS)

Li, G. Q.; Wang, S. J.; Lao, L. L.; Turnbull, A. D.; Chu, M. S.; Brennan, D. P.; Groebner, R. J.; Zhao, L.

2008-01-01

The ideal MHD stability for double transport barrier (DTB or DB) plasmas with varying edge and internal barrier width and height was investigated, using the ideal MHD stability code GATO. A moderate ratio of edge transport barriers (ETB) height to internal transport barriers (ITBs) height is found to be beneficial to MHD stability and the βN is limited by global low n instabilities. For moderate ITB width DB plasmas, if the ETB is weak, the stability is limited by n = 1 (n is the toroidal mode number) global mode; whereas if the ETB is strong it is limited by intermediate-n edge peeling-ballooning modes. Broadening the ITB can improve stability if the ITB half width wi lsim 0.3. For very broad ITB width plasmas the stability is limited by stability to a low n (n > 1) global mode.

The endothelin system has a significant role in the pathogenesis and progression of Mycobacterium tuberculosis infection.

PubMed

Correa, Andre F; Bailão, Alexandre M; Bastos, Izabela M D; Orme, Ian M; Soares, Célia M A; Kipnis, Andre; Santana, Jaime M; Junqueira-Kipnis, Ana Paula

2014-12-01

Tuberculosis (TB) remains a major global health problem, and although multiple studies have addressed the relationship between Mycobacterium tuberculosis and the host on an immunological level, few studies have addressed the impact of host physiological responses. Proteases produced by bacteria have been associated with important alterations in the host tissues, and a limited number of these enzymes have been characterized in mycobacterial species. M. tuberculosis produces a protease called Zmp1, which appears to be associated with virulence and has a putative action as an endothelin-converting enzyme. Endothelins are a family of vasoactive peptides, of which 3 distinct isoforms exist, and endothelin 1 (ET-1) is the most abundant and the best-characterized isoform. The aim of this work was to characterize the Zmp1 protease and evaluate its role in pathogenicity. Here, we have shown that M. tuberculosis produces and secretes an enzyme with ET-1 cleavage activity. These data demonstrate a possible role of Zmp1 for mycobacterium-host interactions and highlights its potential as a drug target. Moreover, the results suggest that endothelin pathways have a role in the pathogenesis of M. tuberculosis infections, and ETA or ETB receptor signaling can modulate the host response to the infection. We hypothesize that a balance between Zmp1 control of ET-1 levels and ETA/ETB signaling can allow M. tuberculosis adaptation and survival in the lung tissues. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Toxicokinetics of ethers used as fuel oxygenates.

PubMed

Dekant, W; Bernauer, U; Rosner, E; Amberg, A

2001-10-15

The toxicokinetics and biotransformation of methyl-tert.butyl ether (MTBE), ethyl-tert.butyl ether (ETBE) and tert.amyl-methyl ether (TAME) in rats and humans are summarized. These ethers are used as gasoline additives in large amounts, and thus, a considerable potential for human exposure exists. After inhalation exposure MTBE, ETBE and TAME are rapidly taken up by both rats and humans; after termination of exposure, clearance by exhalation and biotransformation to urinary metabolites is rapid in rats. In humans, clearance by exhalation is slower in comparison to rats. Biotransformation of MTBE and ETBE is both qualitatively and quantitatively similar in humans and rats after inhalation exposure under identical conditions. The extent of biotransformation of TAME is also quantitatively similar in rats and humans; the metabolic pathways, however, are different. The results suggest that reactive and potentially toxic metabolites are not formed during biotransformation of these ethers and that toxic effects of these compounds initiated by covalent binding to cellular macromolecules are unlikely.

Role of endothelin-1 and its receptors, ETA and ETB, in the survival of human vascular endothelial cells.

PubMed

Mikhail, Marianne; Vachon, Pierre H; D'Orléans-Juste, Pedro; Jacques, Danielle; Bkaily, Ghassan

2017-10-01

Our previous work showed the presence of endothelin-1 (ET-1) receptors, ET A and ET B , in human vascular endothelial cells (hVECs). In this study, we wanted to verify whether ET-1 plays a role in the survival of hVECs via the activation of its receptors ET A and (or) ET B (ET A R and ET B R, respectively). Our results showed that treatment of hVECs with ET-1 prevented apoptosis induced by genistein, an effect that was mimicked by treatment with ET B R-specific agonist IRL1620. Furthermore, blockade of ET B R with the selective ET B R antagonist A-192621 prevented the anti-apoptotic effect of ET-1 in hVECs. However, activation of ET A receptor alone did not seem to contribute to the anti-apoptotic effect of ET-1. In addition, the anti-apoptotic effect of ET B R was found to be associated with caspase 3 inhibition and does not depend on the density of this type of receptor. In conclusion, our results showed that ET-1 possesses an anti-apoptotic effect in hVECs and that this effect is mediated, to a great extent, via the activation of ET B R. This study revealed a new role for ET B R in the survival of hVECs.

Does Deep Bradycardia Increase the Risk of Arrhythmias and Syncope in Endurance Athletes?

PubMed

Matelot, D; Schnell, F; Khodor, N; Endjah, N; Kervio, G; Carrault, G; Thillaye du Boullay, N; Carre, F

2016-09-01

The aim of this study was to evaluate whether endurance athletes who exhibit deep bradycardia are more prone to arrhythmias and reflex syncope than their non-bradycardic peers. 46 healthy men (ages 19-35) were divided into 3 groups based on whether they were sedentary (SED,<2 h/week) or endurance trained (ET,>6 h/week), and non-bradycardic (NB, resting heart rate (HR)≥60 bpm) or bradycardic (B, resting HR<50 bpm). Resting HR was lower in ETB vs. ETNB and SED (43.8±3.1, 61.3±3.3, 66.1±5.9 bpm, respectively; p<0.001). Thus, 16 SED, 13 ETNB and 17 ETB underwent resting echocardiography, maximal exercise test, tilt test (TT) and 24 h-Holter ECG. Subjects were followed-up during 4.7±1.1 years for training, syncope and cardiac events. Our results showed that incidence of arrhythmias and hypotensive susceptibility did not differ between groups. During follow-up, no episode of syncope or near-syncope was reported. However, cardio-inhibitory syncope occurrence tended to be higher in ETB. Left ventricular end-diastolic diameter index was increased in ETB vs. ETNB and was correlated with resting HR (r=- 0.64; p<0.001). As a result, athletes with deep bradycardia do not present more arrhythmias and more hypotensive susceptibility than their non-bradycardic peers. Cardiac enlargement and autonomic alteration both seem to be involved in an athlete's bradycardia. © Georg Thieme Verlag KG Stuttgart · New York.

Endothelin-1–Rho kinase interactions impair lung structure and cause pulmonary hypertension after bleomycin exposure in neonatal rat pups

PubMed Central

Tseng, Nancy; Seedorf, Gregory; Kuhn, Katherine; Abman, Steven H.

2016-01-01

Bronchopulmonary dysplasia (BPD) is the chronic lung disease associated with premature birth, characterized by impaired vascular and alveolar growth. In neonatal rats bleomycin decreases lung growth and causes pulmonary hypertension (PH), which is poorly responsive to nitric oxide. In the developing lung, through Rho kinase (ROCK) activation, ET-1 impairs endothelial cell function; however, whether ET-1–ROCK interactions contribute to impaired vascular and alveolar growth in experimental BPD is unknown. Neonatal rats were treated daily with intraperitoneal bleomycin with and without selective ETA (BQ123/BQ610) and ETB (BQ788) receptor blockers, nonselective ET receptor blocker (ETRB) (bosentan), or fasudil (ROCK inhibitor). At day 14, lungs were harvested for morphometrics, and measurements of Fulton's index (RV/LV+S), medial wall thickness (MWT), and vessel density. Lung ET-1 protein and ROCK activity (phospho-MYPT-1:total MYPT-1 ratio) were also measured by Western blot analysis. Bleomycin increased lung ET-1 protein expression by 65%, RV/LV+S by 60%, mean linear intercept (MLI) by 212%, and MWT by 140% and decreased radial alveolar count (RAC) and vessel density by 40 and 44%, respectively (P < 0.01 for each comparison). After bleomycin treatment, fasudil and bosentan partially restored RAC and vessel density and decreased MLI, RV/LV+S, and MWT to normal values. Bleomycin increased ROCK activity by 120%, which was restored to normal values by bosentan but not selective ETRB. We conclude that ET-1–ROCK interactions contribute to decreased alveolar and vascular growth and PH in experimental BPD. We speculate that nonselective ETRB and ROCK inhibitors may be effective in the treatment of infants with BPD and PH. PMID:27760762

Involvement of endothelin and ET(A) endothelin receptor in mechanical allodynia in mice given orthotopic melanoma inoculation.

PubMed

Fujita, Masahide; Andoh, Tsugunobu; Saiki, Ikuo; Kuraishi, Yasushi

2008-02-01

We investigated whether endothelin (ET) would be involved in skin cancer pain in mice. Orthotopic inoculation of B16-BL6 melanoma cells into the plantar region of the hind paw produced marked mechanical allodynia in C57BL/6 mice. Intraplantar injections of the ET(A)-receptor antagonist BQ-123 (0.3 - 3 nmol/site), but not the ET(B)-receptor antagonist BQ-788 (1 and 3 nmol/site), inhibited mechanical allodynia in mice with grown melanoma. In naive mice, an intraplantar injection of tumor extract (1 and 3 mg/site), which was prepared from the grown melanoma in the paw, produced mechanical allodynia, which was inhibited by BQ-123 and BQ-788 at doses of 3 and 10 nmol/site. An intraplantar injection of ET-1 (1 and 10 pmol/site) elicited licking behavior, which was increased in the melanoma-bearing hind paw. BQ-123 (3 and 10 nmol/site) inhibited licking induced by ET-1 (10 pmol/site). The level of mRNA of ET(A), but not ET(B), receptor, was significantly increased in the dorsal root ganglia on the inoculated side. Cultured B16-BL6 cells contained ET, and the melanoma mass increased the concentration of ET as it grew bigger. These results suggest that ET-1 and ET(A) receptor are at least partly involved in the induction of pain induced by melanoma cell inoculation.

Understanding roles of E  ×  B flow and magnetic shear on the formation of internal and edge transport barriers using two-field bifurcation concept

NASA Astrophysics Data System (ADS)

Chatthong, B.; Onjun, T.

2016-01-01

A set of heat and particle transport equations with the inclusion of E  ×  B flow and magnetic shear is used to understand the formation and behaviors of edge transport barriers (ETBs) and internal transport barriers (ITBs) in tokamak plasmas based on two-field bifurcation concept. A simple model that can describe the E  ×  B flow shear and magnetic shear effect in tokamak plasma is used for anomalous transport suppression with the effect of bootstrap current included. Consequently, conditions and formations of ETB and ITB can be visualized and studied. It can be seen that the ETB formation depends sensitively on the E  ×  B flow shear suppression with small dependence on the magnetic shear suppression. However, the ITB formation depends sensitively on the magnetic shear suppression with a small dependence on the E  ×  B flow shear suppression. Once the H-mode is achieved, the s-curve bifurcation diagram is modified due to an increase of bootstrap current at the plasma edge, resulting in reductions of both L-H and H-L transition thresholds with stronger hysteresis effects. It is also found that both ITB and ETB widths appear to be governed by heat or particle sources and the location of the current peaking. In addition, at a marginal flux just below the L-H threshold, a small perturbation in terms of heat or density fluctuation can result in a transition, which can remain after the perturbation is removed due to the hysteresis effect.

Endothelin ETA Receptor Blockade, by Activating ETB Receptors, Increases Vascular Permeability and Induces Exaggerated Fluid Retention.

PubMed

Vercauteren, Magali; Trensz, Frederic; Pasquali, Anne; Cattaneo, Christophe; Strasser, Daniel S; Hess, Patrick; Iglarz, Marc; Clozel, Martine

2017-05-01

Endothelin (ET) receptor antagonists have been associated with fluid retention. It has been suggested that, of the two endothelin receptor subtypes, ET B receptors should not be blocked, because of their involvement in natriuresis and diuresis. Surprisingly, clinical data suggest that ET A -selective antagonists pose a greater risk of fluid overload than dual antagonists. The purpose of this study was to evaluate the contribution of each endothelin receptor to fluid retention and vascular permeability in rats. Sitaxentan and ambrisentan as ET A -selective antagonists and bosentan and macitentan as dual antagonists were used as representatives of each class, respectively. ET A -selective antagonism caused a dose-dependent hematocrit/hemoglobin decrease that was prevented by ET B -selective receptor antagonism. ET A -selective antagonism led to a significant blood pressure reduction, plasma volume expansion, and a greater increase in vascular permeability than dual antagonism. Isolated vessel experiments showed that ET A -selective antagonism increased vascular permeability via ET B receptor overstimulation. Acutely, ET A -selective but not dual antagonism activated sympathetic activity and increased plasma arginine vasopressin and aldosterone concentrations. The hematocrit/hemoglobin decrease induced by ET A -selective antagonism was reduced in Brattleboro rats and in Wistar rats treated with an arginine vasopressin receptor antagonist. Finally, the decrease in hematocrit/hemoglobin was larger in the venous than in the arterial side, suggesting fluid redistribution. In conclusion, by activating ET B receptors, endothelin receptor antagonists (particularly ET A -selective antagonists) favor edema formation by causing: 1) fluid retention resulting from arginine vasopressin and aldosterone activation secondary to vasodilation, and 2) increased vascular permeability. Plasma volume redistribution may explain the clinical observation of a hematocrit/hemoglobin decrease even in the absence of signs of fluid retention. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Endothelin-2, the forgotten isoform: emerging role in the cardiovascular system, ovarian development, immunology and cancer

PubMed Central

Ling, Lowell; Maguire, Janet J; Davenport, Anthony P

2013-01-01

Endothelin-2 [ET-2; also known as vasoactive intestinal contractor (VIC), in rodents] differs from endothelin-1 (ET-1) by only two amino acids, and unlike the third isoform, endothelin-3 (ET-3), it has the same affinity as ET-1 for both ETA and ETB receptors. It is often assumed that ET-2 would mimic the actions of the more abundant ET-1 and current pharmacological interventions used to inhibit the ET system would also block the actions of ET-2. These assumptions have focused research on ET-1 with ET-2 studied in much less detail. Recent research suggests that our understanding of the ET family requires re-evaluation. Although ET-2 is very similar in structure as well as pharmacology to ET-1, and may co-exist in the same tissue compartments, there is converging evidence for an important and distinct ET-2 pathway. Specifically is has been demonstrated that ET-2 has a key role in ovarian physiology, with ET-2-mediated contraction proposed as a final signal facilitating ovulation. Furthermore, ET-2 may also have a pathophysiological role in heart failure, immunology and cancer. Comparison of ET-2 versus ET-1 mRNA expression suggests this may be accomplished at the level of gene expression but differences may also exist in peptide synthesis by enzymes such as endothelin converting enzymes (ECEs) and chymase, which may allow the two pathways to be distinguished pharmacologically and become separate drug targets. LINKED ARTICLES This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1 PMID:22118774

Nuclear Membranes ETB Receptors Mediate ET-1-induced Increase of Nuclear Calcium in Human Left Ventricular Endocardial Endothelial Cells.

PubMed

Jules, Farah; Avedanian, Levon; Al-Khoury, Johny; Keita, Ramatoulaye; Normand, Alexandre; Bkaily, Ghassan; Jacques, Danielle

2015-07-01

In fetal human left ventricular endocardial endothelial cells (EECLs), both plasma membrane (PM) ET(A)R and ET(B)R were reported to mediate ET-1-induced increase of intracellular calcium [Ca](i); however, this effect was mediated by ET(A)R in right EECs (EECRs). In this study, we verified whether, as for the PM, nuclear membranes (NMs) ET-1 receptors activation in EECLs and EECRs induce an increase of nuclear calcium ([Ca](n)) and if this effect is mediated through the same receptor type as in PM. Using a plasmalemma-perforated technique and 3D confocal microscopy, our results showed that, as in PM intact cells, superfusion of nuclei of both cell types with cytosolic ET-1 induced a concentration-dependent sustained increase of [Ca](n). In EECRs, the ET(A)R antagonist prevented the effect of ET-1 on [Ca](n) without affecting EECLs. However, in both cell types, the effect of cytosolic ET-1 on [Ca](n) was prevented by the ETBR antagonist. In conclusion, both NMs' ET(A)R and ET(B)R mediated the effect of cytosolic ET-1 on [Ca](n) in EECRs. In contrast, only NMs' ET(B)R activation mediated the effect of cytosolic ET-1 in EECLs. Hence, the type of NMs' receptors mediating the effect of ET-1 on [Ca](n) are different from those of PM mediating the increase in [Ca](i).

Vapor intrusion risk of fuel ether oxygenates methyl tert-butyl ether (MTBE), tert-amyl methyl ether (TAME) and ethyl tert-butyl ether (ETBE): A modeling study.

PubMed

Ma, Jie; Xiong, Desen; Li, Haiyan; Ding, Yi; Xia, Xiangcheng; Yang, Yongqi

2017-06-15

Vapor intrusion of synthetic fuel additives represents a critical yet still neglected problem at sites contaminated by petroleum fuel releases. This study used an advanced numerical model to investigate the vapor intrusion potential of fuel ether oxygenates methyl tert-butyl ether (MTBE), tert-amyl methyl ether (TAME), and ethyl tert-butyl ether (ETBE). Simulated indoor air concentration of these compounds can exceed USEPA indoor air screening level for MTBE (110μg/m 3 ). Our results also reveal that MTBE has much higher chance to cause vapor intrusion problems than TAME and ETBE. This study supports the statements made by USEPA in the Petroleum Vapor Intrusion (PVI) Guidance that the vertical screening criteria for petroleum hydrocarbons may not provide sufficient protectiveness for fuel additives, and ether oxygenates in particular. In addition to adverse impacts on human health, ether oxygenate vapor intrusion may also cause aesthetic problems (i.e., odour and flavour). Overall, this study points out that ether oxygenates can cause vapor intrusion problems. We recommend that USEPA consider including the field measurement data of synthetic fuel additives in the existing PVI database and possibly revising the PVI Guidance as necessary. Copyright © 2017 Elsevier B.V. All rights reserved.

Off-Axis Driven Current Effects on ETB and ITB Formations based on Bifurcation Concept

NASA Astrophysics Data System (ADS)

Pakdeewanich, J.; Onjun, T.; Chatthong, B.

2017-09-01

This research studies plasma performance in fusion Tokamak system by investigating parameters such as plasma pressure in the presence of an edge transport barrier (ETB) and an internal transport barrier (ITB) as the off-axis driven current position is varied. The plasma is modeled based on the bifurcation concept using a suppression function that can result in formation of transport barriers. In this model, thermal and particle transport equations, including both neoclassical and anomalous effects, are solved simultaneously in slab geometry. The neoclassical coefficients are assumed to be constant while the anomalous coefficients depend on gradients of local pressure and density. The suppression function, depending on flow shear and magnetic shear, is assumed to affect only on the anomalous channel. The flow shear can be calculated from the force balance equation, while the magnetic shear is calculated from the given plasma current. It is found that as the position of driven current peak is moved outwards from the plasma center, the central pressure is increased. But at some point it stars to decline, mostly when the driven current peak has reached the outer half of the plasma. The higher pressure value results from the combination of ETB and ITB formations. The drop in central pressure occurs because ITB stats to disappear.

Experimental validation of non-uniformity effect of the radial electric field on the edge transport barrier formation in JT-60U H-mode plasmas

PubMed Central

Kamiya, K.; Itoh, K.; Itoh, S.-I.

2016-01-01

The turbulent structure formation, where strongly-inhomogeneous turbulence and global electromagnetic fields are self-organized, is a fundamental mechanism that governs the evolution of high-temperature plasmas in the universe and laboratory (e.g., the generation of edge transport barrier (ETB) of the H-mode in the toroidal plasmas). The roles of inhomogeneities of radial electric field (Er) are known inevitable. In this mechanism, whether the first derivative of Er (shear) or the second derivative of Er (curvature) works most is decisive in determining the class of nontrivial solutions (which describe the barrier structure). Here we report the experimental identification of the essential role of the Er-curvature on the ETB formation, for the first time, based on the high-spatiotemporal resolution spectroscopic measurement. We found the decisive importance of Er-curvature on ETB formation during ELM-free phase, but there is only a low correlation with the Er-shear value at the peak of normalized ion temperature gradient. Furthermore, in the ELMing phase, the effect of curvature is also quantified in terms of the relationship between pedestal width and thickness of the layer of inhomogeneous Er. This is the fundamental basis to understand the structure of transport barriers in fusion plasmas. PMID:27480931

The Effects of Tooth Brushing on Whole Salivary Flow Rate in Older Adults

PubMed Central

Trottier, K.; Garrick, R.; Mascarenhas, T.; Jang, Y.

2018-01-01

Objectives (1) To determine whether manual (MTB), or electric, tooth brushing (ETB) modulates whole salivary flow rate in older adults who are free of systemic disease. (2) To determine the duration of the brushing-related modulation of salivary flow rate. (3) To compare salivary flow rate modulation associated with MTB and ETB. Method Twenty-one adults aged 60 years and older participated in two experimental sessions during which they used a manual, or electric, toothbrush to brush their teeth, tongue, and palate. Whole salivary flow rates were determined using the draining method before, during, and after brushing. Differences in salivary flow rates across time periods, and between conditions, were examined using paired samples t-tests applying a Holm-Bonferroni sequential procedure (pcorr < 0.0045). The relationship between tooth brushing and age with respect to maximum salivary flow rate increase was examined using Pearson's correlation coefficient (p < 0.05). Results/Conclusion Whole salivary flow rates increased during, and for up to 5 minutes following, tooth brushing in adults aged 60 years and older who were free of systemic disease. The salivary effects of MTB and ETB were not significantly different. A moderate, positive correlation was observed between tooth-brushing-related maximum salivary flow rate increase and age. PMID:29682540

Subthreshold concentration of endothelin-1-enhanced, capsaicin-induced bronchoconstriction in anaesthetized guinea-pigs.

PubMed

Kanazawa, H; Fujiwara, H; Hirata, K; Yoshikawa, J

1998-12-01

An increasing number of studies have been performed to address a possible role for endothelin-1 (ET-1) as a significant mediator in asthma. However, the effects of subthreshold concentrations of ET-1, which cannot elicit bronchial smooth muscle contraction itself, in asthma has yet to be determined. This study determined these effects of ET-1 on capsaicin-induced bronchoconstriction in anaesthetized guinea-pigs. Aerosolized ET-1 administered at doses of 10(-9) M and higher induced a dose-dependent increase in pulmonary resistance, but ET-1 at 10(-10) M did not have any bronchoconstrictive effect. However, this subthreshold concentration of ET-1 potentiated capsaicin-induced bronchoconstriction. In addition, the potentiation of capsaicin-induced bronchoconstriction by this subthreshold concentration of ET-1 was completely abolished by BQ788 (ET(B) receptor antagonist), but not BQ123 (ET(A) receptor antagonists). Immunoreactive substance P (SP) levels in bronchoalveolar lavage fluid after capsaicin administration were significantly higher than those after solvent administration. However, ET-1 alone did not significantly stimulate immunoreactive SP release and ET-1 (10(-10) M) did not potentiate capsaicin-induced immunoreactive SP release. In contrast, ET-1 (10(-10) M) potentiated exogenous neurokinin A- and SP-induced bronchoconstriction. These findings suggest that a subthreshold concentration of endothelin-1 does not potentiate the tachykinin release induced by capsaicin but the airway smooth muscle contraction through endothelin-B receptors.

Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism.

PubMed

Angus, James A; Hughes, Richard J A; Wright, Christine E

2017-12-01

Dual endothelin ET A and ET B receptor antagonists are approved therapy for pulmonary artery hypertension (PAH). We hypothesized that ET B receptor-mediated clearance of endothelin-1 at specific vascular sites may compromise this targeted therapy. Concentration-response curves (CRC) to endothelin-1 or the ET B agonist sarafotoxin S6c were constructed, with endothelin receptor antagonists, in various rat and mouse isolated arteries using wire myography or in rat isolated trachea. In rat small mesenteric arteries, bosentan displaced endothelin-1 CRC competitively indicative of ET A receptor antagonism. In rat small pulmonary arteries, bosentan 10 μmol L -1 left-shifted the endothelin-1 CRC, demonstrating potentiation consistent with antagonism of an ET B receptor-mediated endothelin-1 clearance mechanism. Removal of endothelium or L-NAME did not alter the EC 50 or Emax of endothelin-1 nor increase the antagonism by BQ788. In the presence of BQ788 and L-NAME, bosentan displayed ET A receptor antagonism. In rat trachea (ET B ), bosentan was a competitive ET B antagonist against endothelin-1 or sarafotoxin S6c. Modeling showed the importance of dual receptor antagonism where the potency ratio of ET A to ET B antagonism is close to unity. In conclusion, the rat pulmonary artery is an example of a special vascular bed where the resistance to antagonism of endothelin-1 constriction by ET dual antagonists, such as bosentan or the ET B antagonist BQ788, is possibly due to the competition of potentiation of endothelin-1 by blockade of ET B -mediated endothelin-1 clearance located on smooth muscle and antagonism of ET A - and ET B -mediated contraction. This conclusion may have direct application for the efficacy of endothelin-1 antagonists for treating PAH. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (Etbe) ...

EPA Pesticide Factsheets

In September 2016, the U.S. Environmental Protection Agency's (USEPA) released the draft Integrated Risk Information System (IRIS) Toxicological Review of Ethyl Tertiary Butyl Ether (ETBE). Consistent with the 2013 IRIS Enhancements, draft IRIS assessments are released prior to external peer review for a 60-day public comment period and discussed at an upcoming public science meeting. Accordingly, the toxicological review, supplementary information, and other materials pertaining to this draft assessment are posted on this site. This material is being released for public viewing and comment prior to a public meeting, providing an opportunity for the IRIS Program to engage in early discussions with stakeholders and the public on data that may be used to identify adverse health effects and characterize exposure-response relationships.

Epidemiological investigation of nosocomial outbreak of staphylococcal skin diseases in neonatal ward.

PubMed

Kurlenda, J; Grinholc, M; Krzysztoń-Russjan, J; Wiśniewska, K

2009-05-01

During a 1-month period, eight neonates developed staphylococcal skin disease diagnosed as a bullous impetigo in the maternity unit of the Provincial Hospital in Gdansk. An epidemiological investigation based on phenotyping and genotyping methods was performed. All neonates involved in the outbreak, their mothers and 15 staff members were screened for carriage of Staphylococcus aureus by nasal swabs. Isolated strains were compared with strains cultured from affected skin and purulent conjunctiva of infected newborns. Isolates were analyzed for the presence of the etA and etB genes using polymerase chain reaction and genotyped by pulsed-field gel electrophoresis (PFGE) and coa gene polymorphism. The analyzed S. aureus strains were methicillin-sensitive and could be divided into two groups according to antibiotyping, phage typing, coa polymorphism and PFGE pattern. The first group consisted of etA and etB negative strains, and the second one involved only the etB positive ones. Our results have shown that there were two different clusters of infection caused by two populations of S. aureus strains. Among the 15 medical staff members screened we have found seven carriers. However, phage typing revealed that distinct strains unrelated to the outbreak isolates were carried. Although we have not been able to establish the source of bacteria involved in the outbreak, our results suggest that for both groups, mothers could be the source of the infecting strains.

Endothelin-A Receptor Antagonism after Renal Angioplasty Enhances Renal Recovery in Renovascular Disease

PubMed Central

Tullos, Nathan; Stewart, Nicholas J.; Surles, Bret

2015-01-01

Percutaneous transluminal renal angioplasty/stenting (PTRAS) is frequently used to treat renal artery stenosis and renovascular disease (RVD); however, renal function is restored in less than one half of the cases. This study was designed to test a novel intervention that could refine PTRAS and enhance renal recovery in RVD. Renal function was quantified in pigs after 6 weeks of chronic RVD (induced by unilateral renal artery stenosis), established renal damage, and hypertension. Pigs with RVD then underwent PTRAS and were randomized into three groups: placebo (RVD+PTRAS), chronic endothelin-A receptor (ET-A) blockade (RVD+PTRAS+ET-A), and chronic dual ET-A/B blockade (RVD+PTRAS+ET-A/B) for 4 weeks. Renal function was again evaluated after treatments, and then, ex vivo studies were performed on the stented kidney. PTRAS resolved renal stenosis, attenuated hypertension, and improved renal function but did not resolve renal microvascular rarefaction, remodeling, or renal fibrosis. ET-A blocker therapy after PTRAS significantly improved hypertension, microvascular rarefaction, and renal injury and led to greater recovery of renal function. Conversely, combined ET-A/B blockade therapy blunted the therapeutic effects of PTRAS alone or PTRAS followed by ET-A blockade. These data suggest that ET-A receptor blockade therapy could serve as a coadjuvant intervention to enhance the outcomes of PTRAS in RVD. These results also suggest that ET-B receptors are important for renal function in RVD and may contribute to recovery after PTRAS. Using clinically available compounds and techniques, our results could contribute to both refinement and design of new therapeutic strategies in chronic RVD. PMID:25377076

First-in-Man Demonstration of Direct Endothelin-Mediated Natriuresis and Diuresis

PubMed Central

Hunter, Robert W.; Moorhouse, Rebecca; Farrah, Tariq E.; MacIntyre, Iain M.; Asai, Takae; Gallacher, Peter J.; Kerr, Debbie; Melville, Vanessa; Czopek, Alicja; Morrison, Emma E.; Ivy, Jess R.; Dear, James W.; Bailey, Matthew A.; Goddard, Jane; Webb, David J.

2017-01-01

Endothelin (ET) receptor antagonists are potentially novel therapeutic agents in chronic kidney disease and resistant hypertension, but their use is complicated by sodium and water retention. In animal studies, this side effect arises from ETB receptor blockade in the renal tubule. Previous attempts to determine whether this mechanism operates in humans have been confounded by the hemodynamic consequences of ET receptor stimulation/blockade. We aimed to determine the effects of ET signaling on salt transport in the human nephron by administering subpressor doses of the ET-1 precursor, big ET-1. We conducted a 2-phase randomized, double-blind, placebo-controlled crossover study in 10 healthy volunteers. After sodium restriction, subjects received either intravenous placebo or big ET-1, in escalating dose (≤300 pmol/min). This increased plasma concentration and urinary excretion of ET-1. Big ET-1 reduced heart rate (≈8 beats/min) but did not otherwise affect systemic hemodynamics or glomerular filtration rate. Big ET-1 increased the fractional excretion of sodium (from 0.5 to 1.0%). It also increased free water clearance and tended to increase the abundance of the sodium–potassium–chloride cotransporter (NKCC2) in urinary extracellular vesicles. Our protocol induced modest increases in circulating and urinary ET-1. Sodium and water excretion increased in the absence of significant hemodynamic perturbation, supporting a direct action of ET-1 on the renal tubule. Our data also suggest that sodium reabsorption is stimulated by ET-1 in the thick ascending limb and suppressed in the distal renal tubule. Fluid retention associated with ET receptor antagonist therapy may be circumvented by coprescribing potassium-sparing diuretics. PMID:28507171

Selective improvement of pulmonary arterial hypertension with a dual ETA/ETB receptors antagonist in the apolipoprotein E-/- model of PAH and atherosclerosis.

PubMed

Renshall, Lewis; Arnold, Nadine; West, Laura; Braithwaite, Adam; Pickworth, Josephine; Walker, Rachel; Alfaidi, Mabruka; Chamberlain, Janet; Casbolt, Helen; Thompson, A A Roger; Holt, Cathy; Iglarz, Marc; Francis, Sheila; Lawrie, Allan

2018-01-01

Idiopathic pulmonary arterial hypertension (IPAH) is increasingly diagnosed in elderly patients who also have an increased risk of co-morbid atherosclerosis. Apolipoprotein E-deficient (ApoE -/- ) mice develop atherosclerosis with severe PAH when fed a high-fat diet (HFD) and have increased levels of endothelin (ET)-1. ET-1 receptor antagonists (ERAs) are used for the treatment of PAH but less is known about whether ERAs are beneficial in atherosclerosis. We therefore examined whether treatment of HFD-ApoE -/- mice with macitentan, a dual ET A /ET B receptor antagonist, would have any effect on both atherosclerosis and PAH. ApoE -/- mice were fed chow or HFD for eight weeks. After four weeks of HFD, mice were randomized to a four-week treatment of macitentan by food (30 mg/kg/day dual ET A /ET B antagonist), or placebo groups. Echocardiography and closed-chest right heart catheterization were used to determine PAH phenotype and serum samples were collected for cytokine analysis. Thoracic aortas were harvested to assess vascular reactivity using wire myography, and histological analyses were performed on the brachiocephalic artery and aortic root to assess atherosclerotic burden. Macitentan treatment of HFD-fed ApoE -/- mice was associated with a beneficial effect on the PAH phenotype and led to an increase in endothelial-dependent relaxation in thoracic aortae. Macitentan treatment was also associated with a significant reduction in interleukin 6 (IL-6) concentration but there was no significant effect on atherosclerotic burden. Dual blockade of ET A /ET B receptors improves endothelial function and improves experimental PAH but had no significant effect on atherosclerosis.

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

PubMed

Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

2014-04-01

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p < 0.05), while the frequency of sea, sed, eta, and etb genes were higher among MRSA isolates (p > 0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

The Development of Differential Use of Inner and Outer Face Features in Familiar Face Identification.

ERIC Educational Resources Information Center

Campbell, Ruth; And Others

1995-01-01

Studied 4- to 10-year-olds' familiarity judgments of peers. Found that, contrary to adults, external facial features were key. Also found that the switch to adult recognition pattern takes place after the ninth year. (ETB)

U.S. EPA, Pesticides, Label, DITHIOPYR 0.172 PLUS, 7/10/2009

EPA Pesticide Factsheets

2011-04-13

... MS. NC, se, TX Crowfootgrass. ... V::~:;esi:o~~:~~t~~:~~t~s::~w~oison ;::::lyrfc~~~;~et~:b= ~~ ~s:::,,ds:: :: :gO~~f6~~ (~; ...

Contamination of Ethiopian paper currency notes from various food handlers with E. coli.

PubMed

Hiko, Adem; Abdata, Kasahun; Muktar, Yimer; Woyesa, Mezene; Mohammed, Abdela

2016-01-01

Contamination rate of Ethiopian paper currency notes handled by various food handlers with Escherichia coli and antimicrobial susceptibility of the isolates was assessed. A total of 384 Ethiopian Birr (ETB) notes were randomly sampled from meat handlers at butchers, bread and the related food handlers at cafeteria, fruit and vegetables handlers at supermarket, and milk sellers both at open market and dairy station. Fifty control new currencies were also sampled from Commercial Bank of Ethiopia. Both surfaces of the currency were swabbed using wet sterile cotton. The swab was overnight incubated in buffered peptone water. A loop full was streaked on eosin methylene blue agar and followed by biochemical test on presumptive E. coli colonies. Randomly selected isolates were exposed to chloramphenicol (C-30 µg), neomycin (N-30 µg), oxytetracycline (OT-30 µg), polymyxin-B (PB-300 IU) and trimethoprim-sulfamethoxazole (SXT-1.25/23.75/µg) susceptibility using disc diffusion techniques. E. coli was not isolated from currency used as control. A total of 288 (75 %) currency notes were found carrying E. coli. E. coli prevalence was ranges from 67.2 % at open market milk sellers to 87.2 % at dairy station milk sellers; from 64.8 % on ETB 100 to 82.9 % on ETB 1. Differences were not observed in E. coli prevalence on currency notes from among almost all food handlers (P > 0.05). Susceptibility of tested isolates to each chloramphenicol, oxytetracycline and trimethoprim-sulfamethoxazole was 100 %, and to polymyxin-B was 97.3 %. High resistance (83.7 %) was observed to neomycin. The finding indicates, contaminated food can be a source of E. coli for further contamination of currency which again transfer through various foods ready for consumption.

Thermodynamic aspects of reformulation of automotive fuels

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

Zudkevitch, D.; Murthy, A.K.S.; Gmehling, J.

1995-09-01

A study of procedures for measuring and predicting the RVP and the initial vapor emissions of reformulated gasoline blends which contain one or more oxygenated compounds, viz., Ethanol, MTBE, ETBE, and TAME is discussed. Two computer simulation methods were programmed and tested. In one method, Method A, the D-86 distillation data on the blend are used for predicting the blend`s RVP from a simulation of the Mini RVPE (RVP Equivalent) experiment. The other method, Method B, relies on analytical information (PIANO analyzes) on the nature of the base gasoline and utilizes classical thermodynamics for simulating the same RVPE, Mini experiment.more » Method B, also, predicts the composition and other properties of the initial vapor emission from the fuel. The results indicate that predictions made with both methods agree very well with experimental values. The predictions with Method B illustrate that the admixture of an oxygenate to a gasoline blend changes the volatility of the blend and, also, the composition of the vapor emission. From the example simulations, a blend with 10 vol % ethanol increases the RVP by about 0.8 psi. The accompanying vapor emission will contain about 15% ethanol. Similarly, the vapor emission of a fuel blend with 11 vol % MTBE was calculated to contain about 11 vol % MTBE. Predictions of the behavior of blends with ETBE and ETBE+Ethanol are also presented and discussed. Recognizing that quite some efforts have been invested in developing empirical correlations for predicting RVP, the writers consider the purpose of this paper to be pointing out that the methods of classical thermodynamics are adequate and that there is a need for additional work in developing certain fundamental data that are still lacking.« less

Electron transport in nano-scaled piezoelectronic devices

NASA Astrophysics Data System (ADS)

Jiang, Zhengping; Kuroda, Marcelo A.; Tan, Yaohua; Newns, Dennis M.; Povolotskyi, Michael; Boykin, Timothy B.; Kubis, Tillmann; Klimeck, Gerhard; Martyna, Glenn J.

2013-05-01

The Piezoelectronic Transistor (PET) has been proposed as a post-CMOS device for fast, low-power switching. In this device, the piezoresistive channel is metalized via the expansion of a relaxor piezoelectric element to turn the device on. The mixed-valence compound SmSe is a good choice of PET channel material because of its isostructural pressure-induced continuous metal insulator transition, which is well characterized in bulk single crystals. Prediction and optimization of the performance of a realistic, nano-scaled PET based on SmSe requires the understanding of quantum confinement, tunneling, and the effect of metal interface. In this work, a computationally efficient empirical tight binding (ETB) model is developed for SmSe to study quantum transport in these systems and the scaling limit of PET channel lengths. Modulation of the SmSe band gap under pressure is successfully captured by ETB, and ballistic conductance shows orders of magnitude change under hydrostatic strain, supporting operability of the PET device at nanoscale.

Proof Construction: Adolescent Development from Inductive to Deductive Problem-Solving Strategies.

ERIC Educational Resources Information Center

Foltz, Carol; And Others

1995-01-01

Studied 100 adolescents' approaches to problem-solving proofs and reasoning competence tasks. Found that a formal level of reasoning competence is associated with a deductive approach. Results support the notion of a cognitive development progression from an inductive approach to a deductive approach. (ETB)

Endothelin receptor antagonist macitentan or deletion of mouse mast cell protease 4 delays lesion development in atherosclerotic mice.

PubMed

Houde, Martin; Desbiens, Louisane; Schwertani, Adel; Pejler, Gunnar; Iglarz, Marc; D'Orléans-Juste, Pedro

2016-08-15

To determine the impact of mixed endothelin receptor antagonist and mouse mast cell protease-4 (mMCP-4) in the development of atherosclerosis in the mouse model. Apolipoprotein E (ApoE) KO mice were crossed with mMCP-4 KO mice to generate ApoE/mMCP-4 double KO mice. Atherosclerosis was induced with a normal- or high-fat diet for 12, 27 or 52weeks. Macitentan (30mg/kg/day), a dual ETA/ETB receptor antagonist, was given orally for 6weeks (27week protocol). At sacrifice, aortas and brachiocephalic arteries (BCAs) were collected. En face Sudan IV staining was performed on aortas and BCA sections were subjected to Masson's trichrome stain and α-smooth muscle actin labeling. Under normal diet, both macitentan treatment and the absence of mMCP-4 reduced the development of aortic atherosclerotic lesions in 27-week old ApoE KO mice, but mMCP-4 deletion failed to maintain this effect on 52-week old mice. Under high-fat diet (WD), macitentan, but not the absence of mMCP-4, reduced aortic lesion development in ApoE KO mice. On BCA lesions of 27-week old WD mice, macitentan treatment had a small impact while mMCP-4 deletion showed improved features of plaque stability. These results suggest that the inhibition of mMCP-4 reduces lesion spreading in the earlier phases of atherosclerosis development and can help stabilise the more advanced plaque. Macitentan treatment was more effective to prevent lesion spreading but did not improve plaque features to the same extent. Copyright © 2016 Elsevier Inc. All rights reserved.

A case for biofuels in aviation

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

NONE

1996-12-31

In the last 15 years, the technical and the economic feasibility of biomass based fuels for general aviation piston engines has been proven. Exhaustive ground and flight tests performed at the Renewable Aviation Fuels Development Center (RAFDC) using ethanol, ethanol/methanol blends, and ETBE have proven these fuels to be superior to aviation gasoline (avgas) in all aspects of performance except range. Two series of Lycoming engines have been certified. Record flights, including a transatlantic flight on pure ethanol, were made to demonstrate the reliability of the fuel. Aerobatic demonstrations with aircraft powered by ethanol, ethanol/methanol, and ETBE were flown atmore » major airshows around the world. the use of bio-based fuels for aviation will benefit energy security, improve the balance of trade, domestic economy, and environmental quality. The United States has the resources to supply the aviation community`s needs with a domestically produced fuel using current available technology. The adoption of a renewable fuel in place of conventional petroleum-based fuels for aviation piston and turbine engines is long overdue.« less

Virulence Factor Genes in Staphylococcus aureus Isolated From Diabetic Foot Soft Tissue and Bone Infections.

PubMed

Víquez-Molina, Gerardo; Aragón-Sánchez, Javier; Pérez-Corrales, Cristian; Murillo-Vargas, Christian; López-Valverde, María Eugenia; Lipsky, Benjamin A

2018-03-01

The aim of this study is to describe the presence of genes encoding for 4 virulence factors (pvl, eta, etb, and tsst), as well as the mecA gene conferring resistance to beta-lactam antibiotics, in patients with diabetes and a staphylococcal foot infection. We have also analyzed whether isolates of Staphylococcus aureus from bone infections have a different profile for these genes compared with those from exclusively soft tissue infections. In this cross-sectional study of a prospectively recruited series of patients admitted to the Diabetic Foot Unit, San Juan de Dios Hospital, San José, Costa Rica with a moderate or severe diabetic foot infection (DFI), we collected samples from infected soft tissue and from bone during debridement. During the study period (June 1, 2014 to May 31, 2016), we treated 379 patients for a DFI. S aureus was isolated from 101 wound samples, of which 43 were polymicrobial infections; we only included the 58 infections that were monomicrobial S aureus for this study. Infections were exclusively soft tissue in 17 patients (29.3%) while 41 (70.7%) had bone involvement (osteomyelitis). The mecA gene was detected in 35 cases (60.3%), pvl gene in 4 cases (6.9%), and tsst gene in 3 (5.2%). We did not detect etA and etB in any of the cases. There were no differences in the profile of S aureus genes encoding for virulence factors (pvl, etA, etB, and tsst) recovered from DFIs between those with just soft tissue compared to those with osteomyelitis. However, we found a significantly higher prevalence of pvl+ strains of S aureus associated with soft tissue compared with bone infections. Furthermore, we observed a significantly longer time to healing among patients infected with mecA+ (methicillin-resistant) S aureus (MRSA).

Model based design of electronic throttle control

NASA Astrophysics Data System (ADS)

Cherian, Fenin; Ranjan, Ashish; Bhowmick, Pathikrit; Rammohan, A.

2017-11-01

With the advent of torque based Engine Management Systems, the precise control and robust performance of the throttle body becomes a key factor in the overall performance of the vehicle. Electronic Throttle Control provides benefits such as improved air-fuel ratio for improving the vehicle performance and lower exhausts emissions to meet the stringent emission norms. Modern vehicles facilitate various features such as Cruise Control, Traction Control, Electronic Stability Program and Pre-crash systems. These systems require control over engine power without driver intervention, which is not possible with conventional mechanical throttle system. Thus these systems are integrated to function with the electronic throttle control. However, due to inherent non-linearities in the throttle body, the control becomes a difficult task. In order to eliminate the influence of this hysteresis at the initial operation of the butterfly valve, a control to compensate the shortage must be added to the duty required for starting throttle operation when the initial operation is detected. Therefore, a lot of work is being done in this field to incorporate the various nonlinearities to achieve robust control. In our present work, the ETB was tested to verify the working of the system. Calibration of the TPS sensors was carried out in order to acquire accurate throttle opening angle. The response of the calibrated system was then plotted against a step input signal. A linear model of the ETB was prepared using Simulink and its response was compared with the experimental data to find out the initial deviation of the model from the actual system. To reduce this deviation, non-linearities from existing literature were introduced to the system and a response analysis was performed to check the deviation from the actual system. Based on this investigation, an introduction of a new nonlinearity parameter can be used in future to reduce the deviation further making the control of the ETB more precise and accurate.

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

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

Kajitani, Takashi; Tamamori-Adachi, Mimi; Okinaga, Hiroko

Highlights: {yields} Steroid hormones repress expression of PTHrP in the cell lines where the corresponding nuclear receptors are expressed. {yields} Nuclear receptors are required for suppression of PTHrP expression by steroid hormones, except for androgen receptor. {yields} Androgen-induced suppression of PTHrP expression appears to be mediated by estrogen receptor. -- Abstract: Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here wemore » studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor {alpha}, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.« less

The lung in liver disease: old problem, new concepts.

PubMed

Fallon, Michael B; Zhang, Junlan

2013-01-01

Liver dysfunction has been recognized to influence the lung in many different clinical situations, although the mechanisms for these effects are not well understood. One increasingly recognized interaction, the hepatopulmonary syndrome (HPS) occurs in the context of cirrhosis and results when alveolar microvascular dilation causes arterial gas exchange abnormalities and hypoxemia. HPS occurs in up to 30% of patients with cirrhosis and significantly increases mortality in affected patients. Currently, liver transplantation is the only curative therapy. Experimental biliary cirrhosis induced by common bile duct ligation (CBDL) in the rat reproduces the pulmonary vascular and gas exchange abnormalities of human HPS and has been contrasted with other experimental models of cirrhosis in which HPS does not develop. Microvascular dilation, intravascular monocyte infiltration, and angiogenesis in the lung have been identified as pathologic features that drive gas exchange abnormalities in experimental HPS. Our recent studies have identified biliary epithelium and activation and interaction between the endothelin-1 (ET-1)/endothelial endothelin B (ETB) receptor and CX3CL1/CX3CR1 pathways as important mechanisms for the observed pathologic events. These studies define novel interactions between the lung and liver in cirrhosis and may lead to effective medical therapies.

ABT-627, an endothelin ET(A) receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain.

PubMed

Jarvis, M F; Wessale, J L; Zhu, C Z; Lynch, J J; Dayton, B D; Calzadilla, S V; Padley, R J; Opgenorth, T J; Kowaluk, E A

2000-01-24

Tactile allodynia, the enhanced perception of pain in response to normally non-painful stimulation, represents a common complication of diabetic neuropathy. The activation of endothelin ET(A) receptors has been implicated in diabetes-induced reductions in peripheral neurovascularization and concomitant endoneurial hypoxia. Endothelin receptor activation has also been shown to alter the peripheral and central processing of nociceptive information. The present study was conducted to evaluate the antinociceptive effects of the novel endothelin ET(A) receptor-selective antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N, N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), in the streptozotocin-induced diabetic rat model of neuropathic pain. Rats were injected with 75 mg/kg streptozotocin (i. p.), and drug effects were assessed 8-12 weeks following streptozotocin treatment to allow for stabilization of blood glucose levels (>/=240 mg/dl) and tactile allodynia thresholds (

Androgen receptor expression in breast cancer in relation to molecular phenotype: results from the Nurses' Health Study.

PubMed

Collins, Laura C; Cole, Kimberly S; Marotti, Jonathan D; Hu, Rong; Schnitt, Stuart J; Tamimi, Rulla M

2011-07-01

Previous studies have demonstrated that androgen receptor is expressed in many breast cancers, but its expression in relation to the various breast cancer subtypes as defined by molecular profiling has not been studied in detail. We constructed tissue microarrays from 3093 breast cancers that developed in women enrolled in the Nurses' Health Study. Tissue microarray sections were immunostained for estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6, epidermal growth factor receptor (EGFR) and androgen receptor (ER). Immunostain results were used to categorize each cancer as luminal A or B, HER2 and basal like. The relationships between androgen receptor expression and molecular subtype were analyzed. Overall, 77% of the invasive breast carcinomas were androgen receptor positive. Among 2171 invasive cancers, 64% were luminal A, 15% luminal B, 6% HER2 and 11% basal like. The frequency of androgen receptor expression varied significantly across the molecular phenotypes (P<0.0001). In particular, androgen receptor expression was commonly observed in luminal A (91%) and B (68%) cancers, but was less frequently seen in HER2 cancers (59%). Despite being defined by the absence of ER and PR expression and being considered hormonally unresponsive, 32% of basal-like cancers expressed androgen receptor. Among 246 cases of ductal carcinoma in situ, 86% were androgen receptor positive, but the frequency of androgen receptor expression differed significantly across the molecular phenotypes (P=0.001), and high nuclear grade lesions were less likely to be androgen receptor positive compared with lower-grade lesions. Androgen receptor expression is most commonly seen in luminal A and B invasive breast cancers. However, expression of androgen receptor is also seen in approximately one-third of basal-like cancers, providing further evidence that basal-like cancers represent a heterogeneous group. Our findings raise the possibility that targeting the androgen receptor pathway may represent a novel therapeutic approach to the management of patients with basal-like cancers.

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

PubMed

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

2012-04-10

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

Effects of endothelin on phospholipases and generation of second messengers in cat iris sphincter and SV-CISM-2 cells.

PubMed

Abdel-Latif, A A; Ding, K H; Akhtar, R A; Yousufzai, S Y

1996-09-01

In both immortalized cat iris sphincter smooth muscle cells (SV-CISM-2 cells) and cat iris sphincter, endothelin-1 (ET-1) markedly increased the activities of phospholipase A2 (PLA2), as measured by the release of arachidonic acid (AA), phospholipase C (PLC), as measured by the production of inositol trisphosphate (IP3), and phospholipase D (PLD), as measured by the formation of phosphatidylethanol (PEt). In SV-CISM-2 cells, ET-1 induced AA release, IP3 production and PEt formation in a dose- and time-dependent manner. The dose-response studies showed that the peptide is more potent in activating PLD (EC50 = 1.2 nM) than in activating PLC (EC50 = 1.5 nM) or PLA2 (EC50 = 1.7 nM). The time course studies revealed that ET-1 activated the phospholipases in a temporal sequence in which PLA2 was stimulated first (t1/2 = 12 s), followed by PLC (t1/2 = 48 s) and lastly PLD (t1/2 = 106 s). In SV-CISM-2 cells, in contrast to the intact iris sphincter, sarafotoxin-c, an ETB receptor agonist, had no effect on the phospholipases, and indomethacin, a cyclooxygenase inhibitor, had no effect on the stimulatory effect of ET-1 on the phospholipases. These results suggest that in this smooth muscle cell line, ET-1 interacts with the ETA receptor subtype to activate, via G proteins, phospholipases A2, C and D in a temporal sequence.

Buprenorphine-elicited alteration of adenylate cyclase activity in human embryonic kidney 293 cells coexpressing κ-, μ-opioid and nociceptin receptors

PubMed Central

Wang, Pei-Chen; Ho, Ing-Kang; Lee, Cynthia Wei-Sheng

2015-01-01

Buprenorphine, a maintenance drug for heroin addicts, exerts its pharmacological function via κ- (KOP), μ-opioid (MOP) and nociceptin/opioid receptor-like 1 (NOP) receptors. Previously, we investigated its effects in an in vitro model expressing human MOP and NOP receptors individually or simultaneously (MOP, NOP, and MOP+NOP) in human embryonic kidney 293 cells. Here, we expanded this cell model by expressing human KOP, MOP and NOP receptors individually or simultaneously (KOP, KOP+MOP, KOP+NOP and KOP+MOP+NOP). Radioligand binding with tritium-labelled diprenorphine confirmed the expression of KOP receptors. Immunoblotting and immunocytochemistry indicated that the expressed KOP, MOP and NOP receptors are N-linked glycoproteins and colocalized in cytoplasmic compartments. Acute application of the opioid receptor agonists— U-69593, DAMGO and nociceptin— inhibited adenylate cyclase (AC) activity in cells expressing KOP, MOP and NOP receptors respectively. Buprenorphine, when applied acutely, inhibited AC activity to ~90% in cells expressing KOP+MOP+NOP receptors. Chronic exposure to buprenorphine induced concentration-dependent AC superactivation in cells expressing KOP+NOP receptors, and the level of this superactivation was even higher in KOP+MOP+NOP-expressing cells. Our study demonstrated that MOP receptor could enhance AC regulation in the presence of coexpressed KOP and NOP receptors, and NOP receptor is essential for concentration-dependent AC superactivation elicited by chronic buprenorphine exposure. PMID:26153065

75 FR 63827 - Integrated Risk Information System (IRIS); Request for Chemical Substance Nominations for 2011...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-10-18

........... 156-60-5 1,4-dioxane (oral) 123-91-1 ethylene glycol monobutyl ether 111-76-2 (EGBE...-dichlorobenzene 106-46-7 1,2-dichloroethane (ethylene 107-06-2 dichloride). dichloromethane (methylene 75-09-2... 131-18-0 ethanol 64-17-5 ethyl tertiary butyl ether (ETBE). 637-92-3 ethylbenzene 100-41-4 ethylene...

IRIS Toxicological Review of Ethyl Tertiary Butyl Ether (Etbe) ...

EPA Pesticide Factsheets

In August 2013, EPA released the draft literature searches and associated search strategies, evidence tables, and exposure response arrays for ETBE to obtain input from stakeholders and the public prior to developing the draft IRIS assessment. Specifically, EPA was interested in comments on the following: Draft literature search strategies The approach for identifying studies The screening process for selecting pertinent studies The resulting list of pertinent studies Preliminary evidence tables The process for selecting studies to include in evidence tables The quality of the studies in the evidence tables The literature search strategy, which describes the processes for identifying scientific literature, contains the studies that EPA considered and selected to include in the evidence tables. The preliminary evidence tables and exposure-response arrays present the key study data in a standardized format. The evidence tables summarize the available critical scientific literature. The exposure-response figures provide a graphical representation of the responses at different levels of exposure for each study in the evidence table. The draft Toxicological Review of Ethyl Tertiary Butyl Ether provides scientific support and rationale for the hazard and dose-response assessment pertaining to chronic exposure to ethyl tertiary butyl ether.

The revenue generated from clinical chemistry and hematology laboratory services as determined using activity-based costing (ABC) model.

PubMed

Adane, Kasaw; Abiy, Zenegnaw; Desta, Kassu

2015-01-01

The rapid and continuous growth of health care cost aggravates the frequently low priority and less attention given in financing laboratory services. The poorest countries have the highest out-of-pocket spending as a percentage of income. Higher charges might provide a greater potential for revenue. If fees raise quality sufficiently, it can enhance usage. Therefore, estimating the revenue generated from laboratory services could help in capacity building and improved quality service provision. Panel study design was used to determine revenue generated from clinical chemistry and hematology services at Tikur Anbessa Specialized Teaching Hospital, Addis Ababa, Ethiopia. Activity-Based Costing (ABC) model was used to determine the true cost of tests performed from October 2011 to December 2011 in the hospital. The principle of Activity-based Costing is that activities consume resources and activities consumed by services which incur the costs and hence service takes the cost of resources. All resources with costs are aggregated with the established casual relationships. The process maps designed was restructured in consultation with the senior staffs working and/or supervising the laboratory and pretested checklists were used for observation. Moreover, office documents, receipts and service bills were used while collecting data. The amount of revenue collected from services was compared with the cost of each subsequent test and the profitability or return on investment (ROI) of services was calculated. Data were collected, entered, cleaned, and analyzed using Microsoft Excel 2007 software program and Statistical Software Package for Social Sciences version 19 (SPSS). Paired sample t test was used to compare the price and cost of each test. P-value less than 0.05 were considered as statistically significant. A total of 25,654 specimens were analyzed during 3 months of regular working hours. The total numbers of clinical chemistry and hematology tests performed during the study period were 45,959 (66.1 %) and 23,570 (33.9 %), respectively. Only 274, 386 (25.3 %) Ethiopian Birr (ETB) was recovered from the total cost of 1,086,008.09 ETB incurred on clinical chemistry and hematology laboratory tests. The result showed that, about 133,821 (12.32 %) ETB was revenue not collected from out-of-pocket payments that was paid for the services as a result of under pricing. The result showed that 18 out of 20 laboratory tests were under priced. The cost burden related to free Anti Retro-viral Therapy (ART) services was 285,979.82 (26.3 %) ETB. The cost per test estimated was significantly different to the existing price. About 90 % of the tests were under priced. This information could warn the hospital to reconsider resetting prices of these tests profitability ration less than 1. The revenue collected could help to build capacity, upscale quality, and sustainable service delivery.

Quantification of three steroid hormone receptors of the leopard gecko (Eublepharis macularius), a lizard with temperature-dependent sex determination: their tissue distributions and the effect of environmental change on their expressions.

PubMed

Endo, Daisuke; Park, Min Kyun

2003-12-01

Sex steroid hormones play a central role in the reproduction of all vertebrates. These hormones function through their specific receptors, so the expression levels of the receptors may reflect the responsibility of target organs. However, there was no effective method to quantify the expression levels of these receptors in reptilian species. In this study, we established the competitive-PCR assay systems for the quantification of the mRNA expression levels of three sex steroid hormone receptors in the leopard gecko. These assay systems were successfully able to detect the mRNA expression level of each receptor in various organs of male adult leopard geckoes. The expression levels of mRNA of these receptors were highly various depending on the organs assayed. This is the first report regarding the tissue distributions of sex steroid hormone receptor expressions in reptile. The effects of environmental conditions on these hormone receptor expressions were also examined. After the low temperature and short photoperiod treatment for 6 weeks, only the androgen receptor expression was significantly increased in the testes. The competitive-PCR assay systems established in this report should be applicable for various studies of the molecular mechanism underlying the reproductive activity of the leopard gecko.

Prognostic value of sex-hormone receptor expression in non-muscle-invasive bladder cancer.

PubMed

Nam, Jong Kil; Park, Sung Woo; Lee, Sang Don; Chung, Moon Kee

2014-09-01

We investigated sex-hormone receptor expression as predicting factor of recurrence and progression in patients with non-muscle invasive bladder cancer. We retrospectively evaluated tumor specimens from patients treated for transitional cell carcinoma of the bladder at our institution between January 2006 and January 2011. Performing immunohistochemistry using a monoclonal androgen receptor antibody and monoclonal estrogen receptor-beta antibody on paraffin-embedded tissue sections, we assessed the relationship of immunohistochemistry results and prognostic factors such as recurrence and progression. A total of 169 patients with bladder cancer were evaluated in this study. Sixty-threepatients had expressed androgen receptors and 52 patients had estrogen receptor beta. On univariable analysis, androgen receptor expression was significant lower in recurrence rates (p=0.001), and estrogen receptor beta expression was significant higher in progression rates (p=0.004). On multivariable analysis, significant association was found between androgen receptor expression and lower recurrence rates (hazard ratio=0.500; 95% confidence interval, 0.294 to 0.852; p=0.011), but estrogen receptor beta expression was not significantly associated with progression rates. We concluded that the possibility of recurrence was low when the androgen receptor was expressed in the bladder cancer specimen and it could be the predicting factor of the stage, number of tumors, carcinoma in situ lesion and recurrence.

Allergic sensitization modifies the pulmonary expression of 5-hydroxytryptamine receptors in guinea pigs.

PubMed

Córdoba-Rodríguez, Guadalupe; Vargas, Mario H; Ruiz, Víctor; Carbajal, Verónica; Campos-Bedolla, Patricia; Mercadillo-Herrera, Paulina; Arreola-Ramírez, José Luis; Segura-Medina, Patricia

2016-03-01

There is mounting evidence that 5-hydroxytryptamine (5-HT) plays a role in asthma. However, scarce information exists about the pulmonary expression of 5-HT receptors and its modification after allergic sensitization. In the present work, we explored the expression of 5-HT1A, 5-HT2A, 5-HT3, 5-HT4, 5-ht5a, 5-HT6, and 5-HT7 receptors in lungs from control and sensitized guinea pigs through qPCR and Western blot. In control animals, mRNA from all receptors was detectable in lung homogenates, especially from 5-HT2A and 5-HT4 receptors. Sensitized animals had decreased mRNA expression of 5-HT2A and 5-HT4 receptors and increased that of 5-HT7 receptor. In contrast, they had increased protein expression of 5-HT2A receptor in bronchial epithelium and of 5-HT4 receptor in lung parenchyma. The degree of airway response to the allergic challenge was inversely correlated with mRNA expression of the 5-HT1A receptor. In summary, our results showed that major 5-HT receptor subtypes are constitutively expressed in the guinea pig lung, and that allergic sensitization modifies the expression of 5-HT2A, 5-HT4, and 5-HT7 receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

PubMed Central

WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

2013-01-01

Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

Correlation between erythropoietin receptor(s) and estrogen and progesterone receptor expression in different breast cancer cell lines.

PubMed

Trošt, Nina; Hevir, Neli; Rižner, Tea Lanišnik; Debeljak, Nataša

2013-03-01

Erythropoietin (EPO) receptor (EPOR) expression in breast cancer has been shown to correlate with the expression of estrogen receptor (ESR) and progesterone receptor (PGR) and to be associated with the response to tamoxifen in ESR+/PGR+ tumors but not in ESR- tumors. In addition, the correlation between EPOR and G protein-coupled estrogen receptor 1 [GPER; also known as G protein-coupled receptor 30 (GPR30)] has been reported, suggesting the prognostic potential of EPOR expression. Moreover, the involvement of colony stimulating factor 2 receptor, β, low‑affinity (CSF2RB) and ephrin type-B receptor 4 (EPHB4) as EPOR potential receptor partners in cancer has been indicated. This study analyzed the correlation between the expression of genes for EPO, EPOR, CSF2RB, EPHB4, ESR, PGR and GPER in the MCF-7, MDA-MB-361, T-47D, MDA-MB-231, Hs578Bst, SKBR3, MCF-10A and Hs578T cell lines. The cell lines were also treated with recombinant human EPO (rHuEPO) in order to determine its ability to activate the Jak/STAT5, MAPK and PI3K signaling pathways and modify cell growth characteristics. Expression analysis stratified the cell lines in 2 main clusters, hormone-dependent cell lines expressing ESR and PGR and a hormone-independent cluster. A significant correlation was observed between the expression levels of ESR and PGR and their expression was also associated with that of GPER. Furthermore, the expression of GPER was associated with that of EPOR, suggesting the connection between this orphan G protein and EPO signaling. A negative correlation between EPOR and CSF2RB expression was observed, questioning the involvement of these two receptors in the hetero-receptor formation. rHuEPO treatment only influenced the hormone-independent cell lines, since only the MDA-MB-231, SKBR3 and Hs578T cells responded to the treatment. The correlation between the expression of the analyzed receptors suggests that the receptors may interact in order to activate signaling pathways or to evade their inhibition. Therefore, breast cancer classification upon ESR, PGR and human epidermal growth factor receptor 2 (HER2) may not be sufficient for the selection of suitable treatment protocol. The expression of EPOR, GPER and EPHB4 may be considered as additional classification factors.

Survey of Genes Encoding Staphylococcal Enterotoxins, Toxic Shock Syndrome Toxin 1, and Exfoliative Toxins in Members of the Staphylococcus sciuri Group

PubMed Central

Dakić, Ivana; Vuković, Dragana; Stepanović, Srdjan; Hauschild, Tomasz; Ježek, Petr; Petráš, Petr; Morrison, Donald

2005-01-01

Genes encoding staphylococcal enterotoxins (sea to see, seg, and seh), toxic shock syndrome toxin 1 (tst), and exfoliative toxins (eta and etb) were not detected in a large panel of 48 Staphylococcus sciuri group isolates tested. This strongly suggests that production of the staphylococcal exotoxins by these bacteria is highly unlikely. PMID:16145164

26 CFR 1.40-1 - Questions and answers relating to the meaning of the term “qualified mixture” in section 40(b)(1).

Code of Federal Regulations, 2014 CFR

2014-04-01

...) if such product is produced in a chemical reaction between alcohol and either gasoline or a special... ether (ETBE), a compound derived from ethanol (a qualified alcohol), in a chemical reaction in which... the alcohol is no longer present as a separate chemical in the final product, provided that there is...

26 CFR 1.40-1 - Questions and answers relating to the meaning of the term “qualified mixture” in section 40(b)(1).

Code of Federal Regulations, 2010 CFR

2010-04-01

...) if such product is produced in a chemical reaction between alcohol and either gasoline or a special... ether (ETBE), a compound derived from ethanol (a qualified alcohol), in a chemical reaction in which... the alcohol is no longer present as a separate chemical in the final product, provided that there is...

26 CFR 1.40-1 - Questions and answers relating to the meaning of the term “qualified mixture” in section 40(b)(1).

Code of Federal Regulations, 2011 CFR

2011-04-01

...) if such product is produced in a chemical reaction between alcohol and either gasoline or a special... ether (ETBE), a compound derived from ethanol (a qualified alcohol), in a chemical reaction in which... the alcohol is no longer present as a separate chemical in the final product, provided that there is...

26 CFR 1.40-1 - Questions and answers relating to the meaning of the term “qualified mixture” in section 40(b)(1).

Code of Federal Regulations, 2012 CFR

2012-04-01

...) if such product is produced in a chemical reaction between alcohol and either gasoline or a special... ether (ETBE), a compound derived from ethanol (a qualified alcohol), in a chemical reaction in which... the alcohol is no longer present as a separate chemical in the final product, provided that there is...

26 CFR 1.40-1 - Questions and answers relating to the meaning of the term “qualified mixture” in section 40(b)(1).

Code of Federal Regulations, 2013 CFR

2013-04-01

...) if such product is produced in a chemical reaction between alcohol and either gasoline or a special... ether (ETBE), a compound derived from ethanol (a qualified alcohol), in a chemical reaction in which... the alcohol is no longer present as a separate chemical in the final product, provided that there is...

Expression of Cannabinoid Receptors in Human Osteoarthritic Cartilage: Implications for Future Therapies.

PubMed

Dunn, Sara L; Wilkinson, Jeremy Mark; Crawford, Aileen; Bunning, Rowena A D; Le Maitre, Christine L

2016-01-01

Introduction: Cannabinoids have shown to reduce joint damage in animal models of arthritis and reduce matrix metalloproteinase expression in primary human osteoarthritic (OA) chondrocytes. The actions of cannabinoids are mediated by a number of receptors, including cannabinoid receptors 1 and 2 (CB1 and CB2), G-protein-coupled receptors 55 and 18 (GPR55 and GPR18), transient receptor potential vanilloid-1 (TRPV1), and peroxisome proliferator-activated receptors alpha and gamma (PPARα and PPARγ). However, to date very few studies have investigated the expression and localization of these receptors in human chondrocytes, and expression during degeneration, and thus their potential in clinical applications is unknown. Methods: Human articular cartilage from patients with symptomatic OA was graded histologically and the expression and localization of cannabinoid receptors within OA cartilage and underlying bone were determined immunohistochemically. Expression levels across regions of cartilage and changes with degeneration were investigated. Results: Expression of all the cannabinoid receptors investigated was observed with no change with grade of degeneration seen in the expression of CB1, CB2, GPR55, PPARα, and PPARγ. Conversely, the number of chondrocytes within the deep zone of cartilage displaying immunopositivity for GPR18 and TRPV1 was significantly decreased in degenerate cartilage. Receptor expression was higher in chondrocytes than in osteocytes in the underlying bone. Conclusions: Chondrocytes from OA joints were shown to express a wide range of cannabinoid receptors even in degenerate tissues, demonstrating that these cells could respond to cannabinoids. Cannabinoids designed to bind to receptors inhibiting the catabolic and pain pathways within the arthritic joint, while avoiding psychoactive effects, could provide potential arthritis therapies.

Distribution of cellular HSV-1 receptor expression in human brain.

PubMed

Lathe, Richard; Haas, Juergen G

2017-06-01

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus linked to a range of acute and chronic neurological disorders affecting distinct regions of the brain. Unusually, HSV-1 entry into cells requires the interaction of viral proteins glycoprotein D (gD) and glycoprotein B (gB) with distinct cellular receptor proteins. Several different gD and gB receptors have been identified, including TNFRSF14/HVEM and PVRL1/nectin 1 as gD receptors and PILRA, MAG, and MYH9 as gB receptors. We investigated the expression of these receptor molecules in different areas of the adult and developing human brain using online transcriptome databases. Whereas all HSV-1 receptors showed distinct expression patterns in different brain areas, the Allan Brain Atlas (ABA) reported increased expression of both gD and gB receptors in the hippocampus. Specifically, for PVRL1, TNFRFS14, and MYH9, the differential z scores for hippocampal expression, a measure of relative levels of increased expression, rose to 2.9, 2.9, and 2.5, respectively, comparable to the z score for the archetypical hippocampus-enriched mineralocorticoid receptor (NR3C2, z = 3.1). These data were confirmed at the Human Brain Transcriptome (HBT) database, but HBT data indicate that MAG expression is also enriched in hippocampus. The HBT database allowed the developmental pattern of expression to be investigated; we report that all HSV1 receptors markedly increase in expression levels between gestation and the postnatal/adult periods. These results suggest that differential receptor expression levels of several HSV-1 gD and gB receptors in the adult hippocampus are likely to underlie the susceptibility of this brain region to HSV-1 infection.

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

PubMed

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

2014-03-01

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

Time-course of 5-HT(6) receptor mRNA expression during memory consolidation and amnesia.

PubMed

Huerta-Rivas, A; Pérez-García, G; González-Espinosa, C; Meneses, A

2010-01-01

Growing evidence indicates that antagonists of the 5-hydroxytryptamine (serotonin) receptor(6) (5-HT(6)) improve memory and reverse amnesia although the mechanisms involved are poorly understood. Hence, in this paper RT-PCR was used to evaluate changes in mRNA expression of 5-HT(6) receptor in trained and untrained rats treated with the 5-HT(6) receptor antagonist SB-399885 and amnesic drugs scopolamine or dizocilpine. Changes in mRNA expression of 5-HT(6) receptor were investigated at different times in prefrontal cortex, hippocampus and striatum. Data indicated that memory in the Pavlovian/instrumental autoshaping task was a progressive process associated to reduced mRNA expression of 5-HT(6) receptor in the three structures examined. SB-399885 improved long-term memory at 48h, while the muscarinic receptor antagonist scopolamine or the non-competitive NMDA receptor antagonist dizocilpine impaired it at 24h. Autoshaping training and treatment with SB-399885 increased 5-HT(6) receptor mRNA expression in (maximum increase) prefrontal cortex and striatum, 24 or 48h. The scopolamine-induced amnesia suppressed 5-HT(6) receptor mRNA expression while the dizocilpine-induced amnesia did not modify 5-HT(6) receptor mRNA expression. SB-399885 and scopolamine or dizocilpine were able to reestablish memory and 5-HT(6) receptor mRNA expression. These data confirmed previous memory evidence and of more interest is the observation that training, SB-399885 and amnesic drugs modulated 5-HT(6) receptor mRNA expression in prefrontal cortex, hippocampus and striatum. Further investigation in different memory tasks, times and amnesia models together with more complex control groups might provide further clues. Copyright 2009 Elsevier Inc. All rights reserved.

Molecular analysis of nicotinic receptor expression in autism.

PubMed

Martin-Ruiz, C M; Lee, M; Perry, R H; Baumann, M; Court, J A; Perry, E K

2004-04-07

Autism is a developmental disorder of unknown aetiopathology and lacking any specific pharmacological therapeutic intervention. Neurotransmitters such as serotonin, gamma-aminobutyric acid (GABA) and acetylcholine have been implicated. Abnormalities in nicotinic acetylcholine receptors have been identified including cortical loss of binding to the alpha4/beta2 subtype and increase in cerebellar alpha7 binding. Receptor expression (mRNA) has not so far been systematically examined. This study aims to further explore the role of nicotinic receptors in autism by analysing nicotinic receptor subunit mRNA in conjunction with protein levels and receptor binding in different brain areas. Quantitative RT-PCR for alpha4, alpha7 and beta2 subunit mRNA expression levels; alpha3, alpha4, alpha7 and beta2 subunit protein expression immunochemistry and specific radioligand receptor binding were performed in adult autism and control brain samples from cerebral cortex and cerebellum. Alpha4 and beta2 protein expression and receptor binding density as well as alpha4 mRNA levels were lower in parietal cortex in autism, while alpha7 did not change for any of these parameters. In cerebellum, alpha4 mRNA expression was increased, whereas subunit protein and receptor levels were decreased. Alpha7 receptor binding in cerebellum was increased alongside non-significant elevations in mRNA and protein expression levels. No significant changes were found for beta2 in cerebellum. The data obtained, using complementary measures of receptor expression, indicate that reduced gene expression of the alpha4beta2 nicotinic receptor in the cerebral cortex is a major feature of the neurochemical pathology of autism, whilst post-transcriptional abnormalities of both this and the alpha7 subtype are apparent in the cerebellum. The findings point to dendritic and/or synaptic nicotinic receptor abnormalities that may relate to disruptions in cerebral circuitry development.

Expression of the macrophage scavenger receptor, a multifunctional lipoprotein receptor, in microglia associated with senile plaques in Alzheimer's disease.

PubMed Central

Christie, R. H.; Freeman, M.; Hyman, B. T.

1996-01-01

The macrophage scavenger receptor is a multifunctional receptor whose ligands include oxidized low density lipoprotein (LDL), as well as several other polyanionic macromolecules. Although the capacity of the receptor to bind modified LDL has implicated it in the process of atherosclerosis, its physiological role remains uncertain. We have examined human brain for expression of macrophage scavenger receptor as part of ongoing studies of lipoprotein receptors in the central nervous system. The receptor is expressed on microglia, but not on astrocytes, neurons, or vessel-associated structures. In Alzheimer disease, there is strong expression of the scavenger receptor in association with senile plaques. Images Figure 2 Figure 3 Figure 4 PMID:8579103

Platelet dysfunction associated with the novel Trp29Cys thromboxane A₂ receptor variant.

PubMed

Mumford, A D; Nisar, S; Darnige, L; Jones, M L; Bachelot-Loza, C; Gandrille, S; Zinzindohoue, F; Fischer, A-M; Mundell, S J; Gaussem, P

2013-03-01

Genetic variations that affect the structure of the thromboxane A2 receptor (TP receptor) provide insights into the function of this key platelet and vascular receptor, but are very rare in unselected populations. To determine the functional consequences of the TP receptor Trp29Cys (W29C) substitution. We performed a detailed phenotypic analysis of an index case (P1) with reduced platelet aggregation and secretion responses to TP receptor pathway activators, and a heterozygous TP receptor W29C substitution. An analysis of the variant W29C TP receptor expressed in heterologous cells was performed. Total TP receptor expression in platelets from P1 was similar to that of controls, but there was reduced maximum binding and reduced affinity of binding to the TP receptor antagonist [(3) H]SQ29548. HEK293 cells transfected with W29C TP receptor cDNA showed similar total TP receptor expression to wild-type (WT) controls. However, the TP receptor agonist U46619 was less potent at inducing rises in cytosolic free Ca(2+) in HEK293 cells expressing the W29C TP receptor than in WT controls, indicating reduced receptor function. Immunofluorescence microscopy and cell surface ELISA showed intracellular retention and reduced cell surface expression of the W29C TP receptor in HEK293 cells. Consistent with the platelet phenotype, both maximum binding and the affinity of binding of [(3) H]SQ29548 to the W29C TP receptor were reduced compared to WT controls. These findings extend the phenotypic description of the very rare disorder TP receptor deficiency, and show that the W29C substitution reduces TP receptor function by reducing surface receptor expression and by disrupting ligand binding. © 2012 International Society on Thrombosis and Haemostasis.

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

PubMed Central

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Equine insulin receptor and insulin-like growth factor-1 receptor expression in digital lamellar tissue and insulin target tissues.

PubMed

Kullmann, A; Weber, P S; Bishop, J B; Roux, T M; Norby, B; Burns, T A; McCutcheon, L J; Belknap, J K; Geor, R J

2016-09-01

Hyperinsulinaemia is implicated in the pathogenesis of endocrinopathic laminitis. Insulin can bind to different receptors: two insulin receptor isoforms (InsR-A and InsR-B), insulin-like growth factor-1 receptor (IGF-1R) and InsR/IGF-1R hybrid receptor (Hybrid). Currently, mRNA expression of these receptors in equine tissues and the influence of body type and dietary carbohydrate intake on expression of these receptors is not known. The study objectives were to characterise InsR-A, InsR-B, IGF-1R and Hybrid expression in lamellar tissue (LT) and insulin responsive tissues from horses and examine the effect of dietary nonstructural carbohydrate (NSC) on mRNA expression of these receptors in LT, skeletal muscle, liver and two adipose tissue (AT) depots of lean and obese ponies. In vivo experiment. Lamellar tissue samples were evaluated by quantitative reverse transcription polymerase chain reaction (RT-qPCR) for receptor mRNA expression (n = 8) and immunoblotting for protein expression (n = 3). Archived LT, skeletal muscle, liver and AT from lean and obese mixed-breed ponies fed either a low (~7% NSC as dry matter; 5 lean, 5 obese) or high NSC diet (~42% NSC as dry matter; 6 lean, 6 obese) for 7 days were evaluated by RT-qPCR to determine the effect of body condition and diet on expression of the receptors in different tissues. Significance was set at P≤0.05. Lamellar tissue expresses both InsR isoforms, IGF-1R and Hybrid. LT IGF-1R gene expression was greater than either InsR isoform and InsR-A expression was greater than InsR-B (P≤0.05). Obesity significantly lowered IGF-1R, InsR-A and InsR-B mRNA expression in LT and InsR-A in tailhead AT. High NSC diet lowered expression of all three receptor types in liver; IGF-1R and InsR-A in LT and InsR-A in tailhead AT. Lamellar tissue expresses IGF-1R, InsR isoforms and Hybrids. The functional characteristics of these receptors and their role in endocrinopathic laminitis warrants further investigation. © 2015 EVJ Ltd.

Variation of M3 muscarinic receptor expression in different prostate tissues and its significance.

PubMed

Song, Wei; Yuan, Mingzhen; Zhao, Shengtian

2009-08-01

To detect the expression of the muscarinic receptor (M receptor) in different prostate tissues and analyze the role of its subtype in prostatic oncogenesis. Thirty-six cases of normal prostate and benign prostatic hyperplasia, and 8 cases of prostatic tumor, were used in this study from the Shandong University, Shandong, China, between 2003-2006. The protein expressions of M1, M2, and M3 receptors in each group were determined by Western-blotting. The gene expressions of the M3 receptor and vascular endothelial growth factors (VEGF) in each group were determined by reverse transcriptase-polymerase chain reaction. The protein and gene expressions of the M3 receptor in the prostatic carcinoma group were higher than that of benign prostatic hyperplasia group (p=0.0001) and normal prostate group (p=0.0001). The M3 receptor and VEGF showed positive straight-line correlations of gene expressions with the 3 groups (r=0.4999, p=0.0001). The M3 receptor may have a close relationship with prostatic oncogenesis.

Expression of NK cell receptors on decidual T cells in human pregnancy.

PubMed

Tilburgs, Tamara; van der Mast, Barbara J; Nagtzaam, Nicole M A; Roelen, Dave L; Scherjon, Sicco A; Claas, Frans H J

2009-06-01

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Bombesin-like peptide receptors in human bronchial epithelial cells.

PubMed

Kane, M A; Toi-Scott, M; Johnson, G L; Kelley, K K; Boose, D; Escobedo-Morse, A

1996-01-01

Northern blot and RNAse protection assays previously failed to detect bombesin-like peptide (BLP) receptors in normal human lung tissue, but by RT/PCR cultured human bronchial epithelial (HBE) cells expressed all three BLP receptor subtypes, predominantly neuromedin B (NMB) receptor. By RT/PCR, we found expression of all three BLP receptor subtypes by human lung tissue and confirmed NMB receptor expression in six out of six HBE samples. However, transformed HBE BEAS B2B cells expressed only gastrin-releasing peptide (GRP) receptors; saturable, high-affinity (Kd = 3.5 nM) specific [125I]GRP binding confirmed functional GRP receptor, with M(r) = 75 kDa and immunologic cross-reactivity with GRP receptor from human small-cell lung carcinoma (SCLC) NCI-H345 cells. Altered regulation of BLP receptors may accompany transformation of normal lung cells to cancer.

Catestatin (chromogranin A344-364) is a novel cardiosuppressive agent: inhibition of isoproterenol and endothelin signaling in the frog heart

PubMed Central

Mazza, Rosa; Gattuso, Alfonsina; Mannarino, Cinzia; Brar, Bhawanjit K.; Barbieri, Sandra Francesca; Tota, Bruno; Mahata, Sushil K.

2008-01-01

The catecholamine release-inhibitory catestatin [Cts; human chromogranin (Cg) A352-372, bovine CgA344-364] is a vasoreactive and anti-hypertensive peptide derived from CgA. Using the isolated avascular frog heart as a bioassay, in which the interactions between the endocardial endothelium and the subjacent myocardium can be studied without the confounding effects of the vascular endothelium, we tested the direct cardiotropic effects of bovine Cts and its interaction with β-adrenergic (isoproterenol, ISO) and endothelin-1 (ET-1) signaling. Cts dose-dependently decreased stroke volume and stroke work, with a threshold concentration of 11 nM, approaching the in vivo level of the peptide. Cts reduced contractility by inhibiting phosphorylation of phospholamban (PLN). Furthermore, the Cts effect was abolished by pretreatment with either nitric oxide synthase (NG-monomethyl-l-arginine) or guanylate cyclase (ODQ) inhibitors, or an ETB receptor (ETBR) antagonist (BQ-788). Cts also noncompetitively inhibited the positive inotropic action of ISO. In addition, Cts inhibited the positive inotropic effect of ET-1, mediated by ETA receptors, and did not alter the negative inotropic ET-1 influence mediated by ETBR. Cts action through ETBR was further suggested when, in the presence of BQ-788, Cts failed to inhibit the positive inotropism of both ISO and ET-1 stimulation and PLN phosphorylation. We concluded that the cardiotropic actions of Cts, including the β-adrenergic and ET-1 antagonistic effects, support a novel role of this peptide as an autocrine-paracrine modulator of cardiac function, particularly when the stressed heart becomes a preferential target of both adrenergic and ET-1 stimuli. PMID:18469147

Development and Validation of Extract the Base: An English Derivational Morphology Test for Third through Fifth Grade Monolingual Students and Spanish-Speaking English Language Learners

ERIC Educational Resources Information Center

Goodwin, Amanda P.; Huggins, A. Corinne; Carlo, Maria; Malabonga, Valerie; Kenyon, Dorry; Louguit, Mohammed; August, Diane

2012-01-01

This study describes the development and validation of the Extract the Base test (ETB), which assesses derivational morphological awareness. Scores on this test were validated for 580 monolingual students and 373 Spanish-speaking English language learners (ELLs) in third through fifth grade. As part of the validation of the internal structure,…

A field trial of production and financial consequences of helminthosis control in sheep production in Ethiopia.

PubMed

Tibbo, M; Aragaw, K; Philipsson, J; Malmfors, B; Näsholm, A; Ayalew, W; Rege, J E O

2008-04-17

We used a partial-budget analysis to evaluate profitability of different management strategies of three genotypes of sheep in a 2 x 2 x 3 factorial experiment conducted at Debre Berhan research station in the central highlands of Ethiopia. This involved two anthelmintic-treatment levels (treated vs. non-treated), two supplementary nutrition levels (protein-energy supplementation yes/no) and three genotypes: indigenous Menz (n=40), 50% Awassi x 50% Menz crosses (n=38) and 75% Awassi x 25% Menz crosses (n=31). All sheep were exposed to natural sub-clinical helminthosis challenge. Supplemented sheep were offered a concentrate mix daily on an individual basis. Anthelmintic-treated sheep were drenched with fenbendazole against nematodes and with triclabendazole against trematodes. Data were collected during the experimental period (for 10 months from approximately 1 year of age) on feed intake, live weight, eggs per gram (EPG) of faeces, packed-cell volume (PCV), wool weight, and adult-worm burden. Actual market input and output prices were recorded. Supplemented sheep had significantly higher marginal profit (MP) per sheep than non-supplemented sheep (ETB 33 vs. 4). Likewise, anthelmintic treated sheep performed significantly better than their non-treated contemporaries (MP=ETB 28 vs. 8). The 75% Awassi crosses were least profitable.

Global two-fluid turbulence simulations of L-H transitions and edge localized mode dynamics in the COMPASS-D tokamak

NASA Astrophysics Data System (ADS)

Thyagaraja, A.; Valovič, M.; Knight, P. J.

2010-04-01

It is shown that the transition from L-mode to H-mode regimes in tokamaks can be reproduced using a two-fluid, fully electromagnetic, plasma model when a suitable particle sink is added at the edge. Such a model is implemented in the CUTIE code [A. Thyagaraja et al., Eur. J. Mech. B/Fluids 23, 475 (2004)] and is illustrated on plasma parameters that mimic those in the COMPASS-D tokamak with electron cyclotron resonance heating [Fielding et al., Plasma Phys. Contr. Fusion 42, A191 (2000)]. In particular, it is shown that holding the heating power, current, and magnetic field constant and increasing the fuelling rate to raise the plasma density leads spontaneously to the formation of an edge transport barrier (ETB) which occurs going from low to higher density experimentally. In the following quiescent period in which the stored energy of the plasma rises linearly with time, a dynamical transition occurs in the simulation with the appearance of features resembling strong edge localized modes. The simulation qualitatively reproduces many features observed in the experiment. Its relative robustness suggests that some, at least of the observed characteristics of ETBs and L-H transitions, can be captured in the global electromagnetic turbulence model.

User experience analysis of e-TB Manager, a nationwide electronic tuberculosis recording and reporting system in Ukraine.

PubMed

Konduri, Niranjan; Sawyer, Kelly; Nizova, Nataliya

2017-04-01

Ukraine has successfully implemented e-TB Manager nationwide as its mandatory national tuberculosis registry after first introducing it in 2009. Our objective was to perform an end-of-programme evaluation after formal handover of the registry administration to Ukraine's Centre for Disease Control in 2015. We conducted a nationwide, cross-sectional, anonymous, 18-point user experience survey, and stratified the registry's transaction statistics to demonstrate usability. Contrary to initial implementation experience, older users (aged >50 years), often with limited or no computer proficiency prior to using the registry, had significantly better user experience scores for at least six of the 12 measures compared to younger users (aged 18-29 years). Using the registry for >3 years was associated with significantly higher scores for having capacity, adequacy of training received and satisfaction with the registry. Of the 5.9 million transactions over a 4-year period, nine out of 24 oblasts (regions) and Kiev city accounted for 62.5% of all transactions, and corresponded to 59% of Ukraine's tuberculosis burden. There were 437 unique active users in 486 rayons (districts) of Ukraine, demonstrating extensive reach. Our key findings complement the World Health Organization and European Respiratory Society's agenda for action on digital health to help implement the End TB Strategy.

Overlapping but distinct topology for zebrafish V2R-like olfactory receptors reminiscent of odorant receptor spatial expression zones.

PubMed

Ahuja, Gaurav; Reichel, Vera; Kowatschew, Daniel; Syed, Adnan S; Kotagiri, Aswani Kumar; Oka, Yuichiro; Weth, Franco; Korsching, Sigrun I

2018-05-23

The sense of smell is unrivaled in terms of molecular complexity of its input channels. Even zebrafish, a model vertebrate system in many research fields including olfaction, possesses several hundred different olfactory receptor genes, organized in four different gene families. For one of these families, the initially discovered odorant receptors proper, segregation of expression into distinct spatial subdomains within a common sensory surface has been observed both in teleost fish and in mammals. However, for the remaining three families, little to nothing was known about their spatial coding logic. Here we wished to investigate, whether the principle of spatial segregation observed for odorant receptors extends to another olfactory receptor family, the V2R-related OlfC genes. Furthermore we thought to examine, how expression of OlfC genes is integrated into expression zones of odorant receptor genes, which in fish share a single sensory surface with OlfC genes. To select representative genes, we performed a comprehensive phylogenetic study of the zebrafish OlfC family, which identified a novel OlfC gene, reduced the number of pseudogenes to 1, and brought the total family size to 60 intact OlfC receptors. We analyzed the spatial pattern of OlfC-expressing cells for seven representative receptors in three dimensions (height within the epithelial layer, horizontal distance from the center of the olfactory organ, and height within the olfactory organ). We report non-random distributions of labeled neurons for all OlfC genes analysed. Distributions for sparsely expressed OlfC genes are significantly different from each other in nearly all cases, broad overlap notwithstanding. For two of the three coordinates analyzed, OlfC expression zones are intercalated with those of odorant receptor zones, whereas in the third dimension some segregation is observed. Our results show that V2R-related OlfC genes follow the same spatial logic of expression as odorant receptors and their expression zones intermingle with those of odorant receptor genes. Thus, distinctly different expression zones for individual receptor genes constitute a general feature shared by teleost and tetrapod V2R/OlfC and odorant receptor families alike.

Expression of very low density lipoprotein receptor mRNA in circulating human monocytes: its up-regulation by hypoxia.

PubMed

Nakazato, K; Ishibashi, T; Nagata, K; Seino, Y; Wada, Y; Sakamoto, T; Matsuoka, R; Teramoto, T; Sekimata, M; Homma, Y; Maruyama, Y

2001-04-01

Although very low density lipoprotein (VLDL) receptor expression by macrophages has been shown in the vascular wall, it is not clear whether or not circulating monocytes express the VLDL receptor. We investigated the expression of VLDL receptor mRNA in human peripheral blood monocytes and monocyte-derived macrophages by reverse transcriptase polymerase chain reaction (RT-PCR) and nucleotide sequencing after subcloning of PCR product. VLDL receptor mRNA was detected both in peripheral blood monocytes and monocyte-derived macrophages. Expression of VLDL receptor mRNA was upregulated by hypoxia in monocytes, whereas treatment with oxidized LDL, interleukin-1beta or monocyte chemoattractant protein-1 did not affect the levels of VLDL receptor mRNA in monocytes and macrophages. The present study shows a novel response of VLDL receptor mRNA to hypoxia, suggesting a role for VLDL receptor in the metabolism of lipoproteins in the vascular wall and the development of atherosclerosis.

Hormone Receptor Expression Analyses in Neoplastic and Non-Neoplastic Canine Mammary Tissue by a Bead Based Multiplex Branched DNA Assay: A Gene Expression Study in Fresh Frozen and Formalin-Fixed, Paraffin-Embedded Samples.

PubMed

Mohr, Annika; Lüder Ripoli, Florenza; Hammer, Susanne Conradine; Willenbrock, Saskia; Hewicker-Trautwein, Marion; Kiełbowicz, Zdzisław; Murua Escobar, Hugo; Nolte, Ingo

2016-01-01

Immunohistochemistry (IHC) is currently considered the method of choice for steroid hormone receptor status evaluation in human breast cancer and, therefore, it is commonly utilized for assessing canine mammary tumors. In case of low hormone receptor expression, IHC is limited and thus is complemented by molecular analyses. In the present study, a multiplex bDNA assay was evaluated as a method for hormone receptor gene expression detection in canine mammary tissues. Estrogen receptor (ESR1), progesterone receptor (PGR), prolactin receptor (PRLR) and growth hormone receptor (GHR) gene expressions were evaluated in neoplastic and non-neoplastic canine mammary tissues. A set of 119 fresh frozen and 180 formalin-fixed, paraffin-embedded (FFPE) was comparatively analyzed and used for assay evaluation. Furthermore, a possible association between the hormone receptor expression in different histological subtypes of canine malignant mammary tumors and the castration status, breed and invasive growth of the tumor were analyzed. The multiplex bDNA assay proved to be more sensitive for fresh frozen specimens. Hormone receptor expression found was significantly decreased in malignant mammary tumors in comparison to non-neoplastic tissue and benign mammary tumors. Among the histological subtypes the lowest gene expression levels of ESR1, PGR and PRLR were found in solid, anaplastic and ductal carcinomas. In summary, the evaluation showed that the measurement of hormone receptors with the multiplex bDNA assay represents a practicable method for obtaining detailed quantitative information about gene expression in canine mammary tissue for future studies. Still, comparison with IHC or quantitative real-time PCR is needed for further validation of the present method.

Angiotensin II AT1 receptor blocker candesartan prevents the fast up-regulation of cerebrocortical benzodiazepine-1 receptors induced by acute inflammatory and restraint stress

PubMed Central

Sánchez-Lemus, Enrique; Honda, Masaru; Saavedra, Juan M.

2012-01-01

Centrally acting Angiotensin II AT1 receptor blockers (ARBs) protect from stress-induced disorders and decrease anxiety in a model of inflammatory stress, the systemic injection of bacterial endotoxin lipopolysaccharide (LPS). In order to better understand the anxiolytic effect of ARBs, we treated rats with LPS (50 µg/kg) with or without three days of pretreatment with the ARB candesartan (1 mg/kg/day), and studied cortical benzodiazepine (BZ) and corticotrophin-releasing factor (CRF) receptors. We compared the cortical BZ and CRF receptors expression pattern induced by LPS with that produced in restraint stress. Inflammation stress produced a generalized increase in cortical BZ1 receptors and reduced mRNA expression of the GABAA receptor γ2 subunit in cingulate cortex; changes were prevented by candesartan pretreatment. Moreover, restraint stress produced similar increases in cortical BZ1 receptor binding, and candesartan prevented these changes. Treatment with candesartan alone increased cortical BZ1 binding, and decreased γ2 subunit mRNA expression in the cingulate cortex. Conversely, we did not find changes in CRF1 receptor expression in any of the cortical areas studied, either after inflammation or restraint stress. Cortical CRF2 receptor binding was undetectable, but CRF2 mRNA expression was decreased by inflammation stress, a change prevented by candesartan. We conclude that stress promotes rapid and widespread changes in cortical BZ1 receptor expression; and that the stress-induced BZ1 receptor expression is under the control of AT1 receptor activity. The results suggest that the anti-anxiety effect of ARBs may be associated with their capacity to regulate stress-induced alterations in cortical BZ1 receptors. PMID:22503782

Regional expression of prostaglandin E2 and F2alpha receptors in human myometrium, amnion, and choriodecidua with advancing gestation and labor.

PubMed

Grigsby, Peta L; Sooranna, Suren R; Adu-Amankwa, Bernice; Pitzer, Brad; Brockman, Diane E; Johnson, Mark R; Myatt, Leslie

2006-08-01

The change from uterine quiescence to enhanced contractile activity may be due to the differential expression of prostaglandin receptors within the myometrium and fetal membranes, in a temporal and topographically distinct manner. To address this question, we determined the localization and expression of the PGE2 receptor subtypes (PTGER1-4) and the PGF2alpha receptor (PTGFR) in paired upper and lower segment myometrium, amnion, and choriodecidual samples throughout human pregnancy, with and without labor. All receptor subtypes were found throughout the muscle layers in both the upper and lower uterine segments, colocalizing with alpha smooth muscle actin. A change in intracellular localization was observed at term labor, where PTGER1 and PTGER4 were predominately associated with the nucleus. Minimal changes in the expression of the PGE2 and PGF2alpha receptor subtypes were observed with gestational age, labor, or between the upper and lower myometrial segments. Receptor expression in maternal and fetal tissues differed between the receptor subtypes; PTGER1 and PTGER4 were predominately expressed in the fetal membranes, PTGER2 was greatest in the myometrium, whereas PTGER3 and PTGFR were similarly expressed in the myometrium and fetal membranes. Myometrial activation through the prostaglandin receptors is perhaps more subtle and may be mediated by a balance between one or several of the prostaglandin receptor subtypes together with other known contraction associated proteins. Lack of coordination in receptor expression between the myometrium and fetal membranes may indicate different regulatory mechanisms between these tissues, or it may suggest a function for these receptors in the amnion and choriodecidua that is independent of that seen in the myometrium.

Functional expression of Squalus acanthias melanocortin-5 receptor in CHO cells: ligand selectivity and interaction with MRAP.

PubMed

Reinick, Christina L; Liang, Liang; Angleson, Josepha K; Dores, Robert M

2012-04-05

The melanocortin-5 receptor (MC(5)) of the dogfish Squalus acanthias (SacMC(5) receptor) can be functionally expressed in CHO cells in the absence of the co-expression of an exogenous MRAP cDNA. Both human ACTH(1-24) and dogfish ACTH(1-25) were much better stimulators of the SacMC(5) receptor than any of the mammalian or dogfish MSH ligands that were tested. The order of ligand selectivity for the dogfish melanocortins was ACTH(1-25)>αMSH>γ-MSH=δ-MSH>β-MSH. Unlike mammalian MC(5) receptors, the functional expression of the SacMC(5) receptor was not negatively impacted when the receptor was co-expressed with a cartilaginous fish (Callorhinchus milii) MRAP2 cDNA. However, co-expression with either mouse mMRAP1 or zebrafish zfMRAP1 increased the sensitivity of SacMC(5) receptor for hACTH(1-24) by at least one order of magnitude. Hence, SacMC(5) receptor has the potential to interact with MRAP1 orthologs and in this regard behaved more like a melanocortin MC(2) receptor ortholog than a melanocortin MC(5) receptor ortholog. These observations are discussed in light of the evolution of the melanocortin receptor gene family in cartilaginous fish, and the physiological implications of these observations are considered. Copyright © 2012 Elsevier B.V. All rights reserved.

Activation of D4 dopamine receptor decreases AT1 angiotensin II receptor expression in rat renal proximal tubule cells

PubMed Central

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

2014-01-01

The dopaminergic and renin angiotensin systems interact to regulate blood pressure. Disruption of the D4 dopamine receptor gene in mice produces hypertension that is associated with increased renal AT1 receptor expression. We hypothesize that the D4 receptor can inhibit AT1 receptor expression and function in renal proximal tubules (RPTs) cells from Wistar-Kyoto (WKY) rats but the D4 receptor regulation of AT1 receptor is aberrant in RPT cells from spontaneously hypertensive rats (SHRs). The D4 receptor agonist, PD168077, decreased AT1 receptor protein expression in a time and concentration-dependent manner in WKY cells. By contrast, in SHR cells, PD168077 increased AT1 receptor protein expression. The inhibitory effect of D4 receptor on AT1 receptor expression in WKY cells was blocked by a calcium channel blocker, nicardipine, or calcium-free medium, indicating that calcium is involved in the D4 receptor-mediated signaling pathway. Angiotensin II increased Na+-K+ ATPase activity in WKY cells. Pretreatment with PD168077 decreased the stimulatory effect of angiotensin II on Na+-K+ ATPase activity in WKY cells. In SHR cells, the inhibitory effect of D4 receptor on angiotensin II-mediated stimulation of Na+-K+ ATPase activity was aberrant; pretreatment with PD168077 augmented the stimulatory effect of AT1 receptor on Na+-K+ ATPase activity in SHR cells. This was confirmed in vivo; pre-treatment with PD128077 for one week augmented the anti-hypertensive and natriuretic effect of losartan in SHRs but not in WKY rats. We suggest that an aberrant interaction between D4 and AT1 receptors may play a role in the abnormal regulation of sodium excretion in hypertension. PMID:25368031

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

PubMed Central

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

2012-01-01

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

Functional expression of cysteinyl leukotriene receptors on human platelets.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Hashimoto, Kunio; Suzuki, Yasuo; Hirano, Reiji; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Normal peripheral blood leukocytes, such as basophils, eosinophils, B lymphocytes and monocytes/macrophages, have a cysteinyl leukotriene 1 (CysLT1) receptor, while the cysteinyl leukotriene 2 (CysLT2) receptor is expressed in cardiac Purkinje cells, endothelium, brain and leukocytes. However, it is unknown whether or not platelets express the CysLT1 or CysLT2 receptor. In this study we identify and characterize the biological function of the CysLT receptor of human platelets. We determined the CysLT1 or CysLT2 receptor mRNA expression in normal human platelets by RT-PCR and determined protein expression by Western blotting and flow cytometry. Moreover, we examined the effect of cysteinyl leukotrienes (CysLTs) in platelets on the induction of RANTES (Regulated on Activation, Normal T Expressed, and presumably Secreted). We also investigated whether the CysLT1 receptor antagonist pranlukast inhibits CysLT-induced RANTES release. In conclusion, we showed the functional expression of CysLT receptors on human platelets and demonstrated that CysLTs induced the release of significant amounts of RANTES, which suggests a novel role for human platelets in CysLT-mediated allergic inflammation.

Sequential expression of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor in rat hippocampal neurons after fluid percussion injury

PubMed Central

Li, Zhiqiang; Shu, Qingming; Li, Lingzhi; Ge, Maolin; Zhang, Yongliang

2014-01-01

Traumatic brain injury causes gene expression changes in different brain regions. Occurrence and development of traumatic brain injury are closely related, involving expression of three factors, namely cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. However, little is known about the correlation of these three factors and brain neuronal injury. In this study, primary cultured rat hippocampal neurons were subjected to fluid percussion injury according to Scott's method, with some modifications. RT-PCR and semi-quantitative immunocytochemical staining was used to measure the expression levels of cyclooxygenase-2, glutamate receptor-2, and platelet activating factor receptor. Our results found that cyclooxygenase-2 expression were firstly increased post-injury, and then decreased. Both mRNA and protein expression levels reached peaks at 8 and 12 hours post-injury, respectively. Similar sequential changes in glutamate receptor 2 were observed, with highest levels mRNA and protein expression at 8 and 12 hours post-injury respectively. On the contrary, the expressions of platelet activating factor receptor were firstly decreased post-injury, and then increased. Both mRNA and protein expression levels reached the lowest levels at 8 and 12 hours post-injury, respectively. Totally, our findings suggest that these three factors are involved in occurrence and development of hippocampal neuronal injury. PMID:25206921

Expression changes of serotonin receptor gene subtype 5HT3a in peripheral blood mononuclear cells from schizophrenic patients treated with haloperidol and Olanzapin.

PubMed

Shariati, Gholam Reza; Ahangari, Ghasem; Hossein-nezhad, Arash; Asadi, Seyed Mohammad; Pooyafard, Farzaneh; Ahmadkhaniha, Hamid Reza

2009-09-01

Serotonin receptors are involved in pathophysiology of schizophrenia and may mediate other neurotransmitter effects. We investigated serotonin receptors gene expression in peripheral blood mononuclear cells (PBMC) of naïve schizophrenic patients, before and after treatment. Also serotonin receptor gene expression was compared in two treatment groups including Haloperidol and Olanzapine. The PBMC was separated from whole blood by Ficoll-hypaque. The total cellular RNA was extracted and the cDNA was synthesized. This process was followed by real-time PCR using primer pairs specific for 5HT(3a) serotonin receptor mRNA and beta-actin as internal control. The results showed the presence of subtype of serotonin receptor in lymphocytes. Serotonin gene expression showed significant changes in Olanzapine treatment group which correlated with Clinical Global Impression (CGI) score improvement. In conclusion, the present study has shown that human PBMC express serotonin receptors 5HT(3a). Moreover, clinical symptom improvement of Olanzapin may be demonstrated by a change in serotonin receptor gene expression.

Placental expression of D6 decoy receptor in preeclampsia

PubMed Central

Cho, Geum Joon; Lee, Eun Sung; Jin, Hye Mi; Lee, Ji Hye; Kim, Yeun Sun; Seol, Hyun-Joo; Hong, Soon-Cheol; Kim, Hai-Joong

2015-01-01

Objective The purpose of this study was to investigate the expression of the D6 decoy receptor that can bind chemokines and target them for degradation, resulting in inhibition of inflammation in placentas from preeclamptic and normal pregnancies. Methods The current study was carried out in 35 pregnant women (23 patients with preeclampsia and 12 healthy, normotensive pregnant women) during the third trimester of pregnancy. The expressions of D6 decoy receptor in the placenta were determined with real time reverse transcriptase polymerase chain reaction and western blotting. Results The mRNA and protein of D6 decoy receptor were detected in all of placentas from preeclamptic and normal pregnancies. Placental D6 decoy receptor mRNA expression was significantly lower in patients with preeclampsia than in patients with normal pregnancies. Western blot analyses revealed decreased protein expression in cases of preeclampsia. Conclusion The expression of the D6 decoy receptor in preeclamptic placentas was significantly lower than in normal placentas. Further studies are needed to clarify the underlying mechanisms that link decreased expression of placental D6 decoy receptor and preeclampsia. PMID:26430656

Adrenocorticotropin receptors: Functional expression from rat adrenal mRNA in Xenopus laevis oocytes

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

Mertz, L.M.; Catt, K.J.

1991-10-01

The adrenocorticotropin (ACTH) receptor, which binds corticotropin and stimulates adenylate cyclase and steroidogenesis in adrenocortical cells, was expressed in Xenopus laevis oocytes microinjected with rat adrenal poly(A){sup +} RNA. Expression of the ACTH receptor in individual stage 5 and 6 oocytes was monitored by radioimmunoassay of ligand-stimulated cAMP production. Injection of 5-40 ng of adrenal mRNA caused dose-dependent increases in ACTH-responsive cAMP production. Size fractionation of rat adrenal poly(A){sup +}RNA by sucrose density-gradient centrifugation revealed that mRNA encoding the ACTH receptor was present in the 1.1-to 2.0-kilobase fraction. These data indicate that ACTH receptors can be expressed from adrenal mRNAmore » in Xenopus oocytes and are fully functional in terms of ligand specificity and signal generation. The extracellular cAMP response to ACTH is a sensitive and convenient index of receptor expression. This system should permit more complete characterization and expression cloning of the ACTH receptor.« less

The neuropeptides CCK and NPY and the changing view of cell-to-cell communication in the taste bud.

PubMed

Herness, Scott; Zhao, Fang-Li

2009-07-14

The evolving view of the taste bud increasingly suggests that it operates as a complex signal processing unit. A number of neurotransmitters and neuropeptides and their corresponding receptors are now known to be expressed in subsets of taste receptor cells in the mammalian bud. These expression patterns set up hard-wired cell-to-cell communication pathways whose exact physiological roles still remain obscure. As occurs in other cellular systems, it is likely that neuropeptides are co-expressed with neurotransmitters and function as neuromodulators. Several neuropeptides have been identified in taste receptor cells including cholecystokinin (CCK), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and glucagon-like peptide 1 (GLP-1). Of these, CCK and NPY are the best studied. These two peptides are co-expressed in the same presynaptic cells; however, their postsynaptic actions are both divergent and antagonistic. CCK and its receptor, the CCK-1 subtype, are expressed in the same subset of taste receptor cells and the autocrine activation of these cells produces a number of excitatory physiological actions. Further, most of these cells are responsive to bitter stimuli. On the other hand, NPY and its receptor, the NPY-1 subtype, are expressed in different cells. NPY, acting in a paracrine fashion on NPY-1 receptors, results in inhibitory actions on the cell. Preliminary evidence suggests the NPY-1 receptor expressing cell co-expresses T1R3, a member of the T1R family of G-protein coupled receptors thought to be important in detection of sweet and umami stimuli. Thus the neuropeptide expressing cells co-express CCK, NPY, and CCK-1 receptor. Neuropeptides released from these cells during bitter stimulation may work in concert to both modulate the excitation of bitter-sensitive taste receptor cells while concurrently inhibiting sweet-sensitive cells. This modulatory process is similar to the phenomenon of lateral inhibition that occurs in other sensory systems.

Cocaine Modulates the Expression of Opioid Receptors and miR-let-7d in Zebrafish Embryos

PubMed Central

López-Bellido, Roger; Barreto-Valer, Katherine; Sánchez-Simón, Fátima Macho; Rodríguez, Raquel E.

2012-01-01

Prenatal exposure to cocaine, in mammals, has been shown to interfere with the expression of opioid receptors, which can have repercussions in its activity. Likewise, microRNAs, such as let-7, have been shown to regulate the expression of opioid receptors and hence their functions in mammals and in vitro experiments. In light of this, using the zebrafish embryos as a model our aim here was to evaluate the actions of cocaine in the expression of opioid receptors and let-7d miRNA during embryogenesis. In order to determine the effects produced by cocaine on the opioid receptors (zfmor, zfdor1 and zfdor2) and let-7d miRNA (dre-let-7d) and its precursors (dre-let-7d-1 and dre-let-7d-2), embryos were exposed to 1.5 µM cocaine hydrochloride (HCl). Our results revealed that cocaine upregulated dre-let-7d and its precursors, and also increased the expression of zfmor, zfdor1 and zfdor2 during early developmental stages and decreased them in late embryonic stages. The changes observed in the expression of opioid receptors might occur through dre-let-7d, since DNA sequences and the morpholinos of opioid receptors microinjections altered the expression of dre-let-7d and its precursors. Likewise, opioid receptors and dre-let-7d showed similar distributions in the central nervous system (CNS) and at the periphery, pointing to a possible interrelationship between them. In conclusion, the silencing and overexpression of opioid receptors altered the expression of dre-let-7d, which points to the notion that cocaine via dre-let-7 can modulate the expression of opioid receptors. Our study provides new insights into the actions of cocaine during zebrafish embryogenesis, indicating a role of miRNAs, let-7d, in development and its relationship with gene expression of opioid receptors, related to pain and addiction process. PMID:23226419

Effect of raclopride on dopamine D2 receptor mRNA expression in rat brain.

PubMed

Kopp, J; Lindefors, N; Brené, S; Hall, H; Persson, H; Sedvall, G

1992-01-01

Prolonged treatment with dopamine D2 receptor antagonists is known to elevate the density of dopamine D2 receptor binding sites in caudate-putamen and nucleus accumbens in rat and human brain. In this study we used the dopamine D2 receptor antagonist raclopride (3 mumol/kg, s.c.) to determine if a single injection or daily administration of this drug for up to 18 days changed the expression of dopamine D2 receptor mRNA in rat caudate-putamen and accumbens as measured by in situ hybridization. A single injection of raclopride did not significantly change the numerical density of dopamine D2 receptor mRNA-expressing neurons in any of the regions examined. A daily administration of raclopride for 18 days resulted in a 31% increase in the number of cells expressing detectable amounts of dopamine D2 receptor mRNA in dorsolateral caudate-putamen and in a 20% increase in the area of silver grains over individual hybridization-positive neurons in this brain region measured on emulsion-dipped slides. The region-specific increase in the D2 receptor mRNA level in dorsolateral caudate-putamen was confirmed by measurement of the hybridization signal on X-ray film autoradiograms. The levels of D2 receptor mRNA remained unchanged in medial caudate-putamen and accumbens after 18 days' treatment. The region-selective increase in dopamine D2 receptor mRNA expression in dorsolateral caudate-putamen indicates a differential regulation of dopamine D2 receptor mRNA expression in a subpopulation of caudate-putamen neurons by this neuroleptic. We suggest that the increase in dopamine D2 receptor density in caudate-putamen known to follow prolonged dopamine D2 receptor blockade to some extent is regulated at the level of gene expression.

Effect of transient receptor potential vanilloid 6 gene silencing on the expression of calcium transport genes in chicken osteoblasts.

PubMed

Zhang, Jie; Deng, Yifeng; Ma, Huijie; Hou, Jiafa; Zhou, ZhenLei

2015-03-01

Ca2+ plays a major role in the regulation of signal transduction. Transient receptor potential vanilloid 6 is a Ca2+-selective channel that serves as an important rate-limiting step in the facilitation of Ca2+ entry into cells, but little is known about the regulation of transient receptor potential vanilloid 6 in chickens. In this study, we evaluated the effects of transient receptor potential vanilloid 6 gene interference on the expression of calbindin-D28K, Na+/Ca2+ exchangers, and plasma membrane Ca2+ ATPase 1b to investigate the mechanism underlying the regulation of transient receptor potential vanilloid 6. Three hairpin siRNA expression vectors targeting transient receptor potential vanilloid 6 (pSIREN- transient receptor potential vanilloid 6) and a negative control (pSIREN-control) were constructed and transfected into chicken osteoblasts. The mRNA and protein expression levels were evaluated by quantitative reverse transcription polymerase chain reaction and Western blot, respectively. The mRNA expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 45.7% (P<0.01) and 27.9% (P<0.01), respectively, 48 h after transfection with one of the three constructs (pSIREN- transient receptor potential vanilloid 6-3) compared with the level obtained in the untreated group. There was no significant difference in the mRNA expression levels of Na+/Ca2+ exchangers and plasma membrane Ca2+ ATPase 1b. The protein expression levels of transient receptor potential vanilloid 6 and calbindin-D28K were reduced by 40.2% (P<0.01) and 29.8% (P<0.01), respectively, 48 h after transfection with pSIREN-transient receptor potential vanilloid 6-3 compared with the level obtained in the untreated group. In conclusion, the vector-based transient receptor potential vanilloid 6-shRNA can efficiently suppress the mRNA and protein expression of transient receptor potential vanilloid 6 in chicken osteoblasts, and transient receptor potential vanilloid 6 regulates the expression of calbindin-D28K during Ca2+ transport. © 2015 Poultry Science Association Inc.

Cysteinyl Leukotriene 1 Receptor Expression Associated With Bronchial Inflammation in Severe Exacerbations of COPD

PubMed Central

Zhu, Jie; Bandi, Venkata; Qiu, Shengyang; Figueroa, David J.; Evans, Jilly F.; Barnes, Neil; Guntupalli, Kay K.

2012-01-01

Background: Cysteinyl leukotriene 1 (CysLT1) receptor expression is known to be increased in the airway mucosa of patients with asthma, especially during exacerbations; however, nothing is known of its expression in COPD. Methods: We applied immunohistochemistry and in situ hybridization to endobronchial biopsies to determine inflammatory cell CysLT1 receptor protein and mRNA expression in the following: (1) 15 nonsmoker control subjects (NSC), (2) 16 smokers with moderate to severe COPD in its stable phase (S-COPD), and (3) 15 smokers with COPD hospitalized for a severe exacerbation (SE-COPD). Results: The total number of bronchial mucosal inflammatory cells (CD45+) and those expressing CysLT1 receptor protein were significantly greater in SE-COPD (CysLT1 receptor protein: median [range] = 139 [31-634]) as compared with S-COPD (32 [6-114]) or NSC (16 [4-66]) (P < .001 for both). CysLT1 receptor gene expression showed similar differences. A greater proportion of CD451 cells expressed CysLT1 receptor protein in SE-COPD (median [range] = 22% [8-81]) compared with S-COPD (10% [4-32]) (P < .03) or NSC (7% [1-19]) (P < .002). In SE-COPD, the relative frequencies of CysLT1 receptor-expressing cells were as follows: tryptase1 mast cells > CD681 monocytes/macrophage > neutrophils > CD201 B lymphocytes = EG21 eosinophils. Moreover, there were positive correlations between the numbers of cells expressing CysLT1 receptor protein and the numbers of CD451 cells (r = 0.78; P < .003) and tryptase1 mast cells (r = 0.62; P < .02). Conclusions: Bronchial mucosal CysLT1 receptor-positive inflammatory cells are present in the bronchial mucosa in COPD in greatest number in those experiencing a severe exacerbation. PMID:22871757

Individual receptor profiling as a novel tool to support diagnosis of bladder pain syndrome/interstitial cystitis (BPS/IC).

PubMed

Neuhaus, Jochen; Schulte-Baukloh, Heinrich; Stolzenburg, Jens-Uwe; Speroni di Fenizio, Pietro; Horn, Lars-Christian; Rüffert, Henrik; Hartenstein, Siegurd; Burger, Maximilian; Schulze, Matthias; Schwalenberg, Thilo

2012-10-01

Dysregulation of neurotransmitter receptors may contribute to bladder overactivity (OAB) symptoms. To address the question whether specific receptor expression patterns are associated with bladder pain syndrome/interstitial cystitis (BPS/IC), we examined the expression of muscarinic, purinergic and histamine receptors in the detrusor. Detrusor receptor expression was investigated in bladder biopsies of female BPS/IC patients (n = 44; age 60.64 ± 13.78, mean ± SD) and carcinoma patients (n = 11; age 58.91 ± 12.72) undergoing cystectomy. Protein expression of muscarinic (M2, M3), purinergic (P2X1-3) and histamine receptors (H1, H2) was analysed by confocal immunofluorescence, and gene expression was quantified by real-time polymerase chain reaction (qPCR). M2, P2X1, P2X2 and H1 receptor immunoreactivity (-IR) was significantly enhanced in BPS/IC compared to the control group, while there was no difference for M3-, P2X3- and H2-IR. We calculated a score, which separated BPS/IC from control patients with an AUC of 89.46%, showing 84.09% sensitivity and 90.91% specificity. Patients had a 9.25 times enhanced calculated risk for BPS/IC. In addition, two patient subgroups (M2 > M3 and M3 > M2) were observed, which differed in associated purinergic and histamine receptor expression. M2, P2X1, P2X2 and H1 were significantly upregulated in BPS/IC patients, and H2 was occasionally highly overexpressed. There was no significant correlation between receptor protein and gene expression, implying posttranslational mechanisms being responsible for the altered receptor expressions. On the basis of individual receptor profiles, upregulated receptors could be targeted by monotherapy or combination therapy with already approved receptor inhibitors, thereby promoting tailored therapy for patients suffering from BPS/IC-like symptoms.

Expression of Plant Receptor Kinases in Tobacco BY-2 Cells.

PubMed

Shinohara, Hidefumi; Matsubayashi, Yoshikatsu

2017-01-01

Although more than 600 single-transmembrane receptor kinase genes have been found in the Arabidopsis genome, only a few of them have known physiological functions, and even fewer plant receptor kinases have known specific ligands. Ligand-binding analysis must be operated using the functionally expressed receptor form. However, the relative abundance of native receptor kinase molecules in the plasma membrane is often quite low. Here, we present a method for stable and functional expression of plant receptor kinases in tobacco BY-2 cells that allows preparation of microsomal fractions containing the receptor. This procedure provides a sufficient amount of receptor proteins while maintaining its ligand-binding activities.

Chemokine receptor binding and signal transduction in native cells of the central nervous system.

PubMed

Davis, Christopher N; Chen, Shuzhen; Boehme, Stefen A; Bacon, Kevin B; Harrison, Jeffrey K

2003-04-01

Chemokine receptors belong to the superfamily of seven-transmembrane-spanning, G-protein-coupled receptors, and their expression by central nervous system cells is clearly documented. As this gene family has become the target of novel therapeutic development, the analysis of these receptors requires radioligand binding techniques as well as methods that entail assessing receptor stimulation of signal transduction pathways. Herein, we describe specific protocols for measuring radiolabeled chemokine binding to their cognate receptors on cultured glial cells as well as to receptors expressed in heterologous cell systems. Multiple downstream signaling pathways, including intracellular calcium influx and receptor-dependent kinase activation, are associated with chemokine receptor stimulation. Protocols for measuring these signaling events in chemokine-receptor-expressing cells are also presented.

Behavioral analysis of Drosophila transformants expressing human taste receptor genes in the gustatory receptor neurons.

PubMed

Adachi, Ryota; Sasaki, Yuko; Morita, Hiromi; Komai, Michio; Shirakawa, Hitoshi; Goto, Tomoko; Furuyama, Akira; Isono, Kunio

2012-06-01

Transgenic Drosophila expressing human T2R4 and T2R38 bitter-taste receptors or PKD2L1 sour-taste receptor in the fly gustatory receptor neurons and other tissues were prepared using conventional Gal4/UAS binary system. Molecular analysis showed that the transgene mRNAs are expressed according to the tissue specificity of the Gal4 drivers. Transformants expressing the transgene taste receptors in the fly taste neurons were then studied by a behavioral assay to analyze whether transgene chemoreceptors are functional and coupled to the cell response. Since wild-type flies show strong aversion against the T2R ligands as in mammals, the authors analyzed the transformants where the transgenes are expressed in the fly sugar receptor neurons so that they promote feeding ligand-dependently if they are functional and activate the neurons. Although the feeding preference varied considerably among different strains and individuals, statistical analysis using large numbers of transformants indicated that transformants expressing T2R4 showed a small but significant increase in the preference for denatonium and quinine, the T2R4 ligands, as compared to the control flies, whereas transformants expressing T2R38 did not. Similarly, transformants expressing T2R38 and PKD2L1 also showed a similar preference increase for T2R38-specific ligand phenylthiocarbamide (PTC) and a sour-taste ligand, citric acid, respectively. Taken together, the transformants expressing mammalian taste receptors showed a small but significant increase in the feeding preference that is taste receptor and also ligand dependent. Although future improvements are required to attain performance comparable to the endogenous robust response, Drosophila taste neurons may serve as a potential in vivo heterologous expression system for analyzing chemoreceptor function.

F-prostanoid receptor regulation of fibroblast growth factor 2 signaling in endometrial adenocarcinoma cells.

PubMed

Sales, Kurt J; Boddy, Sheila C; Williams, Alistair R W; Anderson, Richard A; Jabbour, Henry N

2007-08-01

Prostaglandin (PG) F(2alpha) is a potent bioactive lipid in the female reproductive tract, and exerts its function after coupling with its heptahelical G-protein-coupled receptor [F-series-prostanoid (FP) receptor] to initiate cell signaling and target gene transcription. In the present study, we found elevated expression of fibroblast growth factor (FGF) 2, FGF receptor 1 (FGFR1), and FP receptor, colocalized within the neoplastic epithelial cells of endometrial adenocarcinomas. We investigated a role for PGF(2alpha)-FP receptor interaction in modulating FGF2 expression and signaling using an endometrial adenocarcinoma cell line stably expressing the FP receptor to the levels detected in endometrial adenocarcinomas (FPS cells) and endometrial adenocarcinoma tissue explants. PGF(2alpha)-FP receptor activation rapidly induced FGF2 mRNA expression, and elevated FGF2 protein expression and secretion into the culture medium in FPS cells and endometrial adenocarcinoma explants. The effect of PGF(2alpha) on the expression and secretion of FGF2 could be abolished by treatment of FPS cells and endometrial tissues with an FP receptor antagonist (AL8810) and inhibitor of ERK (PD98059). Furthermore, we have shown that FGF2 can promote the expression of FGF2 and cyclooxygenase-2, and enhance proliferation of endometrial adenocarcinoma cells via the FGFR1 and ERK pathways, thereby establishing a positive feedback loop to regulate neoplastic epithelial cell function in endometrial adenocarcinomas.

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

PubMed

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

2017-08-01

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

A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

PubMed Central

Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

2015-01-01

Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

Immunocytochemical assessment of sigma-1 receptor and human sterol isomerase in breast cancer and their relationship with a series of prognostic factors

PubMed Central

Simony-Lafontaine, J; Esslimani, M; Bribes, E; Gourgou, S; Lequeux, N; Lavail, R; Grenier, J; Kramar, A; Casellas, P

2000-01-01

The purpose of this study was to immunocytochemically investigate two new markers, the sigma-1 receptor and the human sterol isomerase (hSI), in comparison with a series of clinicopathological and immunocytochemical prognostic factors in a trial including 95 patients with operable primary breast cancers. Our results showed no statistically significant relationship between these two markers and the age of the patients, their menopausal status, the tumour size and its histological grade, the nodal status and the expression of the Ki-67 proliferative marker. However, we evidenced a close correlation between the sigma-1 receptor expression and the hormonal receptor positivity (P = 0.008), essentially due to a link with the progesterone receptor status (P = 0.01). By contrast there was an inverse relationship between hSI expression and the oestrogen receptor and/or progesterone receptor positivity (P = 0.098). A significant relationship was shown between both the sigma-1 receptor, hSI expressions and Bcl2 expression, with P = 0.017 and 0.035 respectively. We also assessed whether the expression of the sigma-1 receptor or hSI might be linked with disease-free survival (DFS) and found that the presence of hSI and the absence of sigma-1 receptor expression were associated with a poorer disease-free survival (P = 0.007). Altogether these results suggest that in primary breast carcinomas in association with the evaluation of the steroid receptor status, the sigma-1 receptor and hSI may be interesting new markers useful to identify those patients who might be able to benefit from an adjuvant therapy. © 2000 Cancer Research Campaign PMID:10864204

Expression of CB2 cannabinoid receptor in Pichia pastoris.

PubMed

Feng, Wenke; Cai, Jian; Pierce, William M; Song, Zhao-Hui

2002-12-01

To facilitate purification and structural characterization, the CB2 cannabinoid receptor is expressed in methylotrophic yeast Pichia pastoris. The expression plasmids were constructed in which the CB2 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase 1 gene. A c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB2 to permit easy detection and purification. In membrane preparations of CB2 gene transformed yeast cells, Western blot analysis detected the expression of CB2 proteins. Radioligand binding assays demonstrated that the CB2 receptors expressed in P. pastoris have a pharmacological profile similar to that of the receptors expressed in mammalian systems. Furthermore, the epitope-tagged receptor was purified by metal chelating chromatography and the purified CB2 preparations were subjected to digestion by trypsin. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions detected 14 peptide fragments derived from the CB2 receptor. ESI mass spectrometry was used to sequence one of these peptide fragments, thus, further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope-tagged, functional CB2 cannabinoid receptor can be expressed in P. pastoris for purification.

Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord

NASA Technical Reports Server (NTRS)

Stegenga, S. L.; Kalb, R. G.

2001-01-01

Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children.

PubMed

Keustermans, Genoveva; van der Heijden, Laila B; Boer, Berlinda; Scholman, Rianne; Nuboer, Roos; Pasterkamp, Gerard; Prakken, Berent; de Jager, Wilco; Kalkhoven, Eric; Janse, Arieke J; Schipper, Henk S

2017-01-01

Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children. 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed. Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction. First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors

PubMed Central

Farroni, Jeffrey S; McCool, Brian A

2004-01-01

Background Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. Results While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The β-amino acid taurine possessed 30–50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for β-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Conclusions Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology. PMID:15301692

Extrinsic factors regulate partial agonist efficacy of strychnine-sensitive glycine receptors.

PubMed

Farroni, Jeffrey S; McCool, Brian A

2004-08-09

Strychnine-sensitive glycine receptors in many adult forebrain regions consist of alpha2 + beta heteromeric channels. This subunit composition is distinct from the alpha1 + beta channels found throughout the adult spinal cord. Unfortunately, the pharmacology of forebrain alpha2beta receptors are poorly defined compared to 'neonatal' alpha2 homomeric channels or 'spinal' alpha1beta heteromers. In addition, the pharmacologic properties of native alpha2beta glycine receptors have been generally distinct from receptors produced by heterologous expression. To identify subtype-specific pharmacologic tools for the forebrain alpha2beta receptors, it is important to identify a heterologous expression system that closely resembles these native glycine-gated chloride channels. While exploring pharmacological properties of alpha2beta glycine receptors compared to alpha2-homomers, we found that distinct heterologous expression systems appeared to differentially influence partial agonist pharmacology. The beta-amino acid taurine possessed 30-50% efficacy for alpha2-containing receptor isoforms when expressed in HEK 293 cells. However, taurine efficacy was dramatically reduced in L-cell fibroblasts. Similar results were obtained for beta-alanine. The efficacy of these partial agonists was also strongly reduced by the beta subunit. There were no significant differences in apparent strychnine affinity values calculated from concentration-response data between expression systems or subunit combinations. Nor did relative levels of expression correlate with partial agonist efficacy when compared within or between several different expression systems. Finally, disruption of the tubulin cytoskeleton reduced the efficacy of partial agonists in a subunit-dependent, but system-independent, fashion. Our results suggest that different heterologous expression systems can dramatically influence the agonist pharmacology of strychnine-sensitive glycine receptors. In the systems examine here, these effects are independent of both absolute expression level and any system-related alterations in the agonist binding site. We conclude that complex interactions between receptor composition and extrinsic factors may play a significant role in determining strychnine-sensitive glycine receptor partial agonist pharmacology.

Evidence of alterations in the expression of orphan receptors GPR26 and GPR39 due to the etiology of the metabolic syndrome.

PubMed

Romero-Nava, Rodrigo; Zhou, De-Shan; García, Noemí; Ruiz-Hernández, Armando; Si, Yin-Chu; Sánchez-Muñoz, Fausto; Huang, Fengyang; Hong, Enrique; Villafaña, Santiago

2017-08-01

Metabolic syndrome (MS) is composed of several metabolic abnormalities that increase the risk of cardiovascular diseases and diabetes. Although there are treatments for the components of MS, this pathology maintains a high mortality, suggesting that there are other mechanisms in which orphan receptors such as GPR26 and GPR39 may be involved. For this reason, the aim of this work was to evaluate the expression of GPR26 and GPR39 orphan receptors in two models of MS (diet and genetics). We used male Wistar rats, which received 70% fructose in drinking water for 9 weeks, and obese Zucker rats. We measured weight, blood pressure, glucose, triglycerides, total cholesterol, HDL cholesterol, LDL cholesterol to determine the MS and the expression of the orphan receptors GPR26 and GPR39 in brain, heart, aorta, liver, and kidney by RT-PCR. The analysis of the expression of the orphan receptors GPR26 and GPR39 showed that the receptors are expressed in some tissues, but the expression of the GPR26 tends to decrease in the heart and aorta, whereas in the brain, no changes were observed, this receptor is not expressed in the liver and kidney of both strains. The expression of GPR39 isoforms depends on the tissue and MS model. We conclude that the orphan receptors GPR26, GPR39v1, and GPR39v2 are expressed in different tissues and their profile expression is dependent on the etiology of the MS.

Significance of increased expression of decoy receptor 3 in chronic liver disease.

PubMed

Kim, S; Kotoula, V; Hytiroglou, P; Zardavas, D; Zhang, L

2009-08-01

Considerable evidence has indicated that apoptosis plays an important role in hepatocyte death in chronic liver disease. However, the cellular and molecular mechanisms underlying liver regeneration in these diseases are largely unknown. Plausibly, certain molecules expressed to counteract apoptosis might provide survival advantage of certain liver cells. Therefore, we investigated a possible expression of decoy receptor 3 of the tumour necrosis factor receptor family in chronic liver diseases since decoy receptor 3 is known to inhibit apoptosis mediated by pro-apoptotic tumour necrosis factor family ligands including Fas ligand. A series of liver biopsies from patients with different stages of fibrosis were subjected to immunohistochemistry and in situ hybridization. Both decoy receptor 3 protein and mRNA were mainly expressed in biliary epithelial cells and infiltrating lymphocytes in the diseased livers. Most noticeably, intense decoy receptor 3 expression was observed in newly developing biliary ductules in regenerative nodules as well as dysplastic nodules of cirrhotic livers. In addition, decoy receptor 3 secretion in hepatocellular carcinoma cells in culture was via the activation of mitogen-activated protein kinases. Decoy receptor 3 was specifically expressed in chronic liver diseases and hepatocellular carcinoma cells, and decoy receptor 3 might facilitate the survival of liver cells by exerting its anti-apoptotic activity during the progression of liver cirrhosis and hepatocarcinogenesis.

The N-terminal region of the dopamine D2 receptor, a rhodopsin-like GPCR, regulates correct integration into the plasma membrane and endocytic routes

PubMed Central

Cho, DI; Min, C; Jung, KS; Cheong, SY; Zheng, M; Cheong, SJ; Oak, MH; Cheong, JH; Lee, BK; Kim, KM

2012-01-01

BACKGROUND AND PURPOSE Functional roles of the N-terminal region of rhodopsin-like GPCR family remain unclear. Using dopamine D2 and D3 receptors as a model system, we probed the roles of the N-terminal region in the signalling, intracellular trafficking of receptor proteins, and explored the critical factors that determine the functionality of the N-terminal region. EXPERIMENTAL APPROACH The N-terminal region of the D2 receptor was gradually shortened or switched with that of the D3 receptor or a non-specific sequence (FLAG), or potential N-terminal glycosylation sites were mutated. Effects of these manipulations on surface expression, internalization, post-endocytic behaviours and signalling were determined. KEY RESULTS Shortening the N-terminal region of the D2 receptor enhanced receptor internalization and impaired surface expression and signalling; ligand binding, desensitization and down-regulation were not affected but their association with a particular microdomain, caveolae, was disrupted. Replacement of critical residues within the N-terminal region with the FLAG epitope failed to restore surface expression but partially restored the altered internalization and signalling. When the N-terminal regions were switched between D2 and D3 receptors, cell surface expression pattern of each receptor was switched. Mutations of potential N-terminal glycosylation sites inhibited surface expression but enhanced internalization of D2 receptors. CONCLUSIONS AND IMPLICATIONS Shortening of N-terminus or mutation of glycosylation sites located within the N-terminus enhanced receptor internalization but impaired the surface expression of D2 receptors. The N-terminal region of the D2 receptor, in a sequence-specific manner, controls the receptor's conformation and integration into the plasma membrane, which determine its subcellular localization, intracellular trafficking and signalling properties. PMID:22117524

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

PubMed Central

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

2012-01-01

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

G protein-coupled estrogen receptor (GPER) expression in normal and abnormal endometrium.

PubMed

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

2012-07-01

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

Hetero-oligomerization of the P2Y11 receptor with the P2Y1 receptor controls the internalization and ligand selectivity of the P2Y11 receptor.

PubMed

Ecke, Denise; Hanck, Theodor; Tulapurkar, Mohan E; Schäfer, Rainer; Kassack, Matthias; Stricker, Rolf; Reiser, Georg

2008-01-01

Nucleotides signal through purinergic receptors such as the P2 receptors, which are subdivided into the ionotropic P2X receptors and the metabotropic P2Y receptors. The diversity of functions within the purinergic receptor family is required for the tissue-specificity of nucleotide signalling. In the present study, hetero-oligomerization between two metabotropic P2Y receptor subtypes is established. These receptors, P2Y1 and P2Y11, were found to associate together when co-expressed in HEK293 cells. This association was detected by co-pull-down, immunoprecipitation and FRET (fluorescence resonance energy transfer) experiments. We found a striking functional consequence of the interaction between the P2Y11 receptor and the P2Y1 receptor where this interaction promotes agonist-induced internalization of the P2Y11 receptor. This is remarkable because the P2Y11 receptor by itself is not able to undergo endocytosis. Co-internalization of these receptors was also seen in 1321N1 astrocytoma cells co-expressing both P2Y11 and P2Y1 receptors, upon stimulation with ATP or the P2Y1 receptor-specific agonist 2-MeS-ADP. 1321N1 astrocytoma cells do not express endogenous P2Y receptors. Moreover, in HEK293 cells, the P2Y11 receptor was found to functionally associate with endogenous P2Y1 receptors. Treatment of HEK293 cells with siRNA (small interfering RNA) directed against the P2Y1 receptor diminished the agonist-induced endocytosis of the heterologously expressed GFP-P2Y11 receptor. Pharmacological characteristics of the P2Y11 receptor expressed in HEK293 cells were determined by recording Ca2+ responses after nucleotide stimulation. This analysis revealed a ligand specificity which was different from the agonist profile established in cells expressing the P2Y11 receptor as the only metabotropic nucleotide receptor. Thus the hetero-oligomerization of the P2Y1 and P2Y11 receptors allows novel functions of the P2Y11 receptor in response to extracellular nucleotides.

Aβ mediates Sigma receptor degradation via CaN/NFAT pathway

PubMed Central

Fang, Min; Zhang, Pei; Zhao, Yanxin; Jin, Aiping; Liu, Xueyuan

2016-01-01

Sigma receptor is an endoplasmic reticulum protein and belongs to non-opioid receptor. Increasing evidence shows that Sigma receptor activation can significantly attenuate AD induced neurological dysfunction and the functional deficiency of Sigma receptor plays an important role in the Aβ induced neuronal loss. This study aimed to investigate the influence of extracellular accumulation of Aβ on the Sigma receptor expression. Our results showed the increase in extracellular Aβ had little influence on the mRNA expression of Sigma receptor, but gradually reduced its protein expression. Co-immunoprecipitation was employed to evaluate the interaction of Sigma receptor with other proteins. Results showed BIP could bind to Sigma receptor to affect the ubiquitination of Sigma receptor. Further investigation showed there was a NFAT binding site at the promoter of BIP. Then, Western blot assay was performed to detect NFAT expression. Results showed extracellular Aβ affected the nuclear translocation of NFAT and the CaN activity of NFAT also increased with the accumulation of extracellular Aβ. In this study, NFAT-BIP luciferase reporter gene system was constructed. Results showed NFAT was able to regulate the transcription of BIP. Thus, we speculate that extracellular Aβ accumulation may activate CaN/NFAT signaling pathway to induce chaperone BIP expression, which results in Sigma receptor ubiquitination and its degradation. PMID:27648137

Captodiamine, a putative antidepressant, enhances hypothalamic BDNF expression in vivo by synergistic 5-HT2c receptor antagonism and sigma-1 receptor agonism.

PubMed

Ring, Rebecca M; Regan, Ciaran M

2013-10-01

The putative antidepressant captodiamine is a 5-HT2c receptor antagonist and agonist at sigma-1 and D3 dopamine receptors, exerts an anti-immobility action in the forced swim paradigm, and enhances dopamine turnover in the frontal cortex. Captodiamine has also been found to ameliorate stress-induced anhedonia, reduce the associated elevations of hypothalamic corticotrophin-releasing factor (CRF) and restore the reductions in hypothalamic BDNF expression. Here we demonstrate chronic administration of captodiamine to have no significant effect on hypothalamic CRF expression through sigma-1 receptor agonism; however, both sigma-1 receptor agonism or 5-HT2c receptor antagonism were necessary to enhance BDNF expression. Regulation of BDNF expression by captodiamine was associated with increased phosphorylation of transcription factor CREB and mediated through sigma-1 receptor agonism but blocked by 5-HT2c receptor antagonism. The existence of two separate signalling pathways was confirmed by immunolocalisation of each receptor to distinct cell populations in the paraventricular nucleus of the hypothalamus. Increased BDNF induced by captodiamine was also associated with enhanced expression of synapsin, but not PSD-95, suggesting induction of long-term structural plasticity between hypothalamic synapses. These unique features of captodiamine may contribute to its ability to ameliorate stress-induced anhedonia as the hypothalamus plays a prominent role in regulating HPA axis activity.

Serotonin systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors and induce anorexia via a leptin-independent pathway in mice.

PubMed

Nonogaki, Katsunori; Ohba, Yukie; Sumii, Makiko; Oka, Yoshitomo

2008-07-18

NEFA/nucleobindin2 (NUCB2), a novel satiety molecule, is associated with leptin-independent melanocortin signaling in the central nervous system. Here, we show that systemic administration of m-chlorophenylpiperazine (mCPP), a serotonin 5-HT1B/2C receptor agonist, significantly increased the expression of hypothalamic NUCB2 in wild-type mice. The increases in hypothalamic NUCB2 expression induced by mCPP were attenuated in 5-HT2C receptor mutant mice. Systemic administration of mCPP suppressed food intake in db/db mice with leptin receptor mutation as well as lean control mice. On the other hand, the expression of hypothalamic NUCB2 and proopiomelanocortin (POMC) was significantly decreased in hyperphagic and non-obese 5-HT2C receptor mutants compared with age-matched wild-type mice. Interestingly, despite increased expression of hypothalamic POMC, hypothalamic NUCB2 expression was decreased in 5-HT2C receptor mutant mice with heterozygous mutation of beta-endorphin gene. These findings suggest that 5-HT systems upregulate the expression of hypothalamic NUCB2 via 5-HT2C receptors, and induce anorexia via a leptin-independent pathway in mice.

Serotonergic Modulation Differentially Targets Distinct Network Elements within the Antennal Lobe of Drosophila melanogaster

PubMed Central

Sizemore, Tyler R.; Dacks, Andrew M.

2016-01-01

Neuromodulation confers flexibility to anatomically-restricted neural networks so that animals are able to properly respond to complex internal and external demands. However, determining the mechanisms underlying neuromodulation is challenging without knowledge of the functional class and spatial organization of neurons that express individual neuromodulatory receptors. Here, we describe the number and functional identities of neurons in the antennal lobe of Drosophila melanogaster that express each of the receptors for one such neuromodulator, serotonin (5-HT). Although 5-HT enhances odor-evoked responses of antennal lobe projection neurons (PNs) and local interneurons (LNs), the receptor basis for this enhancement is unknown. We used endogenous reporters of transcription and translation for each of the five 5-HT receptors (5-HTRs) to identify neurons, based on cell class and transmitter content, that express each receptor. We find that specific receptor types are expressed by distinct combinations of functional neuronal classes. For instance, the excitatory PNs express the excitatory 5-HTRs, while distinct classes of LNs each express different 5-HTRs. This study therefore provides a detailed atlas of 5-HT receptor expression within a well-characterized neural network, and enables future dissection of the role of serotonergic modulation of olfactory processing. PMID:27845422

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

PubMed

Vyas, Ashish Kumar; Ramakrishna, Usha; Sen, Bijoya; Islam, Mojahidul; Ramakrishna, Gayatri; Patra, Sharda; Rastogi, Archana; Sarin, Shiv Kumar; Trehanpati, Nirupma

2018-04-30

Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

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

Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.

1989-07-01

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

Class I odorant receptors, TAS1R and TAS2R taste receptors, are markers for subpopulations of circulating leukocytes

PubMed Central

Malki, Agne; Fiedler, Julia; Fricke, Kristina; Ballweg, Ines; Pfaffl, Michael W.; Krautwurst, Dietmar

2015-01-01

Our cellular immune system has to cope constantly with foodborne substances that enter the bloodstream postprandially. Here, they may activate leukocytes via specific but yet mostly unknown receptors. Ectopic RNA expression out of gene families of chemosensory receptors, i.e., the ∼400 ORs, ∼25 TAS2R bitter-taste receptors, and the TAS1R umami- and sweet-taste receptor dimers by which we typically detect foodborne substances, has been reported in a variety of peripheral tissues unrelated to olfaction or taste. In the present study, we have now discovered, by gene-specific RT-PCR experiments, the mRNA expression of most of the Class I ORs (TAS1R) and TAS2R in 5 different types of blood leukocytes. Surprisingly, we did not detect Class II OR mRNA. By RT-qPCR, we show the mRNA expression of human chemosensory receptors and their cow orthologs in PMN, thus suggesting an evolutionary concept. By immunocytochemistry, we demonstrate that some olfactory and taste receptors are expressed, on average, in 40–60% of PMN and T or B cells and largely coexpress in the same subpopulation of PMN. The mRNA expression and the size of subpopulations expressing certain chemosensory receptors varied largely among individual blood samples, suggesting a regulated expression of olfactory and taste receptors in these cells. Moreover, we show mRNA expression of their downstream signaling molecules and demonstrate that PTX abolishes saccharin- or 2-PEA-induced PMN chemotactic migration, indicating a role for Gi-type proteins. In summary, our data suggest "chemosensory"-type subpopulations of circulating leukocytes. PMID:25624459

Progress toward heterologous expression of active G-protein-coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

PubMed Central

O'Malley, Michelle A; Mancini, J Dominic; Young, Carissa L; McCusker, Emily C; Raden, David; Robinson, Anne S

2009-01-01

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A2a (hA2aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA2aR, as expected, and partially cleaved for hA2bR, hCCR5R, and hD2LR. Ligand-binding assays conducted on the adenosine family (hA1R, hA2aR, hA2bR, and hA3R) of receptors show that hA2aR and hA2bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae. PMID:19760666

Apoptosis gene expression and death receptor signaling in mitomycin-C-treated human tenon capsule fibroblasts.

PubMed

Crowston, Jonathan G; Chang, Lydia H; Constable, Peter H; Daniels, Julie T; Akbar, Arne N; Khaw, Peng T

2002-03-01

To examine the effect of mitomycin-C on the expression of apoptosis genes in human Tenon capsule fibroblasts and to evaluate whether death receptor signaling modulates mitomycin-C cytotoxicity. Bcl-2, Bax, Bcl-x, Fas (CD95) and tumor necrosis factor (TNF) receptor expression was determined by flow cytometry in control and mitomycin-C-treated Tenon fibroblasts. Fibroblast death was quantified using a lactate dehydrogenase release assay. The effect of Fas and TNF-receptor signaling was evaluated using Fas-specific antibodies and soluble TNF-alpha. Tenon fibroblasts constitutively express Bcl-2, Bax, and Bcl-x in culture. Mitomycin-C (0.4 mg/mL) induced a small but consistent increase in the expression of all three proteins. Tenon fibroblasts express low levels of Fas but are resistant to the effects of Fas-receptor ligation. Mitomycin-C (0.01-1.0 mg/mL) led to a significant increase in Fas expression at all concentrations tested (P < 0.01). Pretreatment with mitomycin-C (0.4 mg/mL) rendered fibroblasts susceptible to agonistic anti-Fas monoclonal IgM antibodies (50-500 ng/mL) and led to a further 50% reduction in viable fibroblasts at 48 hours, compared with mitomycin-C alone (P < 0.05). Antibodies that block the Fas receptor did not inhibit mitomycin-C-induced apoptosis. Mitomycin-C alters apoptosis gene expression and primes fibroblasts to the effects of Fas receptor ligation. Factors other than the level of Fas receptor expression modulate the response to Fas receptor signaling. Determining the signals that regulate fibroblast apoptosis may help to refine therapeutic strategies for switching off the subconjunctival healing response and maintaining intraocular pressure control.

TRAIL Death Receptor-4 Expression Positively Correlates With the Tumor Grade in Breast Cancer Patients With Invasive Ductal Carcinoma

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

Sanlioglu, Ahter D.; Department of Medical Biology and Genetics, Akdeniz University Faculty of Medicine, Antalya; Korcum, Aylin F.

2007-11-01

Purpose: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, thismore » study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. Methods and Materials: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. Results: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. Conclusions: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.« less

Protease-activated receptor 2, a receptor involved in melanosome transfer, is upregulated in human skin by ultraviolet irradiation.

PubMed

Scott, G; Deng, A; Rodriguez-Burford, C; Seiberg, M; Han, R; Babiarz, L; Grizzle, W; Bell, W; Pentland, A

2001-12-01

Previous studies have shown that the protease-activated receptor 2 is involved in skin pigmentation through increased phagocytosis of melanosomes by keratinocytes. Ultraviolet irradiation is a potent stimulus for melanosome transfer. We show that protease-activated receptor 2 expression in human skin is upregulated by ultraviolet irradiation. Subjects with skin type I, II, or III were exposed to two or three minimal erythema doses of irradiation from a solar simulator. Biopsies were taken from nonexposed and irradiated skin 24 and 96 h after irradiation and protease-activated receptor 2 expression was detected using immunohistochemical staining. In nonirradiated skin, protease-activated receptor 2 expression was confined to keratinocytes in the lower one-third of the epidermis. After ultraviolet irradiation protease-activated receptor 2 expression was observed in keratinocytes in the upper two-thirds of the epidermis or the entire epidermis at both time points studied. Subjects with skin type I showed delayed upregulation of protease-activated receptor 2 expression, however, compared with subjects with skin types II and III. Irradiated cultured human keratinocytes showed upregulation in protease-activated receptor 2 expression as determined by immunofluorescence microscopy and Western blotting. Cell culture supernatants from irradiated keratinocytes also exhibited a dose-dependent increase in protease-activated receptor-2 cleavage activity. These results suggest an important role for protease-activated receptor-2 in pigmentation in vivo. Differences in protease-activated receptor 2 regulation in type I skin compared with skin types II and III suggest a potential mechanism for differences in tanning in subjects with different skin types.

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

PubMed

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

2017-01-01

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

Expression and potential role of the peptide orexin-A in prostate cancer

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

Valiante, Salvatore; Liguori, Giovanna; Tafuri, Simona

The peptides orexin-A and orexin-B and their G protein-coupled OX1 and OX2 receptors are involved in multiple physiological processes in the central nervous system and peripheral organs. Altered expression or signaling dysregulation of orexins and their receptors have been associated with a wide range of human diseases including narcolepsy, obesity, drug addiction, and cancer. Although orexin-A, its precursor molecule prepro-orexin and OX1 receptor have been detected in the human normal and hyperplastic prostate tissues, their expression and function in the prostate cancer (PCa) remains to be addressed. Here, we demonstrate for the first time the immunohistochemical localization of orexin-A inmore » human PCa specimens, and the expression of prepro-orexin and OX1 receptor at both protein and mRNA levels in these tissues. Orexin-A administration to the human androgen-dependent prostate carcinoma cells LNCaP up-regulates OX1 receptor expression resulting in a decrease of cell survival. Noteworthy, nanomolar concentrations of the peptide counteract the testosterone-induced nuclear translocation of the androgen receptor in the cells: the orexin-A action is prevented by the addition of the OX1 receptor antagonist SB-408124 to the test system. These findings indicate that orexin-A/OX1 receptor interaction interferes with the activity of the androgen receptor which regulates PCa onset and progression, thus suggesting that orexin-A and its receptor might represent novel therapeutic targets to challenge this aggressive cancer. - Highlights: • Orexin-A and OX1 receptor are present in human cancer prostate tissues. • Orexin-A up-regulates OX1 receptor expression in LNCaP cells. • Orexin-A inhibits testosterone-induced nuclear translocation of androgen receptor.« less

Functional Expression of Two Neuronal Nicotinic Acetylcholine Receptors from cDNA Clones Identifies a Gene Family

NASA Astrophysics Data System (ADS)

Boulter, Jim; Connolly, John; Deneris, Evan; Goldman, Dan; Heinemann, Steven; Patrick, Jim

1987-11-01

A family of genes coding for proteins homologous to the α subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha3, alpha4, and beta2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha3 or alpha4 protein in combination with the beta2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by α -bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha3, alpha4, and beta2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha3, alpha4, and beta2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Spatial pattern of receptor expression in the olfactory epithelium.

PubMed Central

Nef, P; Hermans-Borgmeyer, I; Artières-Pin, H; Beasley, L; Dionne, V E; Heinemann, S F

1992-01-01

A PCR-based strategy for amplifying putative receptors involved in murine olfaction was employed to isolate a member (OR3) of the seven-transmembrane-domain receptor superfamily. During development, the first cells that express OR3 appear adjacent to the wall of the telencephalic vesicle at embryonic day 10. The OR3 receptor is uniquely expressed in a subset of olfactory cells that have a characteristic bilateral symmetry in the adult olfactory epithelium. This receptor and its specific pattern of expression may serve a functional role in odor coding or, alternatively, may play a role in the development of the olfactory system. Images PMID:1384038

Feasibility Study of Odor Biosensor Using Dissociate Neuronal Culture with Gene Expression of Ionotropic Odorant Receptors

NASA Astrophysics Data System (ADS)

Tanada, Norio; Sakurai, Takeshi; Mitsuno, Hidefumi; Bakkum, Douglas; Kanzaki, Ryohei; Takahashi, Hirokazu

We propose a highly sensitive and real-time odor biosensor by expressing ionotropic odorant receptors of insects into dissociated cultures of neurons of rats. The odorant-gated ion channel structure of insect odorant receptor is expected to allow easy functional expression into cells. The neuronal dissociated cultures of rats have two significant advantages: a long lifetime comparable to rats, i.e., a few years; and amplification ability from weak ionic currents of odorant receptors into easily detectable action potentials of neurons. In the present work, in order to show the feasibility of the proposed sensor, we attempt to express the pheromone receptors of silkmoth, Bombyx mori, into cultured neurons of rats. We demonstrate that 10% of neuronal cells transfected using Lipofectamine successfully expressed pheromone receptors, and that these cells showed significant increase of calcium signals by 50% at the presentation of pheromone.

Ionotropic crustacean olfactory receptors.

PubMed

Corey, Elizabeth A; Bobkov, Yuriy; Ukhanov, Kirill; Ache, Barry W

2013-01-01

The nature of the olfactory receptor in crustaceans, a major group of arthropods, has remained elusive. We report that spiny lobsters, Panulirus argus, express ionotropic receptors (IRs), the insect chemosensory variants of ionotropic glutamate receptors. Unlike insects IRs, which are expressed in a specific subset of olfactory cells, two lobster IR subunits are expressed in most, if not all, lobster olfactory receptor neurons (ORNs), as confirmed by antibody labeling and in situ hybridization. Ligand-specific ORN responses visualized by calcium imaging are consistent with a restricted expression pattern found for other potential subunits, suggesting that cell-specific expression of uncommon IR subunits determines the ligand sensitivity of individual cells. IRs are the only type of olfactory receptor that we have detected in spiny lobster olfactory tissue, suggesting that they likely mediate olfactory signaling. Given long-standing evidence for G protein-mediated signaling in activation of lobster ORNs, this finding raises the interesting specter that IRs act in concert with second messenger-mediated signaling.

HIV-positive pregnant women attending the prevention of mother-to-child transmission of HIV/AIDS (PMTCT) services in Ethiopia: economic productivity losses across urban-rural settings.

PubMed

Zegeye, Elias Asfaw; Mbonigaba, Josue; Kaye, Sylvia Blanche

2018-06-01

HIV/AIDS impacts significantly on pregnant women and on children in Ethiopia. This impact has a multiplier effect on household economies and on productivity losses, and is expected to vary across rural and urban settings. Applying the human capital approach to data collected from 131 respondents, this study estimated productivity losses per HIV-positive pregnant woman-infant pair across urban and rural health facilities in Ethiopia, which in turn were used to estimate the national productivity loss. The study found that the annual productivity loss per woman-infant pair was Ethiopian birr (ETB) 7,433 or United States dollar (US$) 378 and ETB 625 (US$ 32) in urban and rural settings, respectively. The mean patient days lost per year due to inpatient admission at hospitals/health centres was 11 in urban and 22 in rural health facilities. On average, urban home care-givers spent 20 (SD = 21) days annually providing home care services, while their rural counterparts spent 23 days (SD = 26). The productivity loss accounted for 16% and 7% of household income in urban and rural settings, respectively. These high and varying productivity losses require preventive interventions that are appropriate to each setting to ensure the welfare of women and children in Ethiopia.

User experience analysis of e-TB Manager, a nationwide electronic tuberculosis recording and reporting system in Ukraine

PubMed Central

Sawyer, Kelly; Nizova, Nataliya

2017-01-01

Ukraine has successfully implemented e-TB Manager nationwide as its mandatory national tuberculosis registry after first introducing it in 2009. Our objective was to perform an end-of-programme evaluation after formal handover of the registry administration to Ukraine's Centre for Disease Control in 2015. We conducted a nationwide, cross-sectional, anonymous, 18-point user experience survey, and stratified the registry's transaction statistics to demonstrate usability. Contrary to initial implementation experience, older users (aged >50 years), often with limited or no computer proficiency prior to using the registry, had significantly better user experience scores for at least six of the 12 measures compared to younger users (aged 18–29 years). Using the registry for >3 years was associated with significantly higher scores for having capacity, adequacy of training received and satisfaction with the registry. Of the 5.9 million transactions over a 4-year period, nine out of 24 oblasts (regions) and Kiev city accounted for 62.5% of all transactions, and corresponded to 59% of Ukraine's tuberculosis burden. There were 437 unique active users in 486 rayons (districts) of Ukraine, demonstrating extensive reach. Our key findings complement the World Health Organization and European Respiratory Society's agenda for action on digital health to help implement the End TB Strategy. PMID:28512634

Effect of hypobaric hypoxia on the P2X receptors of pyramidal cells in the immature rat hippocampus CA1 sub-field.

PubMed

Zhao, Yan-Dong; Cheng, Sai-Yu; Ou, Shan; Xiao, Zhi; He, Wen-Juan; Jian-Cui; Ruan, Huai-Zhen

2012-01-01

This study was designed to evaluate the effect of hypobaric hypoxia (HH) on the function and expression of P2X receptors in rat hippocampus CA1 pyramidal cells. The functional changes of P2X receptors were investigated through the cell HH model and the expressional alterations of P2X receptors were observed through the animal HH model. P2X receptors mediated currents were recorded from the freshly dissociated CA1 pyramidal cells of 7-day-old SD rats by whole cell patch clamp recording. The expression and distribution of P2X receptors were observed through immunohistochemistry and western blot at HH 3-day and 7-day. In acute HH conditions, the amplitudes of ATP evoked peak currents were decreased compared to control. The immunohistochemistry and western blot results reflected there was no change in P2X receptors expression after 3 days HH injury, while P2X receptors expression was up-regulated in response to 7 days HH injury. These findings supported the possibility that the function of P2X receptors was sensitive to HH damage and long-term function decrease should result in the expression increase of P2X receptors.

Expression and characterization of human CB1 cannabinoid receptor in methylotrophic yeast Pichia pastoris.

PubMed

Kim, Tae-Kang; Zhang, Rundong; Feng, Wenke; Cai, Jian; Pierce, William; Song, Zhao-Hui

2005-03-01

For the purpose of purification and structural characterization, the CB1 cannabinoid receptors are expressed in methylotrophic yeast Pichia pastoris. The expression plasmid was constructed in which the CB1 gene is under the control of the highly inducible promoter of P. pastoris alcohol oxidase I gene. To facilitate easy detection and purification, a FLAG tag was introduced at the N-terminal, a c-myc epitope and a hexahistidine tag were introduced at the C-terminal of the CB1. In membrane preparations of CB1 gene transformed yeast cells, Western blot analysis detected the expression of CB1 proteins. Radioligand binding assays demonstrated that the tagged CB1 receptors expressed in P. pastoris have a pharmacological profile similar to that of the untagged CB1 receptors expressed in mammalian systems. Furthermore, the tagged CB1 receptors were purified by anti-FLAG M2 affinity chromatography and the identity of the purified CB1 receptor proteins was confirmed by Western blot analysis. MALDI/TOF mass spectrometry analysis of the peptides extracted from tryptic digestions of purified CB1 preparations detected 17 peptide fragments derived from the CB1, thus further confirming the identity of the purified receptor. In conclusion, these data demonstrated for the first time that epitope tagged, functional CB1 cannabinoid receptors can be expressed in P. pastoris for purification and mass spectrometry characterization.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

PubMed

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-02-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

PubMed Central

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-01-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

Differential expression and role of hyperglycemia induced oxidative stress in epigenetic regulation of β1, β2 and β3-adrenergic receptors in retinal endothelial cells

PubMed Central

2014-01-01

Background Aberrant epigenetic profiles are concomitant with a spectrum of developmental defects and diseases. Role of methylation is an increasingly accepted factor in the pathophysiology of diabetes and its associated complications. This study aims to examine the correlation between oxidative stress and methylation of β1, β2 and β3-adrenergic receptors and to analyze the differential variability in the expression of these genes under hyperglycemic conditions. Methods Human retinal endothelial cells were cultured in CSC complete medium in normal (5 mM) or high (25 mM) glucose to mimic a diabetic condition. Reverse transcription PCR and Western Blotting were performed to examine the expression of β1, β2 and β3-adrenergic receptors. For detections, immunocytochemistry was used. Bisulfite sequencing method was used for promoter methylation analysis. Apoptosis was determined by the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Dichlorodihydrofluorescein diacetate (DCFH-DA) assay was used to measure reactive oxygen species (ROS) production in the cells. Results β1 and β3-adrenergic receptors were expressed in retinal endothelial cells while β2-adrenergic receptor was not detectable both at protein and mRNA levels. Hyperglycemia had no significant effect on β1 and β2-adrenergic receptors methylation and expression however β3-adrenergic receptors showed a significantly higher expression (p < 0.05) and methylation (p < 0.01) in high and low glucose concentration respectively. Apoptosis and oxidative stress were inversely correlated with β3-adrenergic receptors methylation with no significant effect on β1 and β2-adrenergic receptors. β2-adrenergic receptor was hypermethylated with halted expression. Conclusion Our study demonstrates that β1 and β3-adrenergic receptors expressed in human retinal endothelial cells. Oxidative stress and apoptosis are inversely proportional to the extent of promoter methylation, suggesting that methylation loss might be due to oxidative stress-induced DNA damage. PMID:24885710

Glucagon Like Peptide-1 Receptor Expression in the Human Thyroid Gland

PubMed Central

Gier, Belinda; Butler, Peter C.; Lai, Chi K.; Kirakossian, David; DeNicola, Matthew M.

2012-01-01

Background: Glucagon like peptide-1 (GLP-1) mimetic therapy induces medullary thyroid neoplasia in rodents. We sought to establish whether C cells in human medullary thyroid carcinoma, C cell hyperplasia, and normal human thyroid express the GLP-1 receptor. Methods: Thyroid tissue samples with medullary thyroid carcinoma (n = 12), C cell hyperplasia (n = 9), papillary thyroid carcinoma (n = 17), and normal human thyroid (n = 15) were evaluated by immunofluorescence for expression of calcitonin and GLP-1 receptors. Results: Coincident immunoreactivity for calcitonin and GLP-1 receptor was consistently observed in both medullary thyroid carcinoma and C cell hyperplasia. GLP-1 receptor immunoreactivity was also detected in 18% of papillary thyroid carcinoma (three of 17 cases). Within normal human thyroid tissue, GLP-1 receptor immunoreactivity was found in five of 15 of the examined cases in about 35% of the total C cells assessed. Conclusions: In humans, neoplastic and hyperplastic lesions of thyroid C cells express the GLP-1 receptor. GLP-1 receptor expression is detected in 18% papillary thyroid carcinomas and in C cells in 33% of control thyroid lobes. The consequence of long-term pharmacologically increased GLP-1 signaling on these GLP-1 receptor-expressing cells in the thyroid gland in humans remains unknown, but appropriately powered prospective studies to exclude an increase in medullary or papillary carcinomas of the thyroid are warranted. PMID:22031513

Expression of cholinergic, insulin, vitamin D receptors and GLUT 3 in the brainstem of streptozotocin induced diabetic rats: effect of treatment with vitamin D₃.

PubMed

Peeyush Kumar, T; Paul, Jes; Antony, Sherin; Paulose, C S

2011-11-01

Complications arising from diabetes mellitus include cognitive deficits, neurophysiological and structural changes in the brain. The current study investigated the expression of cholinergic, insulin, Vitamin D receptor and GLUT 3 in the brainstem of streptozotocin-induced diabetic rats. Radioreceptor binding assays and gene expression were done in the brainstem of male Wistar rats. Our results showed that B(max) of total muscarinic, muscarinic M3 receptors was increased and muscarinic M1 receptor was decreased in diabetic rats compared to control. A significant increase in gene expression of muscarinic M3, α7 nicotinic acetylcholine, insulin, Vitamin D₃ receptors, acetylcholine esterase, choline acetyl transferase and GLUT 3 were observed in the brainstem of diabetic rats. Immunohistochemistry studies of muscarinic M1, M3 and α7 nicotinic acetylcholine receptors confirmed the gene expression at protein level. Vitamin D₃ and insulin treatment reversed diabetes-induced alterations to near control. This study provides an evidence that diabetes can alter the expression of cholinergic, insulin, Vitamin D receptors and GLUT 3 in brainstem. We found that Vitamin D₃ treatment could modulate the Vitamin D receptors and plays a pivotal role in maintaining the glucose transport and expressional level of cholinergic receptors in the brainstem of diabetic rats. Thus, our results suggest a therapeutic role of Vitamin D₃ in managing neurological disorders associated with diabetes.

Study of low-density lipoprotein receptor regulation by oral (steroid) contraceptives: desogestrel, levonorgestrel and ethinyl estradiol in JEG-3 cell line and placental tissue.

PubMed

Ramakrishnan, Gopalakrishnan; Rana, Anita; Das, Chandana; Chandra, Nimai Chand

2007-10-01

The aim of this study was to compare in vitro the role of two oral contraceptives, desogestrel (a less androgenic derivative of levonorgestrel) and levonorgestrel--alone and in combination with ethinyl estradiol--on low-density lipoprotein (LDL) receptor regulation by assessing receptor protein expression and functional effectiveness. Placental tissue and cultured placental cells (JEG-3) were used to study the expression and endocytotic activity of LDL receptor protein. The expression of the receptor was assessed by immunocytochemistry and immunoblot assays with and without contraceptive challenge. Functioning activity of LDL receptor was studied by measuring the rate of uptake of LDL by placental cells. Quantification of LDL was based on the total cholesterol content of the lipoprotein. A combination of desogestrel (20 ng/mL of incubation medium) and ethinyl estradiol (10 ng/mL of incubation medium) maintained the LDL receptor at high level of expression and functioning mode. In contrast, the double-blind preparation of levonorgestrel (20 ng/mL) and ethinyl estradiol (10 ng/mL) had shown much lower expression as well as receptor-mediated LDL uptake. The concentration of contraceptives used in this study was similar to the prevailing concentration of oral contraceptives in clinical use. Higher expression of LDL receptor and enhanced rate of LDL uptake by the receptor protein projects the possibility that there might be less atherosclerosis-related disorders from the combination of desogestrol and ethinyl estradiol.

Utilization of the Tango beta-arrestin recruitment technology for cell-based EDG receptor assay development and interrogation.

PubMed

Wetter, Justin A; Revankar, Chetana; Hanson, Bonnie J

2009-10-01

Cellular assay development for the endothelial differentiation gene (EDG) family of G-protein-coupled receptors (GPCRs) and related lysophospholipid (LP) receptors is complicated by endogenous receptor expression and divergent receptor signaling. Endogenously expressed LP receptors exist in most tissue culture cell lines. These LP receptors, along with other endogenously expressed GPCRs, contribute to off-target signaling that can complicate interpretation of second-messenger-based cellular assay results. These receptors also activate a diverse and divergent set of cellular signaling pathways, necessitating the use of a variety of assay formats with mismatched procedures and functional readouts. This complicates examination and comparison of these receptors across the entire family. The Tango technology uses the conserved beta-arrestin-dependent receptor deactivation process to allow interrogation of the EDG and related receptors with a single functional assay. This method also isolates the target receptor signal, allowing the use of tissue culture cell lines regardless of their endogenous receptor expression. The authors describe the use of this technique to build cell-based receptor-specific assays for all 8 members of the EDG receptor family as well as the related LPA receptors GPR23, GPR92, and GPR87. In addition, they demonstrate the value of this technology for identification and investigation of functionally selective receptor compounds as demonstrated by the immunosuppressive compound FtY720-P and its action at the EDG(1) and EDG(3) receptors.

Expression of transforming growth factor alpha and epidermal growth factor receptor messenger RNA in neoplastic and nonneoplastic human kidney tissue.

PubMed

Mydlo, J H; Michaeli, J; Cordon-Cardo, C; Goldenberg, A S; Heston, W D; Fair, W R

1989-06-15

Using Northern blot analysis, we have demonstrated that mRNA for transforming growth factor alpha (TGF-alpha) was expressed in five malignant kidney tissue specimens but was not detected in their autologous nonneoplastic homologues. In addition, the expression of epidermal growth factor (EGF) receptor mRNA in these malignant tissues was 2- to 3-fold greater than in nontransformed tissues. In two cases examined using immunohistochemistry, we were able to correlate the increased expression of the mRNA with an increase in protein expression. Since TGF-alpha is known to bind to the EGF receptor, the finding of an increased expression of both TGF-alpha and EGF receptor mRNA in kidney tumor tissue suggests that interaction between TGF-alpha and the EGF receptor may play a role in promoting transformation and/or proliferation of kidney neoplasms, perhaps by an autocrine mechanism.

Variant ionotropic receptors are expressed in olfactory sensory neurons of coeloconic sensilla on the antenna of the desert locust (Schistocerca gregaria).

PubMed

Guo, Mei; Krieger, Jürgen; Große-Wilde, Ewald; Mißbach, Christine; Zhang, Long; Breer, Heinz

2013-01-01

The behaviour of the desert locust, Schistocera gregaria, is largely directed by volatile olfactory cues. The relevant odorants are detected by specialized antennal sensory neurons which project their sensory dendrites into hair-like structures, the sensilla. Generally, the responsiveness of the antennal chemosensory cells is determined by specific receptors which may be either odorant receptors (ORs) or variant ionotropic receptors (IRs). Previously, we demonstrated that in locust the co-receptor for ORs (ORco) is only expressed in cells of sensilla basiconica and sensilla trichodea, suggesting that cells in sensilla coeloconica may express different types of chemosensory receptors. In this study, we have identified the genes of S. gregaria which encode homologues of co-receptors for the variant ionotropic receptors, the subtypes IR8a and IR25a. It was found that both subtypes, SgreIR8a and SgreIR25a, are expressed in the antennae of all five nymphal stages and in adults. Attempts to assign the relevant cell types by means of in situ hybridization revealed that SgreIR8a and SgreIR25a are expressed in cells of sensilla coeloconica. Double fluorescence in situ hybridization experiments disclosed that the two IR-subtypes are co-expressed in some cells of this sensillum type. Expression of SgreIR25a was also found in some of the sensilla chaetica, however, neither SgreIR25a nor SgreIR8a was found to be expressed in sensilla basiconica and sensilla trichodea. This observation was substantiated by the results of double FISH experiments demonstrating that cells expressing SgreIR8a or SgreIR25a do not express ORco. These results support the notion that the antenna of the desert locust employs two different populations of OSNs to sense odors; cells which express IRs in sensilla coeloconica and cells which express ORs in sensilla basiconica and sensilla trichodea.

Cloning, phylogeny, and regional expression of a Y5 receptor mRNA in the brain of the sea lamprey (Petromyzon marinus).

PubMed

Pérez-Fernández, Juan; Megías, Manuel; Pombal, Manuel A

2014-04-01

The NPY receptors known as Y receptors are classified into three subfamilies, Y1, Y2, and Y5, and are involved in different physiological functions. The Y5 receptor is the only member of the Y5 subfamily, and it is present in all vertebrate groups, except for teleosts. Both molecular and pharmacological studies show that Y5 receptor is highly conserved during vertebrate evolution. Furthermore, this receptor is widely expressed in the mammalian brain, including the hypothalamus, where it is thought to take part in feeding and homeostasis regulation. Lampreys belong to the agnathan lineage, and they are thought to have branched out between the two whole-genome duplications that occurred in vertebrates. Therefore, they are in a key position for studies on the evolution of gene families in vertebrates. Here we report the cloning, phylogeny, and brain expression pattern of the sea lamprey Y5 receptor. In phylogenetic studies, the lamprey Y5 receptor clusters in a basal position, together with Y5 receptors of other vertebrates. The mRNA of this receptor is broadly expressed in the lamprey brain, being especially abundant in hypothalamic areas. Its expression pattern is roughly similar to that reported for other vertebrates and parallels the expression pattern of the Y1 receptor subtype previously described by our group, as it occurs in mammals. Altogether, these results confirm that a Y5 receptor is present in lampreys, thus being highly conserved during the evolution of vertebrates, and suggest that it is involved in many brain functions, the only known exception being teleosts. Copyright © 2013 Wiley Periodicals, Inc.

The effect of melatonin from slow-release implants on basic and TLR-4-mediated gene expression of inflammatory cytokines and their receptors in the choroid plexus in ewes.

PubMed

Kowalewska, M; Herman, A P; Szczepkowska, A; Skipor, J

2017-08-01

The present study concerns the effect of melatonin from slow-release implants on the expression of genes coding interleukin-1β (Il1B), inerleukin-6 (Il6), tumour necrosis factor α (Tnf) and their receptors: IL-1 receptor type I (Il1r1) and type II (Il1r2), IL-6 receptor (Il6r) and signal transducer (Il6st), TNFα receptor type I (Tnfrsf1a) and II (Tnfrsf1b) and retinoid-related orphan receptor α (RorA) and Rev.-erbα in the ovine choroid plexus (CP) under basal and lipopolysaccharide (LPS)-challenged conditions. Studies were performed on four groups: 1) sham-implanted and placebo-treated, 2) melatonin-implanted (Melovine, 18mg) and placebo-treated, 3) sham-implanted and LPS-treated (400ng/kg of body weight) and 4) melatonin-implanted and LPS-treated. Under basal conditions, we observed weak expression of Tnf, low expression of Il1B, Il6 and Il1r2 and intermediate expression of other cytokines receptors. LPS treatment induced (P≤0.05) expression in all cytokines and their receptors, except Il6r 3h after the administration. Melatonin attenuated (P≤0.05) LPS-induced up-regulation of Il6 but had no effect on other cytokines and their receptors and up-regulated (P≤0.05) Rev.-erbα expression under basal conditions. This indicates that melatonin from slow-release implants suppresses TLR4-mediated Il6 expression in the ovine CP via a mechanism likely involving clock genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Role of a Ubiquitously Expressed Receptor in the Vertebrate Olfactory System

PubMed Central

DeMaria, Shannon; Berke, Allison P.; Van Name, Eric; Heravian, Anisa; Ferreira, Todd

2013-01-01

Odorant cues are recognized by receptors expressed on olfactory sensory neurons, the primary sensory neurons of the olfactory epithelium. Odorant receptors typically obey the “one receptor, one neuron” rule, in which the receptive field of the olfactory neuron is determined by the singular odorant receptor that it expresses. Odor-evoked receptor activity across the population of olfactory neurons is then interpreted by the brain to identify the molecular nature of the odorant stimulus. In the present study, we characterized the properties of a C family G-protein-coupled receptor that, unlike most other odorant receptors, is expressed in a large population of microvillous sensory neurons in the zebrafish olfactory epithelium and the mouse vomeronasal organ. We found that this receptor, OlfCc1 in zebrafish and its murine ortholog Vmn2r1, is a calcium-dependent, low-sensitivity receptor specific for the hydrophobic amino acids isoleucine, leucine, and valine. Loss-of-function experiments in zebrafish embryos demonstrate that OlfCc1 is required for olfactory responses to a diverse mixture of polar, nonpolar, acidic, and basic amino acids. OlfCc1 was also found to promote localization of other OlfC receptor family members to the plasma membrane in heterologous cells. Together, these results suggest that the broadly expressed OlfCc1 is required for amino acid detection by the olfactory system and suggest that it plays a role in the function and/or intracellular trafficking of other olfactory and vomeronasal receptors with which it is coexpressed. PMID:24048853

Novel receptor-targeted contrast agents for optical imaging of tumors

NASA Astrophysics Data System (ADS)

Becker, Andreas; Hessenius, Carsten; Bhargava, Sarah; Ebert, Bernd; Sukowski, Uwe; Rinneberg, Herbert H.; Wiedenmann, Bertram; Semmler, Wolfhard; Licha, Kai

2000-04-01

Many gastroenteropancreatic tumors express receptors for somatostatin (SST) and/or vasoactive intestinal peptide (VIP). These receptors can be used as molecular targets for the delivery of contrast agents for tumor diagnostics. We have synthesized conjugates consisting of a cyanine dye and an SST analogue or VIP for use as contrast agents in optical imaging. Receptor binding and internalization of these compounds were examined with optical methods in transfected RIN38 tumor cells expressing the SST2 receptor or a GFP- labeled VIP (VPAC1) receptor. Furthermore, biodistribution of the conjugates was examined by laser-induced fluorescence imaging in nude mice bearing SST2 or VPAC1 receptor- expressing tumors. After incubation of RIN38 SSTR2 cells in the presence of 100 nM indotricarbocyanine-SST analogue, cell-associated fluorescence increased, whereas no increase was observed when receptor-medicated endocytosis was inhibited. Indodicarbocyanine-VIP accumulated in RIN38 VPAC1 cells and co-localization with the GFP-labeled VPAC1 receptor was observed. After injection of indotricarbocyanine-SST analogue into tumor-bearing nude mice, SST2 receptor-positive tumors could be visualized for a time period from 10 min to at least 48 h. After application of indodicarbocyanine-VIP, a fluorescence signal in VIP1 receptor-expressing tumors was only detected during the first hour. We conclude that cyanine dye-labeled VIP and SST analogue are novel, targeted contrast agents for the optical imaging of tumors expressing the relevant receptor.

The expression of Fc and complement receptors in young, adult and aged mice.

PubMed Central

Vĕtvicka, V; Fornůsek, L; Zídková, J

1985-01-01

Age-dependent changes in the expression of Fc receptors (FcR) for different isotypes of immunoglobulins and receptors for C3b, C5b and C3bi fragments of complement on the membranes of peritoneal macrophages were studied with mice of different ages. An age-related increase in expression of Fc receptors for IgM, IgE, IgA, IgG2b and IgG3, and a decrease in the expression of Fc receptors for IgG1 was observed. The expression of FcR on macrophages of donors of different ages corresponded with Fc-receptor mediated phagocytosis. The highest number of C3b-binding macrophages was found in aged mice, in contrast to low numbers of C3bi-binding macrophages at this age. The percentage of C5b-binding macrophages was lowest in adult animals. We also observed effective inhibition of binding of the C3b component of complement by preincubation of macrophages with aggregated IgG and vice versa. These observations suggest that fluctuation in expression of Fc but not C receptors may be important to the generalized changes that occur in macrophage function during development and ageing. PMID:2931351

Type I Interferon Receptor Expression in Human Pancreatic and Periampullary Cancer Tissue.

PubMed

Booy, Stephanie; Hofland, Leo J; Waaijers, A Marlijn; Croze, Ed; van Koetsveld, Peter M; de Vogel, Lisette; Biermann, Katharina; van Eijck, Casper H J

2015-01-01

Interferons (IFNs) have several anticancer mechanisms. A number of clinical trials have been conducted regarding adjuvant IFN-α therapy in pancreatic cancer. Type I IFNs exert their effect via the type I IFN receptor (IFNAR-1, IFNAR-2c). The aims of the present study were to determine the type I IFN receptor expression in pancreatic and periampullary cancer tissues and to study its relation with clinicopathological factors. Receptor expression was determined by immunohistochemistry in paraffin-embedded cancer tissue of 47 pancreatic and 54 periampullary cancer patients. The results demonstrated that 91.5% of the pancreatic tumors and 88.9% of the periampullary tumors showed expression of IFNAR-1, of which 23.4% and 13.0% were strongly positive, respectively. Regarding IFNAR-2c expression, 68.1% of the pancreatic tumors and 68.5% of the periampullary tumors were positive, of which 4.3% of the pancreatic tumors and none of the periampullary tumors had a strong expression. No statistically significant associations were found between type I IFN receptor expression and clinicopathological factors or survival. Type I IFN receptors are expressed in pancreatic and periampullary cancer tissues although with great intertumoral and intratumoral variability. A small proportion of both tumors showed a strong expression of the IFNAR-1; only a very small percentage of the pancreatic tumors showed strong expression of the IFNAR-2c.

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

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

PubMed

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

2014-03-01

Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. Copyright © 2014 Elsevier B.V. All rights reserved.

Cloning and functional characterization of the DA2 receptor gene in Chinese mitten crab (Eriocheir sinensis)

PubMed Central

Xu, Min-jie; Zhang, Cong; Yang, Zhigang

2018-01-01

Dopamine (DA) plays a modulatory role in numerous physiological processes such as light adaptation and food intake, and exerts these functions through DA receptors (DARs). This study presents, for the first time, isolation and characterization of the dopamine receptor 2(DA2 receptor) cDNA from the intestinal tissue of Eriocheir sinensis, an economically important freshwater aquaculture species in China. The DA2 receptor cDNA sequence, which was obtained by rapid amplification of cDNA ends, is 2369bp long, encode peptide of 589 amino acid, and is highly homologous to related sequences in crustaceans. Analysis of the deduced amino acid sequence and the structure of the DA2 indicated that this receptor is a member of the family of G protein-coupled receptors (GPCRs), as it contains seven transmembrane domains and other common signatures of GPCRs. RT-PCR showed that the expression of the DA2 receptor gene was distributed in various tissues, and high expression levels were observed in the cranial ganglia and the thoracic ganglia. Further study of the effect of photoperiod on DA2 expression showed that constant darkness induced a significant increase in DA2 expression in the cranial ganglia. Finally, analysis of DA2 receptor expression under different feeding statuses showed that there was significantly greater expression in the hepatopancreas and intestines after feeding than before feeding, but there were no differences in expression between the before feeding and during feeding periods in either tissue. Our results indicate that the DA2 receptor structurally belongs to the family of G protein-coupled receptors, and that the cranial ganglia are the main tissues in which the DA2 receptor participates in light adaptation during dark hours. In addition, the DA2 receptor in E. sinensis may be involved in the physiological regulation of the hepatopancreas and digestive tract after the ingestion of food. This study provides a foundation for further exploration of the light adaptation and digestive functions of the DA2 receptor in decapods. PMID:29554147

Expression of PACAP and PAC1 Receptor in Normal Human Thyroid Gland and in Thyroid Papillary Carcinoma.

PubMed

Bardosi, Sebastian; Bardosi, Attila; Nagy, Zsuzsanna; Reglodi, Dora

2016-10-01

Pituitary adenylate cyclase activating polypeptide (PACAP) belongs to the vasoactive intestinal peptide-secretin-glucagon peptide family, isolated first from ovine hypothalamus. The diverse physiological effects of PACAP are known mainly from animal experiments, including several actions in endocrine glands. Alteration of PACAP expression has been shown in several tumors, but changes in expression of PACAP and its specific PAC1 receptor in human thyroid gland pathologies have not yet been investigated. Therefore, the aim of the present study was to investigate expression of PACAP and its PAC1 receptor in human thyroid papillary carcinoma, the most common endocrine malignant tumor. PACAP and PAC1 receptor expressions were investigated from thyroid gland samples of patients with papillary carcinomas. The staining intensity of follicular epithelial cells and thyroid colloid of tumor tissue was compared to that of tumor-free tissue in the same thyroid glands in a semi-quantitative way. Our results reveal that both PACAP(-like) and PAC1 receptor(-like) immunoreactivities are altered in papillary carcinoma. Stronger PACAP immunoreactivity was observed in active follicles. Colloidal PACAP immunostaining was either lacking or very weak, and more tumorous cells displayed strong apical immunoreactivity. Regarding PAC1 receptor, cells of the normal thyroid tissue showed strong granular expression, which was lacking in the tumor cells. The cytoplasm of tumor cells displayed weak, minimal staining, while in a few tumor cells we observed strong PAC1 receptor expression. This pattern was similar to that observed in the PACAP expression, but fewer in number. In summary, we showed alteration of PACAP and PAC1 receptor expression in human thyroid papillary carcinoma, indicating that PACAP regulation is disturbed in tumorous tissue of the thyroid gland. The exact role of PACAP in thyroid tumor growth should be further explored.

Quantitative RT-PCR analysis of estrogen receptor gene expression in laser microdissected prostate cancer tissue.

PubMed

Walton, Thomas J; Li, Geng; McCulloch, Thomas A; Seth, Rashmi; Powe, Desmond G; Bishop, Michael C; Rees, Robert C

2009-06-01

Real-time quantitative RT-PCR analysis of laser microdissected tissue is considered the most accurate technique for determining tissue gene expression. The discovery of estrogen receptor beta (ERbeta) has focussed renewed interest on the role of estrogen receptors in prostate cancer, yet few studies have utilized the technique to analyze estrogen receptor gene expression in prostate cancer. Fresh tissue was obtained from 11 radical prostatectomy specimens and from 6 patients with benign prostate hyperplasia. Pure populations of benign and malignant prostate epithelium were laser microdissected, followed by RNA isolation and electrophoresis. Quantitative RT-PCR was performed using primers for androgen receptor (AR), estrogen receptor beta (ERbeta), estrogen receptor alpha (ERalpha), progesterone receptor (PGR) and prostate specific antigen (PSA), with normalization to two housekeeping genes. Differences in gene expression were analyzed using the Mann-Whitney U-test. Correlation coefficients were analyzed using Spearman's test. Significant positive correlations were seen when AR and AR-dependent PSA, and ERalpha and ERalpha-dependent PGR were compared, indicating a representative population of RNA transcripts. ERbeta gene expression was significantly over-expressed in the cancer group compared with benign controls (P < 0.01). In contrast, PGR expression was significantly down-regulated in the cancer group (P < 0.05). There were no significant differences in AR, ERalpha or PSA expression between the groups. This study represents the first to show an upregulation of ERbeta gene expression in laser microdissected prostate cancer specimens. In concert with recent studies the findings suggest differential production of ERbeta splice variants, which may play important roles in the genesis of prostate cancer. (c) 2009 Wiley-Liss, Inc.

Pharmacological similarities between native brain and heterologously expressed α4β2 nicotinic receptors

PubMed Central

Shafaee, Navid; Houng, McCann; Truong, Anthony; Viseshakul, Nareerat; Figl, Antonio; Sandhu, Sumandeep; Forsayeth, John R; Dwoskin, Linda P; Crooks, Peter A; Cohen, Bruce N

1999-01-01

We studied the pharmacological properties of native rat brain and heterologously expressed rat α4β2 nicotinic receptors immunoprecipitated onto a fixed substrate with the anti-α4 antibody mAb 299.Immunodepletion with the anti-β2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained β2.The association and dissociation rate constants for 30 pM ±[3H]-epibatidine binding to α4β2 receptors expressed in oocytes were 0.02±0.01 and 0.03±0.01 min−1 (±standard error, degrees of freedom=7–8) at 20–23°C.The Hill coefficients for ±[3H]epibatidine binding to the native brain, α4β2 receptors expressed in oocytes, and α4β2 receptors expressed in CV-1 cells (using recombinant adenovirus) were 0.69–0.70 suggesting a heterogeneous receptor population. Fits of the ±[3H]-epibatidine concentration-binding data to a two-site model gave KD s of 8–30 and 560–1,200 pM. The high-affinity sites comprised 73–74% of the native brain and oocyte α4β2 receptor population, 85% of the CV-1 α4β2 receptor population.The expression of rat α4β2 receptors in CV-1 cells using vaccinia viral infection-transfection resulted in a more homogeneous receptor population (Hill coefficient of 1.0±0.2). Fits of the ±[3H]-epibatidine binding data to a single-site model gave a KD of 40±3 pM.DHβE (IC50=260–470 nM) and the novel nicotine analogue NDNI (IC50=7–10 μM) inhibited 30 pM±[3H]-epibatidine binding to the native brain and heterologously expressed α4β2 receptors equally well.The results show that α4β2-containing nicotinic receptors in the rat brain and heterologously expressed rat α4β2 receptors have similar affinities for ±[3H]-epibatidine, DHβE, and NDNI. PMID:10578144

Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling.

PubMed

Tong, Dandan; Liang, Ya-Nan; Stepanova, A A; Liu, Yu; Li, Xiaobo; Wang, Letian; Zhang, Fengmin; Vasilyeva, N V

2017-02-01

Recent research indicates that the C-terminal Eps15 homology domain 1 is associated with epithelial growth factor receptor-mediated endocytosis recycling in non-small-cell lung cancer. The aim of this study was to determine the clinical significance of Eps15 homology domain 1 gene expression in relation to phosphorylation of epithelial growth factor receptor expression in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Eps15 homology domain 1, RAB11FIP3, and phosphorylation of epithelial growth factor receptor expression via immunohistochemistry. The clinical significance was assessed via a multivariate Cox regression analysis, Kaplan-Meier curves, and the log-rank test. Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor were upregulated in 60.46% (185/306) and 53.92% (165/306) of tumor tissues, respectively, as assessed by immunohistochemistry. The statistical correlation analysis indicated that Eps15 homology domain 1 overexpression was positively correlated with the increases in phosphorylation of epithelial growth factor receptor ( r = 0.242, p < 0.001) and RAB11FIP3 ( r = 0.165, p = 0.005) expression. The multivariate Cox proportional hazard model analysis demonstrated that the expression of Eps15 homology domain 1 alone is a significant prognostic marker of breast cancer for the overall survival in the total, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. However, the use of combined expression of Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor markers is more effective for the disease-free survival in the overall population, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. Moreover, the combined markers are also significant prognostic markers of breast cancer in the human epidermal growth factor receptor 2 (+), estrogen receptor (+), and estrogen receptor (-) groups. Eps15 homology domain 1 has a tumor suppressor function, and the combined marker of Eps15 homology domain 1/phosphorylation of epithelial growth factor receptor expression was identified as a better prognostic marker in breast cancer diagnosis. Furthermore, RAB11FIP3 combines with Eps15 homology domain 1 to promote the endocytosis recycling of phosphorylation of epithelial growth factor receptor.

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

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

Nonogaki, Katsunori; Nozue, Kana; Oka, Yoshitomo

2006-12-29

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

Expression of the tachykinin receptor mRNAs in healthy human colon.

PubMed

Jaafari, Nadia; Hua, Guoqiang; Adélaïde, José; Julé, Yvon; Imbert, Jean

2008-12-03

Tachykinins are a family of neuropeptides, involved in a variety of physiological and pathological processes occurring in the gastrointestinal tract. They act via three distinct types of receptors, tachykinin NK(1), NK(2), and NK(3) receptors, which belong to the family of G protein-coupled receptors. The aim of the present study was to characterize, for the first time in the healthy human colon, the TACR(1), TACR(2) and TACR(3) mRNAs encoding the three different tachykinin receptors and to measure their relative expression by quantitative reverse transcription-PCR assay. Our results confirm the broad distribution of the tachykinin receptors but evidenced significant differences in the expression level of their respective mRNAs. A higher expression level of the TACR2 mRNA alpha isoform, the gene encoding the functional tachykinin NK(2) receptor, was observed in comparison to TACR1 and TACR3 mRNAs genes encoding for NK(1) and NK(3) receptors respectively. The prevalence of the TACR2 mRNA alpha isoform strongly suggests a major involvement of tachykinin NK(2) receptor in the regulation of human colonic functions.

Expression of estrogen and progesterone receptors in astrocytomas: a literature review

PubMed Central

Tavares, Cléciton Braga; Gomes-Braga, Francisca das Chagas Sheyla Almeida; Costa-Silva, Danylo Rafhael; Escórcio-Dourado, Carla Solange; Borges, Umbelina Soares; Conde, Airton Mendes; da Conceição Barros-Oliveira, Maria; Sousa, Emerson Brandão; da Rocha Barros, Lorena; Martins, Luana Mota; Facina, Gil; da-Silva, Benedito Borges

2016-01-01

Gliomas are the most common type of primary central nervous system neoplasm. Astrocytomas are the most prevalent type of glioma and these tumors may be influenced by sex steroid hormones. A literature review for the presence of estrogen and progesterone receptors in astrocytomas was conducted in the PubMed database using the following MeSH terms: “estrogen receptor beta” OR “estrogen receptor alpha” OR “estrogen receptor antagonists” OR “progesterone receptors” OR “astrocytoma” OR “glioma” OR “glioblastoma”. Among the 111 articles identified, 13 studies met our inclusion criteria. The majority of reports showed the presence of estrogen and progesterone receptors in astrocytomas. Overall, higher tumor grades were associated with decreased estrogen receptor expression and increased progesterone receptor expression. PMID:27626480

Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

PubMed

Osgood, Doreen; Miller, Miles C; Messier, Arthur A; Gonzalez, Liliana; Silverberg, Gerald D

2017-09-01

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aβ) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aβ transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aβ receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aβ accumulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Immunobiological Aspects of erbB Receptors in Breast Cancer

DTIC Science & Technology

2000-08-01

receptor . The proliferation of cells expressing these chimeric receptors was EGF-dependent, and cells expressing EGFR/Y882F chimeric receptors were...determine Cells were washed twice with cold phosphate-buffered saline which cellular substrates couple with the receptor complex. (PBS) and lysed with 1...turnover, receptor proteins suggests that these substrates are properly lo- and cellular transformation in NEN757 cells (Qian et al., cated for

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

PubMed Central

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M.; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E.; Morales, Miguel A.; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity. PMID:28744222

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion.

PubMed

Valle-Leija, Pablo; Cancino-Rodezno, Angeles; Sánchez-Tafolla, Berardo M; Arias, Erwin; Elinos, Diana; Feria, Jessica; Zetina, María E; Morales, Miguel A; Cifuentes, Fredy

2017-01-01

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.

Sequence genomic organization and expression of two channel catfish Ictalurus punctatus Ghrelin receptors

USDA-ARS?s Scientific Manuscript database

Two ghrelin receptor (GHS-R) genes were isolated from channel catfish tissue and a bacterial artificial chromosome (BAC) library. The two receptors were characterized by determining tissue distribution, ontogeny of receptor mRNA expression, and effects of exogenous homologous ghrelin administration ...

Developmentally Regulated Expression of the Nerve Growth Factor Receptor Gene in the Periphery and Brain

NASA Astrophysics Data System (ADS)

Buck, C. R.; Martinez, Humberto J.; Black, Ira B.; Chao, Moses V.

1987-05-01

Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

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

Phrenic motoneuron expression of serotonergic and glutamatergic receptors following upper cervical spinal cord injury

PubMed Central

Mantilla, Carlos B.; Bailey, Jeffrey P.; Zhan, Wen-Zhi; Sieck, Gary C.

2012-01-01

Following cervical spinal cord injury at C2 (SH hemisection model) there is progressive recovery of phrenic activity. Neuroplasticity in the postsynaptic expression of neurotransmitter receptors may contribute to functional recovery. Phrenic motoneurons express multiple serotonergic (5-HTR) and glutamatergic (GluR) receptors, but the timing and possible role of these different neurotransmitter receptor subtypes in the neuroplasticity following SH are not clear. The current study was designed to test the hypothesis that there is an increased expression of serotonergic and glutamatergic neurotransmitter receptors within phrenic motoneurons after SH. In adult male rats, phrenic motoneurons were labeled retrogradely by intrapleural injection of Alexa 488-conjugated cholera toxin B. In thin (10 μm) frozen sections of the spinal cord, fluorescently-labeled phrenic motoneurons were visualized for laser capture microdissection (LCM). Using quantitative real-time RT-PCR in LCM samples, the time course of changes in 5-HTR and GluR mRNA expression was determined in phrenic motoneurons up to 21 days post-SH. Expression of 5-HTR subtypes 1b, 2a and 2c and GluR subtypes AMPA, NMDA, mGluR1 and mGluR5 was evident in phrenic motoneurons from control and SH rats. Phrenic motoneuron expression of 5-HTR2a increased ~8-fold (relative to control) at 14 days post-SH, whereas NMDA expression increased ~16-fold by 21-days post-SH. There were no other significant changes in receptor expression at any time post-SH. This is the first study to systematically document changes in motoneuron expression of multiple neurotransmitter receptors involved in regulation of motoneuron excitability. By providing information on the neuroplasticity of receptors expressed in a motoneuron pool that is inactivated by a higher-level spinal cord injury, appropriate pharmacological targets can be identified to alter motoneuron excitability. PMID:22227062

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium.

PubMed

Shabir, Saqib; Cross, William; Kirkwood, Lisa A; Pearson, Joanna F; Appleby, Peter A; Walker, Dawn; Eardley, Ian; Southgate, Jennifer

2013-08-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca²⁺. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca²⁺ and in a scratch repair assay. The results confirmed the functional expression of P2Y₄ receptors and excluded nonexpressed receptors/channels (P2X₁, P2X₃, P2X₆, P2Y₆, P2Y₁₁, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X₂, P2X₄, P2Y₁, P2Y₂, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca²⁺ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting.

Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

PubMed Central

Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

2013-01-01

In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

A Subset of Mouse Colonic Goblet Cells Expresses the Bitter Taste Receptor Tas2r131

PubMed Central

Prandi, Simone; Bromke, Marta; Hübner, Sandra; Voigt, Anja; Boehm, Ulrich; Meyerhof, Wolfgang; Behrens, Maik

2013-01-01

The concept that gut nutrient sensing involves taste receptors has been fueled by recent reports associating the expression of taste receptors and taste-associated signaling molecules in the gut and in gut-derived cell lines with physiological responses induced by known taste stimuli. However, for bitter taste receptors (Tas2rs), direct evidence for their functional role in gut physiology is scarce and their cellular expression pattern remained unknown. We therefore investigated Tas2r expression in mice. RT-PCR experiments assessed the presence of mRNA for Tas2rs and taste signaling molecules in the gut. A gene-targeted mouse strain was established to visualize and identify cell types expressing the bitter receptor Tas2r131. Messenger RNA for various Tas2rs and taste signaling molecules were detected by RT-PCR in the gut. Using our knock-in mouse strain we demonstrate that a subset of colonic goblet cells express Tas2r131. Cells that express this receptor are absent in the upper gut and do not correspond to enteroendocrine and brush cells. Expression in colonic goblet cells is consistent with a role of Tas2rs in defense mechanisms against potentially harmful xenobiotics. PMID:24367558

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

PubMed

Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

Constitutive Activity among Orphan Class-A G Protein Coupled Receptors

PubMed Central

Martin, Adam L.; Steurer, Michael A.; Aronstam, Robert S.

2015-01-01

The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

Activation and inhibition of mouse muscle and neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

PubMed

Papke, Roger L; Wecker, Lynn; Stitzel, Jerry A

2010-05-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric alpha7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-beta-erythroidine as selective antagonists in mouse models of alpha3beta4 and alpha4beta2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal alpha and beta subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse alpha5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse alpha4beta2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity.

Activation and Inhibition of Mouse Muscle and Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes

PubMed Central

Wecker, Lynn; Stitzel, Jerry A.

2010-01-01

Transgenic mouse models with nicotinic acetylcholine receptor (nAChR) knockouts and knockins have provided important insights into the molecular substrates of addiction and disease. However, most studies of heterologously expressed neuronal nAChR have used clones obtained from other species, usually human or rat. In this work, we use mouse clones expressed in Xenopus oocytes to provide a relatively comprehensive characterization of the three primary classes of nAChR: muscle-type receptors, heteromeric neuronal receptors, and homomeric α7-type receptors. We evaluated the activation of these receptor subtypes with acetylcholine and cytisine-related compounds, including varenicline. We also characterized the activity of classic nAChR antagonists, confirming the utility of mecamylamine and dihydro-β-erythroidine as selective antagonists in mouse models of α3β4 and α4β2 receptors, respectively. We also conducted an in-depth analysis of decamethonium and hexamethonium on muscle and neuronal receptor subtypes. Our data indicate that, as with receptors cloned from other species, pairwise expression of neuronal α and β subunits in oocytes generates heterogeneous populations of receptors, most likely caused by variations in subunit stoichiometry. Coexpression of the mouse α5 subunit had varying effects, depending on the other subunits expressed. The properties of cytisine-related compounds are similar for mouse, rat, and human nAChR, except that varenicline produced greater residual inhibition of mouse α4β2 receptors than with human receptors. We confirm that decamethonium is a partial agonist, selective for muscle-type receptors, but also note that it is a nondepolarizing antagonist for neuronal-type receptors. Hexamethonium was a relatively nonselective antagonist with mixed competitive and noncompetitive activity. PMID:20100906

TRAIL Death Receptor-4, Decoy Receptor-1 and Decoy Receptor-2 Expression on CD8+ T Cells Correlate with the Disease Severity in Patients with Rheumatoid Arthritis

PubMed Central

2010-01-01

Background Rheumatoid Arthritis (RA) is a chronic autoimmune inflammatory disorder. Although the pathogenesis of disease is unclear, it is well known that T cells play a major role in both development and perpetuation of RA through activating macrophages and B cells. Since the lack of TNF-Related Apoptosis Inducing Ligand (TRAIL) expression resulted in defective thymocyte apoptosis leading to an autoimmune disease, we explored evidence for alterations in TRAIL/TRAIL receptor expression on peripheral T lymphocytes in the molecular mechanism of RA development. Methods The expression of TRAIL/TRAIL receptors on T cells in 20 RA patients and 12 control individuals were analyzed using flow cytometry. The correlation of TRAIL and its receptor expression profile was compared with clinical RA parameters (RA activity scored as per DAS28) using Spearman Rho Analysis. Results While no change was detected in the ratio of CD4+ to CD8+ T cells between controls and RA patient groups, upregulation of TRAIL and its receptors (both death and decoy) was detected on both CD4+ and CD8+ T cells in RA patients compared to control individuals. Death Receptor-4 (DR4) and the decoy receptors DcR1 and DcR2 on CD8+ T cells, but not on CD4+ T cells, were positively correlated with patients' DAS scores. Conclusions Our data suggest that TRAIL/TRAIL receptor expression profiles on T cells might be important in revelation of RA pathogenesis. PMID:20799941

Losartan suppresses the kainate-induced changes of angiotensin AT1 receptor expression in a model of comorbid hypertension and epilepsy.

PubMed

Atanasova, Dimitrinka; Tchekalarova, Jana; Ivanova, Natasha; Nenchovska, Zlatina; Pavlova, Ekaterina; Atanassova, Nina; Lazarov, Nikolai

2018-01-15

Experimental and clinical studies have demonstrated that components of renin-angiotensin system are elevated in the hippocampus in epileptogenic conditions. In the present work, we explored the changes in the expression of angiotensin II receptor, type 1 (AT 1 receptor) in limbic structures, as well as the effect of the AT1 receptor antagonist losartan in a model of comorbid hypertension and epilepsy. The expression of AT 1 receptors was compared between spontaneously hypertensive rats (SHRs) and Wistar rats by using immunohistochemistry in the kainate (KA) model of temporal lobe epilepsy (TLE). The effect of losartan was studied on AT 1 receptor expression in epileptic rats that were treated for a period of 4weeks after status epilepticus. The naive and epileptic SHRs were characterized by stronger protein expression of AT 1 receptor than normotensive Wistar rats in the CA1, CA3a, CA3b, CA3c field and the hilus of the dentate gyrus of the dorsal hippocampus but fewer cells were immunostained in the piriform cortex. Increased AT 1 immunostaining was observed in the basolateral amygdala of epileptic SHRs but not of epileptic Wistar rats. Losartan exerted stronger and structure-dependent suppression of AT 1 receptor expression in SHRs compared to Wistar rats. Our results confirm the important role of AT 1 receptor in epilepsy and suggest that the AT 1 receptor antagonists could be used as a therapeutic strategy for treatment of comorbid hypertension and epilepsy. Copyright © 2017 Elsevier Inc. All rights reserved.

Human urothelial cell lines as potential models for studying cannabinoid and excitatory receptor interactions in the urinary bladder.

PubMed

Bakali, Evangelia; Elliott, Ruth A; Taylor, Anthony H; Lambert, David G; Willets, Jonathon M; Tincello, Douglas G

2014-06-01

To characterize human urothelial cell lines' cannabinoid receptor expression and evaluate their possible use for studying signalling interactions with purinergic and muscarinic receptor activation. PCR was used to detect cannabinoid (CB), muscarinic and purinergic receptor transcripts in HCV29 and UROtsa cells, whilst immunofluorescence evaluated protein expression and localization of cannabinoid receptors. The effect of CB1 agonist (ACEA) on carbachol- and ATP-induced changes in intracellular calcium ([Ca(2+)]i) levels was measured using fluorimetry. The ability of ACEA to reduce intracellular cAMP was investigated in HCV29 cells. CB1 and GPR55 receptor transcripts were detected in HCV29 and UROtsa cells, respectively. Immunofluorescence showed positive staining for CB1 in the HCV29 cells. Both cell lines expressed transcript levels for muscarinic receptors, but carbachol did not raise [Ca(2+)]i levels indicating a lack or low expression of G(q)-coupled muscarinic receptors. Transcripts for purinergic receptors were detected; ATP significantly increased [Ca(2+)]i in HCV29 and UROtsa cells by 395 ± 61 and 705 ± 100 nM (mean ± SEM, n = 6), respectively. ACEA did not alter ATP-induced [Ca(2+)]i or cAMP levels in HCV29 cells. Whilst HCV29 cells expressed CB1 and UROtsa cells expressed GPR55 receptors, these were not functionally coupled to the existing purinergic-driven increase in Ca2+ as such they do not represent a good model to study signalling interactions.

Nuclear receptors HR96 and ultraspiracle from the fall armyworm (Spodoptera frugiperda), developmental expression and induction by xenobiotics.

PubMed

Giraudo, Maeva; Audant, Pascaline; Feyereisen, René; Le Goff, Gaëlle

2013-05-01

The fall armyworm Spodoptera frugiperda is a major polyphagous pest in agriculture and little is known on how this insect can adapt to the diverse and potentially toxic plant allelochemicals that they ingest or to insecticides. To investigate the involvement of nuclear receptors in the response of S. frugiperda to its chemical environment, we cloned SfHR96, a nuclear receptor orthologous to the mammalian xenobiotic receptors, pregnane X receptor (PXR) and constitutive androstane receptor (CAR). We also cloned ultraspiracle (USP), the ortholog of retinoid X receptor (RXR) that serves as partner of dimerization of PXR and CAR. Cloning of SfUSP revealed the presence of two isoforms, SfUSP-1 and SfUSP-2 in this species, that differ in their N-terminal region. The expression of these receptors as well as the ecdysone receptor was studied during specific steps of development in different tissues. SfHR96 was constitutively expressed in larval midgut, fat body and Malpighian tubules throughout the last two instars and pupal stage, as well as in Sf9 cells. EcR and SfUSP-2 showed peaks of expression before larval moults and during metamorphosis, whereas SfUSP-1 was mainly expressed in the pre-pupal stage. Receptor induction was followed after exposure of larvae or cells to 11 chemical compounds. SfHR96 was not inducible by the tested compounds. EcR was significantly induced by the 20-hydroxyecdysone agonist, methoxyfenozide, and SfUSP showed an increase expression when exposed to the juvenile hormone analog, methoprene. The cloning of these nuclear receptors is a first step in understanding the important capacities of adaptation of this insect pest. Copyright © 2013 Elsevier Ltd. All rights reserved.

GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

NASA Technical Reports Server (NTRS)

Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

1995-01-01

The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of schizophrenics, the previously reported upregulation of muscimol binding sites and downregulation of benzodiazepine binding sites in the prefrontal and adjacent cingulate cortex of schizophrenics are possibly due to posttranscriptional modifications of mRNAs and their translated polypeptides.

Expression pattern of G protein‑coupled estrogen receptor 1 (GPER) in human cumulus granulosa cells (CGCs) of patients with PCOS.

PubMed

Zang, Lili; Zhang, Quan; Zhou, Yi; Zhao, Yan; Lu, Linlin; Jiang, Zhou; Peng, Zhen; Zou, Shuhua

2016-06-01

Estradiol mediates its actions by binding to classical nuclear receptors, estrogen receptor α (ER-α) and estrogen receptor β (ER-β), and the non-classical G protein-coupled estrogen receptor 1(GPER). Several gene knockdown models have shown the importance of the receptors for growth of the oocyte and for ovulation. The aim of our study was to identify the pattern of GPER expression in human cumulus granulosa cells (CGCs) from ovarian follicles at different stages of oocyte maturation, and the differences of GPER expression between polycystic ovary syndrome (PCOS) patients and non-PCOS women. Thirty-eight cases of PCOS patients and a control group of thirty-two infertile women without PCOS were used in this study. GPER's location in CGCs was investigated by immunohistochemistry. Quantitative RT-PCR and western blot were used to identify the quantify GPER expression. Here we demonstrated that GPER was expressed in CGCs of both PCOS patients and non-PCOS women, and the expression of GPER was decreased significantly during oocyte maturation. But the expression levels of GPER in CGCs of PCOS patients and non-PCOS women were not significantly different. The data indicate that GPER may play a role during human oocyte maturation through its action in cumulus granulosa cells. AMHRIIs: anti-Mullerian hormone type II receptors; BMI: body mass index; CGCs: cumulus granulosa cells; COH: controlled ovarian hyperstimulation; E2: estradiol; EGFR: epidermal growth factor receptor; ER-α: estrogen receptor; ER-β: estrogen receptor β; FF: follicular fluid; FSH: follicle-stimulating hormone; GCs: granulosa cells; GPER: G protein-coupled estrogen receptor 1; GV: germinal vesicle; GVBD: germinal vesicle breakdown; HCG: human chorionic gonadotropin; IRS: immunoreactive score; IVF-ET: in vitro fertilization and embryo transfer; MI: metaphase I; MII: metaphase II; MAPK: mitogen-activated protein kinase; OCCCs: oocyte corona cumulus complexes; PCOS: polycystic ovarian syndrome; q quantitative real-time PCR: qRT-PCR.

Postnatal Expression of V2 Vasopressin Receptor Splice Variants in the Rat Cerebellum

PubMed Central

Vargas, Karina J.; Sarmiento, José M.; Ehrenfeld, Pamela; Añazco, Carolina C.; Villanueva, Carolina I.; Carmona, Pamela L.; Brenet, Marianne; Navarro, Javier; Müller-Esterl, Werner; Figueroa, Carlos D.; González, Carlos B.

2010-01-01

The V2 vasopressin receptor gene contains an alternative splice site in exon-3, which leads to the generation of two splice variants (V2a and V2b) first identified in the kidney. The open reading frame of the alternatively spliced V2b transcripten codes a truncated receptor, showing the same amino acid sequence as the canonical V2a receptor up to the 6th transmembrane segment, but displaying a distinct sequence to the corresponding 7th transmembrane segment and C-terminal domain relative to the V2a receptor. Here, we demonstrate the postnatal expression of V2a and V2b variants in the rat cerebellum. Most importantly, we showed by in situ hybridization and immunocytochemistry that both V2 splice variants were preferentially expressed in Purkinje cells, from early to late postnatal development. In addition, both variants were transiently expressed in the neuroblastic external granule cells and Bergmann fibers. These results indicate that the cellular distributions of both splice variants are developmentally regulated, and suggest that the transient expression of the V2 receptor is involved in the mechanisms of cerebellar cytodifferentiation by AVP. Finally, transfected CHO-K1 .expressing similar amounts of both V2 splice variants, as that found in the cerebellum, showed a significant reduction in the surface expression of V2a receptors, suggesting that the differential expression of the V2 splice variants regulate the vasopressin signaling in the cerebellum. PMID:19281786

Clathrin-dependent internalization of the angiotensin II AT₁A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells.

PubMed

Morinelli, Thomas A; Walker, Linda P; Velez, Juan Carlos Q; Ullian, Michael E

2015-02-05

The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors. Copyright © 2014 Elsevier B.V. All rights reserved.

Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

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

Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp; Sekiguchi, Toshio; Nagata, Sayaka

2016-02-19

Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding andmore » AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.« less

Effects of YM471, a nonpeptide AVP V1A and V2 receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells

PubMed Central

Tsukada, Junko; Tahara, Atsuo; Tomura, Yuichi; Wada, Koh-ichi; Kusayama, Toshiyuki; Ishii, Noe; Yatsu, Takeyuki; Uchida, Wataru; Taniguchi, Nobuaki; Tanaka, Akihiro

2001-01-01

YM471, (Z)-4′-{4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl}-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V1A, V1B and V2) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [3H]-AVP binding to V1A and V2 receptors with Ki values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V1B and oxytocin receptors with Ki values of 16.4 μM and 31.6 nM, respectively. In CHO cells expressing V1A receptors, YM471 potently inhibited AVP-induced intracellular Ca2+ concentration ([Ca2+]i) increase, exhibiting an IC50 value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca2+]i increase (IC50=193 nM), and did not affect AVP-induced [Ca2+]i increase in CHO cells expressing V1B receptors. Furthermore, in CHO cells expressing V2 receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC50 value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V1A and V2 receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP. PMID:11429400

Effects of YM471, a nonpeptide AVP V(1A) and V(2) receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells.

PubMed

Tsukada, J; Tahara, A; Tomura, Y; Wada Ki; Kusayama, T; Ishii, N; Yatsu, T; Uchida, W; Taniguchi, N; Tanaka, A

2001-07-01

YM471, (Z)-4'-[4,4-difluoro-5-[2-(4-dimethylaminopiperidino)-2-oxoethylidene]-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl]-2-phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V(1A), V(1B) and V(2)) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [(3)H]-AVP binding to V(1A) and V(2) receptors with K(i) values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V(1B) and oxytocin receptors with K(i) values of 16.4 microM and 31.6 nM, respectively. In CHO cells expressing V(1A) receptors, YM471 potently inhibited AVP-induced intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, exhibiting an IC(50) value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin-induced [Ca(2+)](i) increase (IC(50)=193 nM), and did not affect AVP-induced [Ca(2+)](i) increase in CHO cells expressing V(1B) receptors. Furthermore, in CHO cells expressing V(2) receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC(50) value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V(1A) and V(2) receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.

Atypical nuclear localization of VIP receptors in glioma cell lines and patients

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

Barbarin, Alice; Séité, Paule; Godet, Julie

Highlights: • The VIP receptor VPAC1 contains a putative NLS signal. • VPAC1 is predominantly nuclear in GBM cell lines but not VPAC2. • Non-nuclear VPAC1/2 protein expression is correlated with glioma grade. • Nuclear VPAC1 is observed in 50% of stage IV glioma (GBM). - Abstract: An increasing number of G protein-coupled receptors, like receptors for vasoactive intestinal peptide (VIP), are found in cell nucleus. As VIP receptors are involved in the regulation of glioma cell proliferation and migration, we investigated the expression and the nuclear localization of the VIP receptors VPAC1 and VPAC2 in this cancer. First, bymore » applying Western blot and immunofluorescence detection in three human glioblastoma (GBM) cell lines, we observed a strong nuclear staining for the VPAC1 receptor and a weak nuclear VPAC2 receptor staining. Second, immunohistochemical staining of VPAC1 and VPAC2 on tissue microarrays (TMA) showed that the two receptors were expressed in normal brain and glioma tissues. Expression in the non-nuclear compartment of the two receptors significantly increased with the grade of the tumors. Analysis of nuclear staining revealed a significant increase of VPAC1 staining with glioma grade, with up to 50% of GBM displaying strong VPAC1 nuclear staining, whereas nuclear VPAC2 staining remained marginal. The increase in VPAC receptor expression with glioma grades and the enhanced nuclear localization of the VPAC1 receptors in GBM might be of importance for glioma progression.« less

Octopaminergic gene expression and flexible social behaviour in the subsocial burying beetle Nicrophorus vespilloides

PubMed Central

Cunningham, C B; Douthit, M K; Moore, A J

2014-01-01

Flexible behaviour allows organisms to respond appropriately to changing environmental and social conditions. In the subsocial beetle Nicrophorus vespilloides, females tolerate conspecifics when mating, become aggressive when defending resources, and return to social tolerance when transitioning to parenting. Given the association between octopamine and aggression in insects, we hypothesized that genes in the octopaminergic system would be differentially expressed across different social and reproductive contexts. To test this in N. vespilloides, we first obtained the sequences of orthologues of the synthetic enzymes and receptors of the octopaminergic system. We next compared relative gene expression from virgin females, mated females, mated females alone on a resource required for reproduction and mated females on a resource with a male. Expression varied for five receptor genes. The expression of octopamine β receptor 1 and octopamine β receptor 2 was relatively higher in mated females than in other social conditions. Octopamine β receptor 3 was influenced by the presence or absence of a resource and less by social environment. Octopamine α receptor and octopamine/tyramine receptor 1 gene expression was relatively lower in the mated females with a resource and a male. We suggest that in N. vespilloides the octopaminergic system is associated with the expression of resource defence, alternative mating tactics, social tolerance and indirect parental care. PMID:24646461

Posttranscriptional mRNA processing as a mechanism for regulation of human A1 adenosine receptor expression.

PubMed Central

Ren, H; Stiles, G L

1994-01-01

The human A1 adenosine receptor gene contains six exons with exons 1, 2, 3, 4, and part of 5 representing 5' untranslated regions. Reverse transcription-PCR with exon-specific primers showed two distinct transcripts containing either exons 3, 5, and 6 or exons 4, 5, and 6, with exons 3 and 4 being mutually exclusive. No mature mRNAs containing exons 1 and 2 have been detected. All human tissues that express any A1 receptors contain mRNA with exons 4, 5, and 6. Tissues which express high levels of A1 receptors contain mRNA with exons 3, 5, and 6. Exon 4 contains two upstream ATG codons whereas exon 3 contains none. COS cells transfected with expression vectors containing exon 4 (exons 1-6, 3-6, or Ex4-6) express much lower levels of A1 receptors than vectors without exon 4 (exons 3, 5, and 6). Mutation of upstream ATG codons in exon 4 leads to 3- to 7-fold increased A1 receptor expression, up to the level seen with the construct containing exons 3, 5, and 6. Thus, in human tissues "basal" levels of A1 receptors can be expressed by use of mRNA containing exons 4, 5, and 6, but when high levels are needed, alternative transcripts with exons 3, 5, and 6 are produced. Images PMID:8197148

Cytochemical Organization of the Retino-Suprachiasmatic System

DTIC Science & Technology

1994-03-11

strongly expiessed of the, non-NMDA ionotropic receptors . Other AMPA-preferring receptors , GluR3 and -R4, were also found, but to lesser extent...kainate, and NMDA receptor RN was found in the hypothalamus. GluRi and GluR2 were ang the nmst strongly expressed of the non-NvDA ionotropic receptors ...several different glutamate receptors . The expression of many different types of ionotropic glutamate receptors throughout the hypothalamus suggests that

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

PubMed Central

Böttner, Martina; Barrenschee, Martina; Hellwig, Ines; Harde, Jonas; Egberts, Jan-Hendrik; Becker, Thomas; Zorenkov, Dimitri; Schäfer, Karl-Herbert; Wedel, Thilo

2013-01-01

Background & Aims Absence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons. Methods Colonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored. Results mRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression. Conclusions Our results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system. PMID:23805210

Differential relationships between D1 and D2 dopamine receptor expression in the medial preoptic nucleus and sexually-motivated song in male European starlings (Sturnus vulgaris)

PubMed Central

DeVries, M. S.; Cordes, M.A.; Stevenson, S.A.; Riters, L.V.

2015-01-01

Converging data in songbirds support a central role for the medial preoptic nucleus (POM) in motivational aspects of vocal production. Recent data suggest that dopamine in the POM plays a complex modulatory role in the production of sexually-motivated song and that an optimal level of dopamine D1 receptor stimulation is required to facilitate singing behavior. To further explore this possibility, we used quantitative real time PCR to examine relationships between mRNA expression of D1 as well as D2 receptors in the POM (and also the lateral septum and Area X) and sexually-motivated singing behavior in male European starlings. Results showed that both males with the highest and lowest D1 expression in the POM sang significantly less than males with intermediate levels of expression. Furthermore, singing behavior rose linearly in association with increasing levels of D1 expression in POM but dropped abruptly, such that individuals with D1 expression values higher than the mean sang very little. Analysis of birds with low and intermediate levels of D1 expression in POM revealed strong positive correlations between D1 expression and song but negative relationships between D2 receptor expression and song. These findings support prior work suggesting an optimal level of POM D1 receptor stimulation best facilitates sexually-motivated singing behavior. Results also suggest that D2 receptors may work in opposition to D1 receptors in POM to modify vocal production. PMID:26079111

Differences in the length of the carboxyl terminus mediate functional properties of neurokinin-1 receptor

PubMed Central

Lai, Jian-Ping; Lai, Saien; Tuluc, Florin; Tansky, Morris F.; Kilpatrick, Laurie E.; Leeman, Susan E.; Douglas, Steven D.

2008-01-01

The neurokinin-1 receptor (NK1R) has two naturally occurring forms that differ in the length of the carboxyl terminus: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. We examined whether there are differential signaling properties attributable to the carboxyl terminus of this receptor by using stably transfected human embryonic kidney (HEK293) cell lines that express either full-length or truncated NK1R. Substance P (SP) specifically triggered intracellular calcium increase in HEK293 cells expressing full-length NK1R but had no effect in the cells expressing the truncated NK1R. In addition, in cells expressing full-length NK1R, SP activated NF-κB and IL-8 mRNA expression, but in cells expressing the truncated NK1R, SP did not activate NF-κB, and it decreased IL-8 mRNA expression. In cells expressing full-length NK1R, SP stimulated phosphorylation of PKCδ but inhibited phosphorylation of PKCδ in cells expressing truncated NK1R. There are also differences in the timing of SP-induced ERK activation in cells expressing the two different forms of the receptor. Full-length NK1R activation of ERK was rapid (peak within 1–2 min), whereas truncated NK1R-mediated activation was slower (peak at 20–30 min). Thus, the carboxyl terminus of NK1R is the structural basis for differences in the functional properties of the full-length and truncated NK1R. These differences may provide important information toward the design of new NK1R receptor antagonists. PMID:18713853

Human Endometriosis Tissue Microarray Reveals Site-specific Expression of Estrogen Receptors, Progesterone Receptor, and Ki67.

PubMed

Colón-Caraballo, Mariano; García, Miosotis; Mendoza, Adalberto; Flores, Idhaliz

2018-04-07

Most available therapies for endometriosis are hormone-based and generally broadly used without taking into consideration the ovarian hormone receptor expression status. This contrasts strikingly with the standard of care for other hormone-based conditions such as breast cancer. We therefore aimed to characterize the expression of ovarian steroid hormone receptors for estrogen alpha (ESR1), estrogen beta (ESR2), and progesterone (PGR) in different types of endometriotic lesions and eutopic endometrium from women with endometriosis and controls using a tissue microarray (TMA). Nuclear expression levels of the receptors were analyzed by tissue (ie, ectopic vs. eutopic endometrium) and cell type (ie, glands vs. stroma). Ovarian lesions showed the lowest expression of ESR1 and PGR, and the highest expression of ESR2, whereas the fallopian tube lesions showed high expression of the 3 receptors. Differences among endometria included lower expression of ESR1 and higher expression of ESR2 in stroma of proliferative endometrium from patients versus patients, and a trend towards loss of PGR nuclear positivity in proliferative endometrium from patients. The largest ESR2:ESR1 ratios were observed in ovarian lesions and secretory endometrium. The highest proportion of samples with >10% Ki67 positive nuclei was in glands of fallopian tube (54%) and extrapelvic lesions (75%); 60% of glands of secretory endometrium from patients had >10% Ki67 positivity compared with only 15% in controls. Our results provide a better understanding of endometriosis heterogeneity by revealing lesion type-specific differences and case-by-case variability in the expression of ovarian hormone receptors. This knowledge could potentially predict individual responses to hormone therapies, and set the basis for the application of personalized medicine approaches for women with endometriosis.

Non-pharmacological treatment affects neuropeptide expression in neuropathic pain model.

PubMed

Santos, Fabio Martinez; Silva, Joyce Teixeira; Rocha, Igor Rafael Correia; Martins, Daniel Oliveira; Chacur, Marucia

2018-05-15

Chronic constriction injury (CCI) of the sciatic nerve elicits changes in neuropeptide expression on the dorsal root ganglia (DRG). The neural mobilization (NM) technique is a noninvasive method that has been proven clinically effective in reducing pain. The aim of this study was to analyze the expression of substance P, transient receptor potential vanilloid 1 (TRPV1) and opioid receptors in the DRG of rats with chronic constriction injury and to compare it to animals that received NM treatment. CCI was performed on adult male rats. Each animal was submitted to 10 sessions of neural mobilization every other day, starting 14 days after the CCI injury. At the end of the sessions, the DRG (L4-L6) were analyzed using Western blot assays for substance P, TRPV1 and opioid receptors (µ-opioid receptor, δ-opioid receptor and κ-opioid receptor). We observed a decreased substance P and TRPV1 expression (48% and 35%, respectively) and an important increase of µ-opioid receptor expression (200%) in the DRG after NM treatment compared to control animals. The data provide evidence that NM promotes substantial changes in neuropeptide expression in the DRG; these results may provide new options for treating neuropathic pain. Copyright © 2018 Elsevier B.V. All rights reserved.

Molecular identification and functional expression of mu 3, a novel alternatively spliced variant of the human mu opiate receptor gene.

PubMed

Cadet, Patrick; Mantione, Kirk J; Stefano, George B

2003-05-15

Studies from our laboratory have revealed a novel mu opiate receptor, mu 3, which is expressed in both vascular tissues and leukocytes. The mu 3 receptor is selective for opiate alkaloids and is insensitive to opioid peptides. We now identify the mu 3 receptor at the molecular level using a 441-bp conserved region of the mu 1 receptor. Sequence analysis of the isolated cDNA suggests that it is a novel, alternatively spliced variant of the mu opiate receptor gene. To determine whether protein expressed from this cDNA exhibits the biochemical characteristics expected of the mu 3 receptor, the cDNA clone was expressed in a heterologous system. At the functional level, COS-1 cells transfected with the mu 3 receptor cDNA exhibited dose-dependent release of NO following treatment with morphine, but not opioid peptides (i.e., Met-enkephalin). Naloxone was able to block the effect of morphine on COS-1 transfected cells. Nontransfected COS-1 cells did not produce NO in the presence of morphine or the opioid peptides at similar concentrations. Receptor binding analysis with [(3)H]dihydromorphine further supports the opiate alkaloid selectivity and opioid peptide insensitivity of this receptor. These data suggest that this new mu opiate receptor cDNA encodes the mu 3 opiate receptor, since it exhibits biochemical characteristics known to be unique to this receptor (opiate alkaloid selective and opioid peptide insensitive). Furthermore, using Northern blot, RT-PCR, and sequence analysis, we have demonstrated the expression of this new mu variant in human vascular tissue, mononuclear cells, polymorphonuclear cells, and human neuroblastoma cells.

Angiotensin II AT2 receptor decreases AT1 receptor expression and function via nitric oxide/cGMP/Sp1 in renal proximal tubule cells from Wistar–Kyoto rats

PubMed Central

Yang, Jian; Chen, Caiyu; Ren, Hongmei; Han, Yu; He, Duofen; Zhou, Lin; Hopfer, Ulrich; Jose, Pedro A.; Zeng, Chunyu

2013-01-01

Background The renin–angiotensin (Ang) system controls blood pressure, in part, by regulating renal tubular sodium transport. In the kidney, activation of the angiotensin II type 1 (AT1) receptor increases renal sodium reabsorption, whereas the angiotensin II type 2 (AT2) receptor produces the opposite effect. We hypothesized that the AT2 receptor regulates AT1 receptor expression and function in the kidney. Methods and results In immortalized renal proximal tubule (RPT) cells from Wistar–Kyoto rats, CGP42112, an AT2 receptor agonist, decreased AT1 receptor mRNA and protein expression (P < 0.05), as assessed by reverse transcriptase-polymerase chain reaction and immunoblotting. The inhibitory effect of the AT2 receptor on AT1 receptor expression was blocked by the AT2 receptor antagonist, PD123319 (10−6 mol/l), the nitric oxide synthase inhibitor Nw-nitro-l-arginine methyl ester (10−4 mol/l), or the nitric oxide-dependent soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo-[4,3-a] quinoxalin-1-one (10−5 mol/l), indicating that both nitric oxide and cyclic guanosine monophosphate (cGMP) were involved in the signaling pathway. Furthermore, CGP42112 decreased Sp1 serine phosphorylation and reduced the binding of Sp1 to AT1 receptor DNA. Stimulation with Ang II (10−11 mol/l per 30 min) enhanced Na+-K+-ATPase activity in RPT cells, which was prevented by pretreatment with CGP42112 (10−7 mol/l per 24 h) (P < 0.05). The above-mentioned results were confirmed in RPT cells from AT2 receptor knockout mice; AT1 receptor expression and Ang II-stimulated Na+-K+-ATPase activity were greater in these cells than in RPT cells from wild-type mice (P < 0.05). AT1/AT2 receptors co-localized and co-immunoprecipitated in RPT cells; short-term CGP42112 (10−7 mol/l per 30 min) treatment increased AT1/AT2 receptor co-immunoprecipitation (P < 0.05). Conclusions These results indicate that the renal AT2 receptor, via nitric oxide/cGMP/Sp1 pathway, regulates AT1 receptor expression and function, which may be important in the regulation of sodium excretion and blood pressure. PMID:22504846

Expression of Sex Steroid Hormone Receptors in Vagal Motor Neurons Innervating the Trachea and Esophagus in Mouse

PubMed Central

Mukudai, Shigeyuki; Ichi Matsuda, Ken; Bando, Hideki; Takanami, Keiko; Nishio, Takeshi; Sugiyama, Yoichiro; Hisa, Yasuo; Kawata, Mitsuhiro

2016-01-01

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus. PMID:27006520

Inflammation Stimulates the Expression of PCSK9

PubMed Central

Feingold, Kenneth R.; Moser, Arthur H.; Shigenaga, Judy K.; Patzek, Sophie M.; Grunfeld, Carl

2008-01-01

Inflammation induces marked changes in lipid and lipoprotein metabolism. Proprotein convertase subtilisin kexin 9 (PCSK9) plays an important role in regulating LDL receptor degradation. Here we demonstrate that LPS decreases hepatic LDL receptor protein but at the same time hepatic LDL receptor mRNA levels are not decreased. We therefore explored the effect of LPS on PCSK9 expression. LPS results in a marked increase in hepatic PCSK9 mRNA levels (4 hours-2.5 fold increase; 38 hours-12.5 fold increase). The increase in PCSK9 is a sensitive response with 1 ug LPS inducing a ½ maximal response. LPS also increased PCSK9 expression in the kidney. Finally, zymosan and turpentine, other treatments that induce inflammation, also stimulated hepatic expression of PCSK9. Thus, inflammation stimulates PCSK9 expression leading to increased LDL receptor degradation and decreasing LDL receptors thereby increasing serum LDL, which could have beneficial effects on host defense. PMID:18638454

Development of antibodies against the rat brain somatostatin receptor.

PubMed

Theveniau, M; Rens-Domiano, S; Law, S F; Rougon, G; Reisine, T

1992-05-15

Somatostatin (SRIF) is a neurotransmitter in the brain involved in the regulation of motor activity and cognition. It induces its physiological actions by interacting with receptors. We have developed antibodies against the receptor to investigate its structural properties. Rabbit polyclonal antibodies were generated against the rat brain SRIF receptor. These antibodies (F4) were able to immunoprecipitate solubilized SRIF receptors from rat brain and the cell line AtT-20. The specificity of the interaction of these antibodies with SRIF receptors was further demonstrated by immunoblotting. F4 detected SRIF receptors of 60 kDa from rat brain and adrenal cortex and the cell lines AtT-20, GH3, and NG-108, which express high densities of SRIF receptors. They did not detect immunoreactive material from rat liver or COS-1, HEPG, or CRL cells, which do not express functional SRIF receptors. In rat brain, 60-kDa immunoreactivity was detected by F4 in the hippocampus, cerebral cortex, and striatum, which have high densities of SRIF receptors. However, F4 did not interact with proteins from cerebellum and brain stem, which express few SRIF receptors. Immunoreactive material cannot be detected in rat pancreas or pituitary, which have been reported to express a 90-kDa SRIF receptor subtype. The selective detection of 60-kDa SRIF receptors by F4 indicates that the 60- and 90-kDa SRIF receptor subtypes are immunologically distinct. The availability of antibodies that selectively detect native and denatured brain SRIF receptors provides us with a feasible approach to clone the brain SRIF receptor gene(s).

Naturally occurring glucagon-like peptide-2 (GLP-2) receptors in human intestinal cell lines.

PubMed

Sams, Anette; Hastrup, Sven; Andersen, Marie; Thim, Lars

2006-02-17

Although clinical trials with GLP-2 receptor agonists are currently ongoing, the mechanisms behind GLP-2-induced intestinal epithelial growth remain to be understood. To approach the GLP-2 mechanism of action this study aimed to identify intestinal cell lines endogenously expressing the GLP-2 receptor. Here we report the first identification of a cell line endogenously expressing functional GLP-2 receptors. The human intestinal epithelial cell line, FHC, expressed GLP-2 receptor encoding mRNA (RT-PCR) and GLP-2 receptor protein (Western blot). In cultured FHC cells, GLP-2 induced concentration dependent cAMP accumulation (pEC(50)=9.7+/-0.04 (mean+/-S.E.M., n=4)). In addition, a naturally occurring human intestinal fibroblast cell line, 18Co, endogenously expressing GLP-2 receptor encoding mRNA (RT-PCR) and protein (Western blot) was identified. No receptor functionality (binding or G-protein signalling) could be demonstrated in 18Co cells. The identified gut-relevant cell lines provide tools for future clarification of the mechanisms underlying GLP-2-induced epithelial growth.

Dopamine receptors play distinct roles in sexual behavior expression of rats with a different sexual motivational tone.

PubMed

Guadarrama-Bazante, Irma L; Canseco-Alba, Ana; Rodríguez-Manzo, Gabriela

2014-10-01

Dopamine (DA) plays a central role in the expression of male sexual behavior. The effects of DA-enhancing drugs on copulation seem to vary depending on the dose of the agonist used, the type of DA receptor activated, and the sexual condition of the animals. The aim of the present study was to carry out a systematic analysis of the effects of dopaminergic agonists on the expression of male sexual behavior by sexually competent rats in different sexual motivational states, that is when sexually active (sexually experienced) and when temporarily inhibited (sexually exhausted). To this end, the same doses of the nonselective DA receptor agonist apomorphine, the selective D2-like DA receptor agonist quinpirole, and the selective D1-like DA receptor agonist SKF38393 were injected intraperitoneally to sexually experienced or sexually exhausted male rats and their sexual behavior was recorded. Low apomorphine doses induced expression of sexual behavior in sexually satiated rats, but only reduced the intromission latency of sexually experienced rats. SKF38393 facilitated the expression of sexual behavior by sexually exhausted rats, but not that of sexually experienced males and quinpirole did not exert an effect in both types of animal. In line with these results, the apomorphine-induced reversal of sexual exhaustion was blocked by the D1-like receptor antagonist SCH23390. The data suggest that DA receptors play distinct roles in the expression of sexual behavior by male rats depending on their motivational state and that activation of D1-like receptors promotes the expression of sexual behavior in satiated rats.

mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

PubMed

Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

2015-10-01

Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted.

A functional role for CD28 costimulation in tumor recognition by single-chain receptor-modified T cells.

PubMed

Moeller, Maria; Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Tanner, Jane E; Cerutti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2004-05-01

T cells engineered to express single-chain antibody receptors that incorporate TCR-zeta and cluster designation (CD)28 signaling domains (scFv-alpha-erbB2-CD28-zeta) can be redirected in vivo to cancer cells that lack triggering costimulatory molecules. To assess the contribution of CD28 signaling to the function of the scFv-CD28-zeta receptor, we expressed a series of mutated scFv-CD28-zeta receptors directed against erbB2. Residues known to be critical for CD28 signaling were mutated from tyrosine to phenylalanine at position 170 or proline to alanine at positions 187 and 190. Primary mouse T cells expressing either of the mutant receptors demonstrated impaired cytokine (IFN-gamma and GM-CSF) production and decreased proliferation after antigen ligation in vitro and decreased antitumor efficacy in vivo compared with T cells expressing the wild-type scFv-CD28-zeta receptor, suggesting a key signaling role for the CD28 component of the scFv-CD28-zeta receptor. Importantly, cell surface expression, binding capacity and cytolytic activity mediated by the scFv-CD28-zeta receptor were not diminished by either mutation. Overall, this study has definitively demonstrated a functional role for the CD28 component of the scFv-CD28-zeta receptor and has shown that incorporation of costimulatory activity in chimeric scFv receptors is a powerful approach for improving adoptive cancer immunotherapy.

[On the role of selective silencer Freud-1 in the regulation of the brain 5-HT(1A) receptor gene expression].

PubMed

Naumenko, V S; Osipova, D V; Tsybko, A S

2010-01-01

Selective 5-HT(1A) receptor silencer (Freud-1) is known to be one of the main factors for transcriptional regulation of brain serotonin 5-HT(1A) receptor. However, there is a lack of data on implication of Freud-1 in the mechanisms underlying genetically determined and experimentally altered 5-HT(1A) receptor system state in vivo. In the present study we have found a difference in the 5-HT(1A) gene expression in the midbrain of AKR and CBA inbred mouse strains. At the same time no distinction in Freud-1 expression was observed. We have revealed 90.3% of homology between mouse and rat 5-HT(1A) receptor DRE-element, whereas there was no difference in DRE-element sequence between AKR and CBA mice. This indicates the absence of differences in Freud-1 binding site in these mouse strains. In the model of 5-HT(1A) receptor desensitization produced by chronic 5-HT(1A) receptor agonist administration, a significant reduction of 5-HT(1A) receptor gene expression together with considerable increase of Freud-1 expression were found. These data allow us to conclude that the selective silencer of 5-HT(1A) receptor, Freud-1, is involved in the compensatory mechanisms that modulate the functional state of brain serotonin system, although it is not the only factor for 5-HT(1A) receptor transcriptional regulation.

Ly49 Receptors: Innate and Adaptive Immune Paradigms

PubMed Central

Rahim, Mir Munir A.; Tu, Megan M.; Mahmoud, Ahmad Bakur; Wight, Andrew; Abou-Samra, Elias; Lima, Patricia D. A.; Makrigiannis, Andrew P.

2014-01-01

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity. PMID:24765094

Dietary ω-3 Polyunsaturated Fatty Acids Inhibit Tumor Growth in Transgenic ApcMin/+ Mice, Correlating with CB1 Receptor Up-Regulation

PubMed Central

Notarnicola, Maria; Tutino, Valeria; De Nunzio, Valentina; Dituri, Francesco; Caruso, Maria Gabriella; Giannelli, Gianluigi

2017-01-01

Mediterranean diet components, such as olive oil and ω-3 polyunsaturated fatty acids (ω-3 PUFAs), can arrest cell growth and promote cell apoptosis. Recently, olive oil has been demonstrated to modulate type-1 cannabinoid (CB1) receptor gene expression in both human colon cancer cells and rat colon. The aim of this study was to investigate a possible link between olive oil and ω-3 PUFAs effects and CB1 receptor expression in both intestinal and adipose tissue of ApcMin/+ mice. To confirm the role for the CB1 receptor as a negative modulator of cell proliferation in human colon cancer, CB1 receptor gene expression was also detected in tumor tissue and in surrounding normal mucosa of patients with colorectal cancer (CRC). Dietary ω-3 PUFAs significantly inhibited intestinal polyp growth in mice, correlating with CB1 receptor gene and protein expression induction. CB1 receptor gene up-regulation was also detected in adipose tissue, suggesting a close communication between cancer cells and the surrounding environment. Tissue CB1 receptor induction was associated with a concurrent inactivation of the Wnt/β-catenin pathway. Moreover, there was a significant reduction in CB1 receptor gene expression levels in cancer tissue compared to normal surrounding mucosa of patients with CRC, confirming that in cancer the “protective” action of the CB1 receptor is lost. PMID:28245562

Role of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion.

PubMed

Mainali, Dipak; Syed, Aleem; Arora, Neha; Smith, Emily A

2014-12-01

Integrins are ubiquitous transmembrane receptors with adhesion and signaling properties. The influence of insulin receptor and insulin signaling on αPS2CβPS integrins' lateral diffusion was studied using single particle tracking in S2 cells before and after reducing the insulin receptor expression or insulin stimulation. Insulin signaling was monitored by Western blotting for phospho-Akt expression. The expression of the insulin receptor was reduced using RNA interference (RNAi). After insulin receptor RNAi, four significant changes were measured in integrin diffusion properties: (1) there was a 24% increase in the mobile integrin population, (2) 14% of the increase was represented by integrins with Brownian diffusion, (3) for integrins that reside in confined zones of diffusion, there was a 45% increase in the diameter of the confined zone, and (4) there was a 29% increase in the duration integrins spend in confined zones of diffusion. In contrast to reduced expression of the insulin receptor, which alters integrin diffusion properties, insulin stimulation alone or insulin stimulation under conditions of reduced insulin receptor expression have minimal effects on altering the measured integrin diffusion properties. The differences in integrin diffusion measured after insulin receptor RNAi in the presence or absence of insulin stimulation may be the result of other insulin signaling pathways that are activated at reduced insulin receptor conditions. No change in the average integrin diffusion coefficient was measured for any conditions included in this study.

Genomic characterization and regulation of CYP3a13: role of xenobiotics and nuclear receptors.

PubMed

Anakk, Sayeepriyadarshini; Kalsotra, Auinash; Shen, Qi; Vu, Mary T; Staudinger, Jeffrey L; Davies, Peter J A; Strobel, Henry W

2003-09-01

We report that CYP3a13 gene, located on mouse chromosome 5, spans 27.5 Kb and contains 13 exons. The transcription start site is 35 bp upstream of the coding region and results in a 109 bp 5' untranslated region. CYP3a13 promoter shows putative binding sites for retinoid X receptor, pregnane X receptor, and estrogen receptor. CYP3a13 shows a broad tissue distribution with predominant expression in liver. Although CYP3a13 shares 92% nucleotide identity with the female-specific rat CYP3A9, its expression does not exhibit sexual dimorphism. Ligand activation of peroxisomal proliferator-activated receptor-gamma and retinoid X receptor inhibit expression of CYP3a13 at the transcription level in a tissue-specific manner. Another novel finding is hepatic induction of CYP3a13 by dexamethasone occurring only in pregnane X receptor null mice. We also report that pregnane X receptor is essential to maintain robust in vivo basal levels of CYP3a13 in contrast to CYP3a11. CYP3a13 protein expressed in vitro can metabolize clinically active drugs ethylmorphine and erythromycin, as well as benzphetamine. We conclude that CYP3a13 is regulated differentially by various nuclear receptors. In humans this may lead to altered drug metabolism, as many of the newly synthesized ligands/drugs targeted toward these nuclear receptors could influence CYP3A gene expression.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse.

PubMed

Vázquez-Cuevas, F G; Cruz-Rico, A; Garay, E; García-Carrancá, A; Pérez-Montiel, D; Juárez, B; Arellano, R O

2013-01-01

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

kappa-Opioid receptor in humans: cDNA and genomic cloning, chromosomal assignment, functional expression, pharmacology, and expression pattern in the central nervous system.

PubMed Central

Simonin, F; Gavériaux-Ruff, C; Befort, K; Matthes, H; Lannes, B; Micheletti, G; Mattéi, M G; Charron, G; Bloch, B; Kieffer, B

1995-01-01

Using the mouse delta-opioid receptor cDNA as a probe, we have isolated genomic clones encoding the human mu- and kappa-opioid receptor genes. Their organization appears similar to that of the human delta receptor gene, with exon-intron boundaries located after putative transmembrane domains 1 and 4. The kappa gene was mapped at position q11-12 in human chromosome 8. A full-length cDNA encoding the human kappa-opioid receptor has been isolated. The cloned receptor expressed in COS cells presents a typical kappa 1 pharmacological profile and is negatively coupled to adenylate cyclase. The expression of kappa-opioid receptor mRNA in human brain, as estimated by reverse transcription-polymerase chain reaction, is consistent with the involvement of kappa-opioid receptors in pain perception, neuroendocrine physiology, affective behavior, and cognition. In situ hybridization studies performed on human fetal spinal cord demonstrate the presence of the transcript specifically in lamina II of the dorsal horn. Some divergences in structural, pharmacological, and anatomical properties are noted between the cloned human and rodent receptors. Images Fig. 3 Fig. 4 PMID:7624359

Novel down-regulatory mechanism of the surface expression of the vasopressin V2 receptor by an alternative splice receptor variant.

PubMed

Sarmiento, José M; Añazco, Carolina C; Campos, Danae M; Prado, Gregory N; Navarro, Javier; González, Carlos B

2004-11-05

In rat kidney, two alternatively spliced transcripts are generated from the V2 vasopressin receptor gene. The large transcript (1.2 kb) encodes the canonical V2 receptor, whereas the small transcript encodes a splice variant displaying a distinct sequence corresponding to the putative seventh transmembrane domain and the intracellular C terminus of the V2 receptor. This work showed that the small spliced transcript is translated in the rat kidney collecting tubules. However, the protein encoded by the small transcript (here called the V2b splice variant) is retained inside the cell, in contrast to the preferential surface distribution of the V2 receptor (here called the V2a receptor). Cells expressing the V2b splice variant do not exhibit binding to 3H-labeled vasopressin. Interestingly, we found that expression of the splice variant V2b down-regulates the surface expression of the V2a receptor, most likely via the formation of V2a.V2b heterodimers as demonstrated by co-immunoprecipitation and fluorescence resonance energy transfer experiments between the V2a receptor and the V2b splice variant. The V2b splice variant would then be acting as a dominant negative. The effect of the V2b splice variant is specific, as it does not affect the surface expression of the G protein-coupled interleukin-8 receptor (CXCR1). Furthermore, the sequence encompassing residues 242-339, corresponding to the C-terminal domain of the V2b splice variant, also down-regulates the surface expression of the V2a receptor. We suggest that some forms of nephrogenic diabetes insipidus are due to overexpression of the splice variant V2b, which could retain the wild-type V2a receptor inside the cell via the formation of V2a.V2b heterodimers.

Corticosteroid receptor gene expression is related to sex and social behaviour in a social fish.

PubMed

O'Connor, Constance M; Rodela, Tammy M; Mileva, Viktoria R; Balshine, Sigal; Gilmour, Kathleen M

2013-03-01

Circulating corticosteroids have been related to social status in a variety of species. However, our understanding of corticosteroid receptor expression and its relationship with sociality is still in its infancy. Knowledge of variation in receptor expression is critical to understand the physiological relevance of differences in circulating corticosteroid concentrations. In this study, we examined corticosteroid receptor gene expression in relation to dominance rank, sex, and social behaviour in the highly social cichlid fish, Neolamprologus pulcher. We examined the relative gene expression of the three known teleost corticosteroid receptors: glucocorticoid receptor 1 (GR1), glucocorticoid receptor 2 (GR2), and the mineralocorticoid receptor (MR) in liver and brain tissue of dominant and subordinate N. pulcher males and females. Phylogenetic analysis revealed the N. pulcher gene originally described as GR2, clustered with other teleost GR1 genes, while the originally-described N. pulcher GR1 gene clustered with the GR2 genes of other teleosts. Therefore we propose a change in the original nomenclature of the N. pulcher GRs: GR1 (formerly GR2) and GR2 (formerly GR1) and adopt this new nomenclature throughout this manuscript. Liver MR transcript levels were higher in males than females, and positively related to submissive behaviour. Liver GR2 (formerly GR1) transcript levels were also higher in males than females. Collectively, the results demonstrate sex differences in corticosteroid receptor abundance, and suggest tissue- and receptor-specific roles for corticosteroid receptors in mediating aspects of social behaviour. Copyright © 2012. Published by Elsevier Inc.

An expressed sequence tag (EST) data mining strategy succeeding in the discovery of new G-protein coupled receptors.

PubMed

Wittenberger, T; Schaller, H C; Hellebrand, S

2001-03-30

We have developed a comprehensive expressed sequence tag database search method and used it for the identification of new members of the G-protein coupled receptor superfamily. Our approach proved to be especially useful for the detection of expressed sequence tag sequences that do not encode conserved parts of a protein, making it an ideal tool for the identification of members of divergent protein families or of protein parts without conserved domain structures in the expressed sequence tag database. At least 14 of the expressed sequence tags found with this strategy are promising candidates for new putative G-protein coupled receptors. Here, we describe the sequence and expression analysis of five new members of this receptor superfamily, namely GPR84, GPR86, GPR87, GPR90 and GPR91. We also studied the genomic structure and chromosomal localization of the respective genes applying in silico methods. A cluster of six closely related G-protein coupled receptors was found on the human chromosome 3q24-3q25. It consists of four orphan receptors (GPR86, GPR87, GPR91, and H963), the purinergic receptor P2Y1, and the uridine 5'-diphosphoglucose receptor KIAA0001. It seems likely that these receptors evolved from a common ancestor and therefore might have related ligands. In conclusion, we describe a data mining procedure that proved to be useful for the identification and first characterization of new genes and is well applicable for other gene families. Copyright 2001 Academic Press.

The distribution of the orphan bombesin receptor subtype-3 in the rat CNS.

PubMed

Jennings, C A; Harrison, D C; Maycox, P R; Crook, B; Smart, D; Hervieu, G J

2003-01-01

Bombesin receptor subtype 3 (BRS-3) is an orphan G-protein coupled receptor that shares between 47 and 51% homology with other known bombesin receptors. The natural ligand for BRS-3 is currently unknown and little is known about the mechanisms regulating BRS-3 gene expression. Unlike other mammalian bombesin receptors that have been shown to be predominantly expressed in the CNS and gastrointestinal tract, expression of the BRS-3 receptor in the rat brain has previously not been observed. To gain further understanding of the biology of BRS-3, we have studied the distribution of BRS-3 mRNA and protein in the rat CNS. The mRNA expression pattern was studied using reverse transcription followed by quantitative polymerase chain reaction. Using immunohistological techniques, the distribution of BRS-3 protein in the rat brain was investigated using a rabbit affinity-purified polyclonal antiserum raised against an N-terminal peptide. The BRS-3 receptor was found to be widely expressed in the rat brain at both mRNA and protein levels. Particularly strong immunosignals were observed in the cerebral cortex, hippocampal formation, hypothalamus and thalamus. Other regions of the brain such as the basal ganglia, midbrain and reticular formation were also immunopositive for BRS-3. In conclusion, our neuroanatomical data provide evidence that BRS-3 is as widely expressed in the rat brain as other bombesin-like peptide receptors and suggest that this receptor may also have important roles in the CNS, mediating the functions of a so far unidentified ligand.

TAM receptors support neural stem cell survival, proliferation and neuronal differentiation.

PubMed

Ji, Rui; Meng, Lingbin; Jiang, Xin; Cvm, Naresh Kumar; Ding, Jixiang; Li, Qiutang; Lu, Qingxian

2014-01-01

Tyro3, Axl and Mertk (TAM) receptor tyrosine kinases play multiple functional roles by either providing intrinsic trophic support for cell growth or regulating the expression of target genes that are important in the homeostatic regulation of immune responses. TAM receptors have been shown to regulate adult hippocampal neurogenesis by negatively regulation of glial cell activation in central nervous system (CNS). In the present study, we further demonstrated that all three TAM receptors were expressed by cultured primary neural stem cells (NSCs) and played a direct growth trophic role in NSCs proliferation, neuronal differentiation and survival. The cultured primary NSCs lacking TAM receptors exhibited slower growth, reduced proliferation and increased apoptosis as shown by decreased BrdU incorporation and increased TUNEL labeling, than those from the WT NSCs. In addition, the neuronal differentiation and maturation of the mutant NSCs were impeded, as characterized by less neuronal differentiation (β-tubulin III+) and neurite outgrowth than their WT counterparts. To elucidate the underlying mechanism that the TAM receptors play on the differentiating NSCs, we examined the expression profile of neurotrophins and their receptors by real-time qPCR on the total RNAs from hippocampus and primary NSCs; and found that the TKO NSC showed a significant reduction in the expression of both nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), but accompanied by compensational increases in the expression of the TrkA, TrkB, TrkC and p75 receptors. These results suggest that TAM receptors support NSCs survival, proliferation and differentiation by regulating expression of neurotrophins, especially the NGF.

Regulation of dopaminergic neuron firing by heterogeneous dopamine autoreceptors in the substantia nigra pars compacta.

PubMed

Jang, Jin Young; Jang, Miae; Kim, Shin Hye; Um, Ki Bum; Kang, Yun Kyung; Kim, Hyun Jin; Chung, Sungkwon; Park, Myoung Kyu

2011-03-01

Dopamine (DA) receptors generate many cellular signals and play various roles in locomotion, motivation, hormone production, and drug abuse. According to the location and expression types of the receptors in the brain, DA signals act in either stimulatory or inhibitory manners. Although DA autoreceptors in the substantia nigra pars compacta are known to regulate firing activity, the exact expression patterns and roles of DA autoreceptor types on the firing activity are highly debated. Therefore, we performed individual correlation studies between firing activity and receptor expression patterns using acutely isolated rat substantia nigra pars compacta DA neurons. When we performed single-cell RT-PCR experiments, D(1), D(2)S, D(2)L, D(3), and D(5) receptor mRNA were heterogeneously expressed in the order of D(2)L > D(2)S > D(3) > D(5) > D(1). Stimulation of D(2) receptors with quinpirole suppressed spontaneous firing similarly among all neurons expressing mRNA solely for D(2)S, D(2)L, or D(3) receptors. However, quinpirole most strongly suppressed spontaneous firing in the neurons expressing mRNA for both D(2) and D(3) receptors. These data suggest that D(2) S, D(2)L, and D(3) receptors are able to equally suppress firing activity, but that D(2) and D(3) receptors synergistically suppress firing. This diversity in DA autoreceptors could explain the various actions of DA in the brain. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

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

PubMed

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

2018-03-01

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

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis

PubMed Central

2011-01-01

Introduction Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum. Methods Immunohistology and enzyme-linked immunosorbent assays (ELISAs) were used to determine chemokine and chemokine receptor expression in the skin and serum, respectively, of SSc and normal patients. Endothelial cells (ECs) were isolated from SSc skin biopsies and chemokine and chemokine receptor expression was determined by quantitative PCR and immunofluorescence staining. Results Antiangiogenic IP-10/CXCL10 and MIG/CXCL9 were elevated in SSc serum and highly expressed in SSc skin. However, CXCR3, the receptor for these chemokines, was decreased on ECs in SSc vs. normal skin. CXCL16 was elevated in SSc serum and increased in SSc patients with early disease, pulmonary arterial hypertension, and those that died during the 36 months of the study. In addition, its receptor CXCR6 was overexpressed on ECs in SSc skin. At the mRNA and protein levels, CXCR3 was decreased while CXCR6 was increased on SSc ECs vs. human microvascular endothelial cells (HMVECs). Conclusions These results show that while the expression of MIG/CXCL9 and IP-10/CXCL10 are elevated in SSc serum, the expression of CXCR3 is downregulated on SSc dermal ECs. In contrast, CXCL16 and CXCR6 are elevated in SSc serum and on SSc dermal ECs, respectively. In all, these findings suggest angiogenic chemokine receptor expression is likely regulated in an effort to promote angiogenesis in SSc skin. PMID:21303517

Sex-specific hormone receptors in urothelial carcinomas of the human urinary bladder: a comparative analysis of clinicopathological features and survival outcomes according to receptor expression.

PubMed

Tuygun, Can; Kankaya, Duygu; Imamoglu, Abdurrahim; Sertcelik, Ayse; Zengin, Kursad; Oktay, Murat; Sertcelik, Nurettin

2011-01-01

To investigate the expression of sex-specific hormone receptors in normal bladder urothelium and urothelial carcinomas (UCs) of the bladder, and to analyze clinicopathological features and survival outcomes according to receptor expression. We evaluated the clinical data and tumor specimens of 139 patients with bladder cancer (BC). In addition, 72 samples of normal urothelium were included. Immunohistochemistry was performed using streptavidin-biotin peroxidase method, a monoclonal androgen receptor (AR), and an estrogen receptor-β (ERβ) antibody on paraffin-embedded tissue sections. Expression levels of each receptor were assessed by evaluating 500 tumor cells for each case and the percentage of positively-stained nuclei was recorded. None of the 58 male control cases showed any AR and ERβ expression. Five (35, 71%) of the 14 female control cases expressed ERβ. Of the 139 patients with UCs, 71 (51, 07%) expressed AR (62 male vs. 9 female; P = 0.413) and 44 (31, 65%) (39 male vs. 5 female; P = 0.402) showed ERβ expression (P < 0.001). No significant relationship was found between ERβ expression levels and tumor grades, and stages (P = 0.441; P = 0.247). AR expression was significantly lower in T2-tumors (21%) than in Ta-tumors (60%) and T1-tumors (60%) (P < 0.001). It was significantly higher in low-grade papillary UCs (64%) compared with high-grade papillary UCs (44%) and infiltrative high-grade UCs (17%) (P = 0.039; P < 0.001). Data of 79 patients with noninvasive BC were eligible to present, with a median 29 months follow-up. AR expression level did not influence recurrence-free survival (RFS) and progression-free survival (PFS) (P = 0.095; P = 0.110). No significant association was found between ERβ expression level and RFS (P = 0.293). PFS in patients with lower ERβ-expressing tumors was significantly better than that in patients with higher ERβ-expressing tumors (P = 0.035). Multivariate analysis confirmed this significant influence on PFS (P = 0.025). Although ERβ expression had no impact on histopathological tumor characteristics, decrease in its expression may be associated with better PFS rates in patients with noninvasive BC. Conversely, loss of AR expression was associated with higher grade UCs and invasive UCs, but had no prognostic effect on survival. Finally, sex-specific hormone receptors alone cannot be responsible for gender differences in BC rates because they were expressed in similar rates in both sexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Serotonin 5-HT2C receptor-independent expression of hypothalamic NOR1, a novel modulator of food intake and energy balance, in mice

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

Nonogaki, Katsunori, E-mail: knonogaki-tky@umin.ac.jp; Department of Lifestyle Medicine, Biomedical Engineering Center, Tohoku University; Kaji, Takao

2009-08-21

NOR1, Nur77 and Nurr1 are orphan nuclear receptors and members of the NR4A subfamily. Here, we report that the expression of hypothalamic NOR1 was remarkably decreased in mildly obese {beta}-endorphin-deficient mice and obese db/db mice with the leptin receptor mutation, compared with age-matched wild-type mice, whereas there were no genotypic differences in the expression of hypothalamic Nur77 or Nurr1 in these animals. The injection of NOR1 siRNA oligonucleotide into the third cerebral ventricle significantly suppressed food intake and body weight in mice. On the other hand, the decreases in hypothalamic NOR1 expression were not found in non-obese 5-HT2C receptor-deficient mice.more » Moreover, systemic administration of m-chlorophenylpiperazine (mCPP), a 5-HT2C/1B receptor agonist, had no effect on hypothalamic NOR1 expression, while suppressing food intake in {beta}-endorphin-deficient mice. These findings suggest that 5-HT2C receptor-independent proopiomelanocortin-derived peptides regulate the expression of hypothalamic NOR1, which is a novel modulator of feeding behavior and energy balance.« less

Short-Term Hyperprolactinemia Reduces the Expression of Purinergic P2X7 Receptors during Allergic Inflammatory Response of the Lungs.

PubMed

Ochoa-Amaya, Julieta E; Queiroz-Hazarbassanov, Nicolle; Namazu, Lilian B; Calefi, Atilio S; Tobaruela, Carla N; Margatho, Rafael; Palermo-Neto, João; Ligeiro de Oliveira, Ana P; Felicio, Luciano F

2018-06-06

We have previously shown that domperidone-induced short-term hyperprolactinemia reduces the lung's allergic inflammatory response in an ovalbumin antigenic challenge model. Since purinergic receptor P2X7R activity leads to proinflammatory cytokine release and is possibly related to the pathogenesis of allergic respiratory conditions, the present study was designed to investigate a possible involvement of purinergic and prolactin receptors in this phenomenon. To induce hyperprolactinemia, domperidone was injected intraperitoneally in rats at a dose of 5.1 mg × kg-1 per day for 5 days. P2X7 expression was evaluated by lung immunohistochemistry while prolactin receptor expression in bronchoalveolar lavage leukocytes was analyzed through flow cytometry. Previous reports demonstrated that rats subjected to short-term hyperprolactinemia exhibited a decrease in leukocyte counts in bronchoalveolar lavage, especially granulocytes. Here, it is revealed that hyperprolactinemia promotes an increased expression of prolactin receptors in granulocytes. Also, increased expression of purinergic P2X7R observed in allergic animals was significantly reduced by hyperprolactinemia. Both purinergic and prolactin receptor expression changes occur during the anti-asthmatic effect of hyperprolactinemia. © 2018 S. Karger AG, Basel.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions.

PubMed

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity.

Mining microarray datasets in nutrition: expression of the GPR120 (n-3 fatty acid receptor/sensor) gene is down-regulated in human adipocytes by macrophage secretions

PubMed Central

Trayhurn, Paul; Denyer, Gareth

2012-01-01

Microarray datasets are a rich source of information in nutritional investigation. Targeted mining of microarray data following initial, non-biased bioinformatic analysis can provide key insight into specific genes and metabolic processes of interest. Microarrays from human adipocytes were examined to explore the effects of macrophage secretions on the expression of the G-protein-coupled receptor (GPR) genes that encode fatty acid receptors/sensors. Exposure of the adipocytes to macrophage-conditioned medium for 4 or 24 h had no effect on GPR40 and GPR43 expression, but there was a marked stimulation of GPR84 expression (receptor for medium-chain fatty acids), the mRNA level increasing 13·5-fold at 24 h relative to unconditioned medium. Importantly, expression of GPR120, which encodes an n-3 PUFA receptor/sensor, was strongly inhibited by the conditioned medium (15-fold decrease in mRNA at 24 h). Macrophage secretions have major effects on the expression of fatty acid receptor/sensor genes in human adipocytes, which may lead to an augmentation of the inflammatory response in adipose tissue in obesity. PMID:25191551

Hypoxia attenuates purinergic P2X receptor-induced inflammatory gene expression in brainstem microglia

PubMed Central

Smith, Stephanie MC; Mitchell, Gordon S; Friedle, Scott A; Sibigtroth, Christine M; Vinit, Stéphane; Watters, Jyoti J

2013-01-01

Hypoxia and increased extracellular nucleotides are frequently coincident in the brainstem. Extracellular nucleotides are potent modulators of microglial inflammatory gene expression via P2X purinergic receptor activation. Although hypoxia is also known to modulate inflammatory gene expression, little is known about how hypoxia or P2X receptor activation alone affects inflammatory molecule production in brainstem microglia, nor how hypoxia and P2X receptor signaling interact when they occur together. In the study reported here, we investigated the ability of a brief episode of hypoxia (2 hours) in the presence and absence of the nonselective P2X receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine-5′-triphosphate (BzATP) to promote inflammatory gene expression in brainstem microglia in adult rats. We evaluated inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNFα), and interleukin (IL)-6 messenger RNA levels in immunomagnetically isolated brainstem microglia. While iNOS and IL-6 gene expression increased with hypoxia and BzATP alone, TNFα expression was unaffected. Surprisingly, BzATP-induced inflammatory effects were lost after hypoxia, suggesting that hypoxia impairs proinflammatory P2X-receptor signaling. We also evaluated the expression of key P2X receptors activated by BzATP, namely P2X1, P2X4, and P2X7. While hypoxia did not alter their expression, BzATP upregulated P2X4 and P2X7 mRNAs; these effects were ablated in hypoxia. Although both P2X4 and P2X7 receptor expression correlated with increased microglial iNOS and IL-6 levels in microglia from normoxic rats, in hypoxia, P2X7 only correlated with IL-6, and P2X4 correlated only with iNOS. In addition, correlations between P2X7 and P2X4 were lost following hypoxia, suggesting that P2X4 and P2X7 receptor signaling differs in normoxia and hypoxia. Together, these data suggest that hypoxia suppresses P2X receptor-induced inflammatory gene expression, indicating a potentially immunosuppressive role of extracellular nucleotides in brainstem microglia following exposure to hypoxia. PMID:24377098

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

PubMed

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

2007-01-01

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

Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

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

Raufman, Jean-Pierre, E-mail: jraufman@medicine.umaryland.edu; Cheng, Kunrong; Saxena, Neeraj

2011-11-18

Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasionmore » of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination.« less

Estrogen Receptor-α Correlates with Higher Fungal Cell Number in Oral Paracoccidioidomycosis in Women.

PubMed

Caixeta, Clenivaldo Alves; de Carli, Marina Lara; Ribeiro Júnior, Noé Vital; Sperandio, Felipe Fornias; Nonogaki, Suely; Nogueira, Denismar Alves; Pereira, Alessandro Antônio Costa; Hanemann, João Adolfo Costa

2018-05-23

Paracoccidioidomycosis is a neglected tropical fungal infection with great predilection for adult men, indicating the participation of female hormone estrogen in preventing paracoccidioidomycosis development in women. Estrogen has an immunologic effect leading to polarization toward the Th2 immune response, which favors the disease evolution. To evaluate estrogen and progesterone receptors in oral paracoccidioidomycosis lesions and to verify any association with tissue fungi counting in women and men. Thirty-two cases of chronic oral paracoccidioidomycosis were included. Immunohistochemical analyses for anti-estrogen receptor-α, anti-progesterone receptor and anti-Paracoccidioides brasiliensis antibodies were performed. The differences between women and men and the relations among the immunomarkers for each gender were also evaluated. A significant positive correlation was observed between estrogen receptor-α and the amount of fungi in women. In addition, estrogen receptor-α was mildly expressed in the inflammatory cells of female patients, while progesterone receptor was expressed in both genders, with similar expression between women and men. Moreover, fungi counting revealed no differences between genders. Estrogen receptor-α was expressed only in women and showed a positive correlation with the amount of fungi in oral paracoccidioidomycosis, while progesterone receptor was observed in both genders and exhibited no correlation with estrogen receptor-α or fungi counting.

Functional roles of the sweet taste receptor in oral and extraoral tissues

PubMed Central

Laffitte, Anni; Neiers, Fabrice; Briand, Loïc

2014-01-01

Purpose of review This review summarizes and discusses the current knowledge about the physiological roles of the sweet taste receptor in oral and extraoral tissues. Recent findings The expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, including the gut, pancreas, bladder, brain and, more recently, bone and adipose tissues. In the gut, this receptor has been suggested to be involved in luminal glucose sensing, the release of some satiety hormones, the expression of glucose transporters, and the maintenance of glucose homeostasis. More recently, the sweet taste receptor was proposed to regulate adipogenesis and bone biology. Summary The perception of sweet taste is mediated by the T1R2/T1R3 receptor, which is expressed in the oral cavity, wherein it provides input on the caloric and macronutrient contents of ingested food. This receptor recognizes all the chemically diverse compounds perceived as sweet by human beings, including natural sugars and sweeteners. Importantly, the expression of a functional sweet taste receptor has been reported in numerous extragustatory tissues, wherein it has been proposed to regulate metabolic processes. This newly recognized role of the sweet taste receptor makes this receptor a potential novel therapeutic target for the treatment of obesity and related metabolic dysfunctions, such as diabetes and hyperlipidemia. PMID:24763065

Effect of Peptide Receptor Radionuclide Therapy on Somatostatin Receptor Status and Glucose Metabolism in Neuroendocrine Tumors: Intraindividual Comparison of Ga-68 DOTANOC PET/CT and F-18 FDG PET/CT

PubMed Central

Oh, Sowon; Prasad, Vikas; Lee, Dong Soo; Baum, R. P.

2011-01-01

The heterogeneous nature of the neuroendocrine tumors (NET) makes it challenging to find one uniformly applicable management protocol which is especially true for diagnosis. The discovery of the overexpression of somatostatin receptors (SMS-R) on neuroendocrine tumor cells lead to the generalized and rapid acceptance of radiolabeled somatostatin receptor analogs for staging and restaging of NET as well as for Peptide Receptor Radionuclide Therapy (PRRNT) using Y-90 and Lu-177 DOTATATE/DOTATOC. In this present work we tried to look in to the effect of PRRNT on the glucose metabolism assessed by F-18 FDG PET/CT and SMS-R density assessed by Ga-68 DOTANOC PET/CT. We observed a complex relationship between the somatostatin receptor expression and glucose metabolism with only 56% (77/138) of the lesions showing match, while the others show mismatch between the receptor status and metabolism. The match between receptor expression and glucose metabolism increases with the grade of NET. In grade 3 NET, there is a concurrence between the changes in glucose metabolism and somatostatin receptor expression. PRRNT was found to be more effective in lesions with higher receptor expression. PMID:22121482

Cloning, expression and functional role of a nociceptin/orphanin FQ receptor in the porcine gastrointestinal tract.

PubMed

Osinski, M A; Pampusch, M S; Murtaugh, M P; Brown, D R

1999-01-22

The heptadecapeptide nociceptin/orphanin FQ is the cognate ligand for the opioid receptor-like orphanin FQ (OFQ) receptor, a member of the G protein-coupled receptor superfamily. The gastrointestinal tract is a major site of opioid action, and preliminary evidence suggests that an OFQ receptor may be expressed in rat small intestine. We addressed the hypothesis that this receptor is expressed in the gastrointestinal tract of the pig, a model for the human digestive system. A 1205-bp cDNA was isolated from porcine forebrain which contained the 370 amino acid open reading frame encoding the OFQ receptor. The receptor mRNA is likely to arise from a single gene, as determined by Southern blotting of porcine genomic DNA restriction digests using a porcine OFQ receptor cDNA probe. A semi-nested reverse transcriptase-polymerase chain reaction survey of receptor mRNA indicates that it is expressed in the porcine cerebral cortex and kidney, and along the length of the gastrointestinal tract. OFQ decreased initial contractile responses of porcine ileal smooth muscle strips to trains of electrical field stimulation with an IC50 value of 1.3 nM; its effects were resistant to the opioid antagonist, naloxone. The peptide, at concentrations > or =3 nM, also attenuated Isc elevations evoked by electrical transmural stimulation of mucosa-submucosa sheets from porcine ileum. The actions of OFQ appeared to differ from those previously reported for opioid receptor agonists in these tissue preparations. These results indicate that an OFQ receptor is expressed in the porcine intestine which modulates the neural control of intestinal smooth muscle contractility and mucosal transport.

α2-containing GABAA receptors expressed in hippocampal region CA3 control fast network oscillations

PubMed Central

Heistek, Tim S; Ruiperez-Alonso, Marta; Timmerman, A Jaap; Brussaard, Arjen B; Mansvelder, Huibert D

2013-01-01

GABAA receptors are critically involved in hippocampal oscillations. GABAA receptor α1 and α2 subunits are differentially expressed throughout the hippocampal circuitry and thereby may have distinct contributions to oscillations. It is unknown which GABAA receptor α subunit controls hippocampal oscillations and where these receptors are expressed. To address these questions we used transgenic mice expressing GABAA receptor α1 and/or α2 subunits with point mutations (H101R) that render these receptors insensitive to allosteric modulation at the benzodiazepine binding site, and tested how increased or decreased function of α subunits affects hippocampal oscillations. Positive allosteric modulation by zolpidem prolonged decay kinetics of hippocampal GABAergic synaptic transmission and reduced the frequency of cholinergically induced oscillations. Allosteric modulation of GABAergic receptors in CA3 altered oscillation frequency in CA1, while modulation of GABA receptors in CA1 did not affect oscillations. In mice having a point mutation (H101R) at the GABAA receptor α2 subunit, zolpidem effects on cholinergically induced oscillations were strongly reduced compared to wild-type animals, while zolpidem modulation was still present in mice with the H101R mutation at the α1 subunit. Furthermore, genetic knockout of α2 subunits strongly reduced oscillations, whereas knockout of α1 subunits had no effect. Allosteric modulation of GABAergic receptors was strongly reduced in unitary connections between fast spiking interneurons and pyramidal neurons in CA3 of α2H101R mice, but not of α1H101R mice, suggesting that fast spiking interneuron to pyramidal neuron synapses in CA3 contain α2 subunits. These findings suggest that α2-containing GABAA receptors expressed in the CA3 region provide the inhibition that controls hippocampal rhythm during cholinergically induced oscillations. PMID:23109109

ACTIVATION OF MU OPIOID RECEPTORS IN THE STRIATUM DIFFERENTIALLY AUGMENTS METHAMPHETAMINE-INDUCED GENE EXPRESSION AND ENHANCES STEREOTYPIC BEHAVIOR

PubMed Central

Horner, Kristen A.; Hebbard, John C.; Logan, Anna S.; Vanchipurakel, Golda A.; Gilbert, Yamiece E.

2013-01-01

Mu opioid receptors are densely expressed in the patch compartment of striatum and contribute to methamphetamine-induced patch-enhanced gene expression and stereotypy. In order to further elucidate the role of mu opioid receptor activation in these phenomena, we examined whether activation of mu opioid receptors would enhance methamphetamine-induced stereotypy and prodynorphin, c-fos, arc and zif/268 expression in the patch and/or matrix compartments of striatum, as well as the impact of mu opioid receptor activation on the relationship between patch-enhanced gene expression and stereotypy. Male Sprague-Dawley rats were intrastriatally infused with D-Ala(2)-N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO; 1 μg/μl), treated with methamphetamine (0.5 mg/kg) and sacrificed at 45 minutes or 2 hours later. DAMGO augmented methamphetamine-induced zif/268 mRNA expression in the patch and matrix compartments, while prodynorphin expression was increased in the dorsolateral patch compartment. DAMGO pretreatment did not affect methamphetamine-induced arc and c-fos expression. DAMGO enhanced methamphetamine-induced stereotypy and resulted in greater patch versus matrix expression of prodynorphin in the dorsolateral striatum, leading to a negative correlation between the two. These findings indicate that mu opioid receptors contribute to methamphetamine-induced stereotypy, but can differentially influence the genomic responses to methamphetamine. These data also suggest that prodynorphin may offset the overstimulation of striatal neurons by methamphetamine. PMID:22150526

Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain

PubMed Central

de Oliveira-Carlos, Vanessa; Ganz, Julia; Hans, Stefan; Kaslin, Jan; Brand, Michael

2013-01-01

The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches. PMID:24039926

Hepatic Stellate Cells Express Functional CXCR4: Role in Stromal Cell–Derived Factor-1α–Mediated Stellate Cell Activation

PubMed Central

Hong, Feng; Tuyama, Ana; Lee, Ting Fang; Loke, Johnny; Agarwal, Ritu; Cheng, Xin; Garg, Anita; Fiel, M. Isabel; Schwartz, Myron; Walewski, Jose; Branch, Andrea; Schecter, Alison D.; Bansal, Meena B.

2010-01-01

Chemokine interactions with their receptors have been implicated in hepatic stellate cell (HSC) activation. The hepatic expression of CXCR4 messenger RNA is increased in hepatitis C cirrhotic livers and plasma levels of its endogenous ligand, stromal cell–derived factor-1α (SDF-1α), correlate with increased fibrosis in these patients. The expression of CXCR4 by HSCs has not been reported. We therefore examined whether HSCs express CXCR4 in vivo and in vitro and explored whether SDF-1α/CXCR4 receptor engagement promotes HSC activation, fibrogenesis, and proliferation. The hepatic protein expression of both CXCR4 and SDF-1α is increased in hepatitis C cirrhotic livers and immunoflourescent and immunohistochemical staining confirms that HSCs express CXCR4 in vivo. Immortalized human stellate cells as well as primary human HSCs express CXCR4, and cell surface receptor expression increases with progressive culture-induced activation. Treatment of stellate cells with recombinant SDF-1α increases expression of α-smooth muscle actin and collagen I and stimulates a dose-dependent increase in HSC proliferation. Inhibitor studies suggest that SDF-1α/CXCR4-dependent extracellular signal-regulated kinase 1/2 and Akt phosphorylation mediate effects on collagen I expression and stellate cell proliferation. Conclusion HSCs express CXCR4 receptor in vivo and in vitro. CXCR4 receptor activation by SDF-1α is profibrogenic through its effects on HSC activation, fibrogenesis, and proliferation. Extracellular signal-regulated kinase 1/2 and phosphoinositide 3-kinase pathways mediate SDF-1α–induced effects on HSC expression of collagen I and proliferation. The availability of small molecule inhibitors of CXCR4 make this receptor an appealing target for antifibrotic approaches. PMID:19434726

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

PubMed

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

2016-01-01

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

Expression of Steroid Receptors in Ameloblasts during Amelogenesis in Rat Incisors

PubMed Central

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

2016-01-01

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

Software architecture for a distributed real-time system in Ada, with application to telerobotics

NASA Technical Reports Server (NTRS)

Olsen, Douglas R.; Messiora, Steve; Leake, Stephen

1992-01-01

The architecture structure and software design methodology presented is described in the context of telerobotic application in Ada, specifically the Engineering Test Bed (ETB), which was developed to support the Flight Telerobotic Servicer (FTS) Program at GSFC. However, the nature of the architecture is such that it has applications to any multiprocessor distributed real-time system. The ETB architecture, which is a derivation of the NASA/NBS Standard Reference Model (NASREM), defines a hierarchy for representing a telerobot system. Within this hierarchy, a module is a logical entity consisting of the software associated with a set of related hardware components in the robot system. A module is comprised of submodules, which are cyclically executing processes that each perform a specific set of functions. The submodules in a module can run on separate processors. The submodules in the system communicate via command/status (C/S) interface channels, which are used to send commands down and relay status back up the system hierarchy. Submodules also communicate via setpoint data links, which are used to transfer control data from one submodule to another. A submodule invokes submodule algorithms (SMA's) to perform algorithmic operations. Data that describe or models a physical component of the system are stored as objects in the World Model (WM). The WM is a system-wide distributed database that is accessible to submodules in all modules of the system for creating, reading, and writing objects.

Patients'/Clients' Expectation Toward and Satisfaction from Pharmacy Services

PubMed Central

Ayalew, Mohammed Biset; Taye, Kaleab; Asfaw, Daniel; Lemma, Bethlehem; Dadi, Filagot; Solomon, Habtamu; Tazeze, Haile; Tsega, Bayew

2017-01-01

Objective: Satisfaction is becoming a popular health-care quality indicator as it reflects the reality of service or care provided. The aim of this study was to assess the level of patients' expectation toward and satisfaction from pharmacy service provided and to identify associated factor that might affect their expectation and satisfaction. Methods: A cross-sectional study was conducted on 287 patients, who were served in five pharmacies of Gondar University Hospital in May 2015. Data regarding socio-demographic characteristics and parameters that measure patients' expectation and satisfaction were collected through interview using the Amharic version of the questionnaire. Data were entered into SPSS version 21, and descriptive statistics, cross-tabs, and binary logistic regressions were utilized. P < 0.05 was used to declare association. Findings: Among 287 respondents involved in the study, 149 (51.9%) claimed to be satisfied with the pharmacy service and setting. Two hundred and twenty-nine (79.4%) respondents have high expectation toward gaining good services. Even though significant association was observed between the pharmacy type and patients level of satisfaction, sociodemographic characteristics of a patient were not found to predict the level of satisfaction. There is a higher level of expectation among study participants who earn higher income per month (>(2000 Ethiopian birr [ETB]) than those who get less income (<1000 ETB). Conclusion: Although patients have a higher level of expectation toward pharmacy services, their satisfaction from the service was found to be low. PMID:28331862

Health assessment of gasoline and fuel oxygenate vapors: immunotoxicity evaluation.

PubMed

White, Kimber L; Peachee, Vanessa L; Armstrong, Sarah R; Twerdok, Lorraine E; Clark, Charles R; Schreiner, Ceinwen A

2014-11-01

Female Sprague Dawley rats were exposed via inhalation to vapor condensates of either gasoline or gasoline combined with various fuel oxygenates to assess potential immunotoxicity of evaporative emissions. Test articles included vapor condensates prepared from "baseline gasoline" (BGVC), or gasoline combined with methyl tertiary butyl ether (G/MTBE), ethyl t-butyl ether (G/ETBE), t-amyl methyl ether (G/TAME), diisopropyl ether (G/DIPE), ethanol (G/EtOH), or t-butyl alcohol (G/TBA). Target concentrations were 0, 2000, 10,000 or 20,000mg/mg(3) administered for 6h/day, 5days/week for 4weeks. The antibody-forming cell (AFC) response to the T-dependent antigen, sheep erythrocyte (sRBC), was used to determine the effects of the gasoline vapor condensates on the humoral components of the immune system. Exposure to BGVC, G/MTBE, G/TAME, and G/TBA did not result in significant changes in the IgM AFC response to sRBC, when evaluated as either specific activity (AFC/10(6) spleen cells) or as total spleen activity (AFC/spleen). Exposure to G/EtOH and G/DIPE resulted in a dose-dependent decrease in the AFC response, reaching the level of statistical significance only at the high 20,000mg/m(3) level. Exposure to G/ETBE resulted in a statistically significant decrease in the AFC response at the middle (10,000mg/m(3)) and high (20,000mg/m(3)) exposure concentrations. Copyright © 2014 Elsevier Inc. All rights reserved.

Role of decoy molecules in neuronal ischemic preconditioning

PubMed Central

Panneerselvam, Mathivadhani; Patel, Piyush M.; Roth, David M.; Kidd, Michael W.; Chin-Lee, Blake; Head, Brian P.; Niesman, Ingrid R.; Inoue, Satoki; Patel, Hemal H.; Davis, Daniel P.

2011-01-01

Decoy receptors bind with TNF related apoptosis inducing ligands (TRAIL) but do not contain the cytoplasmic domains necessary to transduce apoptotic signals. We hypothesized that decoy receptors may confer neuronal protection against lethal ischemia after ischemic preconditioning (IPC). Mixed cortical neurons were exposed to IPC one day prior to TRAIL treatment or lethal ischemia. IPC increased decoy receptor but reduced death receptor expression compared to lethal ischemia. IPC-induced increase in decoy receptor expression was reduced by prior treatment with CAPE, a nuclear factor-kappa B inhibitor (NFκB). Expression of decoy molecules, dependent on NFκB, may mediate neuronal survival induced by IPC. PMID:21315738

Predominant mucosal expression of 5-HT4(+h) receptor splice variants in pig stomach and colon

PubMed Central

Priem, Evelien KV; De Maeyer, Joris H; Vandewoestyne, Mado; Deforce, Dieter; Lefebvre, Romain A

2013-01-01

AIM: To investigate cellular 5-HT4(-h/+h) receptor distribution, particularly in the epithelial layer, by laser microdissection and polymerase chain reaction (PCR) in porcine gastrointestinal (GI) tissues. METHODS: A stepwise approach was used to evaluate RNA quality and to study cell-specific 5-HT4 receptor mRNA expression in the porcine gastric fundus and colon descendens. After freezing, staining and laser microdissection and pressure catapulting (LMPC), RNA quality was evaluated by the Experion automated electrophoresis system. 5-HT4 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expressions were examined by endpoint reverse transcription (RT)-PCR in mucosal and muscle-myenteric plexus (MMP) tissue fractions, in mucosal and MMP parts of hematoxylin and eosin (HE) stained tissue sections and in microdissected patches of the epithelial and circular smooth muscle cell layer in these sections. Pig gastric fundus tissue sections were also stained immunohistochemically (IHC) for enterochromaffin cells (EC cells; MAB352); these cells were isolated by LMPC and examined by endpoint RT-PCR. RESULTS: After HE staining, the epithelial and circular smooth muscle cell layer of pig colon descendens and the epithelial cell layer of gastric fundus were identified morphologically and isolated by LMPC. EC cells of pig gastric fundus were successfully stained by IHC and isolated by LMPC. Freezing, HE and IHC staining, and LMPC had no influence on RNA quality. 5-HT4 receptor and GAPDH mRNA expressions were detected in mucosa and MMP tissue fractions, and in mucosal and MMP parts of HE stained tissue sections of pig colon descendens and gastric fundus. In the mucosa tissue fractions of both GI regions, the expression of h-exon containing receptor [5-HT4(+h) receptor] mRNA was significantly higher (P < 0.01) compared to 5-HT4(-h) receptor expression, and a similar trend was obtained in the mucosal part of HE stained tissue sections. Large microdissected patches of the epithelial and circular smooth muscle cell layer of pig colon descendens and of the epithelial cell layer of pig gastric fundus, also showed 5-HT4 receptor and GAPDH mRNA expression. No 5-HT4 receptor mRNA expression was detected in gastric LMPC-isolated EC cells from IHC stained tissues, which cells were positive for GAPDH. CONCLUSION: Porcine GI mucosa predominantly expresses 5-HT4(+h) receptor splice variants, suggesting their contribution to the 5-HT4 receptor-mediated mucosal effects of 5-HT. PMID:23840113

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

Progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition.

PubMed

Palm, F; Walter, I; Nowotny, N; Budik, S; Helmreich, M; Aurich, C

2013-01-01

In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected. Copyright © 2013 Elsevier Inc. All rights reserved.

Expression patterns of STAT3, ERK and estrogen-receptor α are associated with development and histologic severity of hepatic steatosis: a retrospective study.

PubMed

Choi, Euno; Kim, Won; Joo, Sae Kyung; Park, Sunyoung; Park, Jeong Hwan; Kang, Yun Kyung; Jin, So-Young; Chang, Mee Soo

2018-04-03

Hepatic steatosis renders hepatocytes vulnerable to injury, resulting in the progression of preexisting liver disease. Previous animal and cell culture studies implicated mammalian target of rapamycin (mTOR), signal transducer and activator of transcription-3 (STAT3), extracellular signal-regulated kinase (ERK) and estrogen-receptor α in the pathogenesis of hepatic steatosis and disease progression. However, to date there have been few studies performed using human liver tissue to study hepatic steatosis. We examined the expression patterns of mTOR, STAT3, ERK and estrogen-receptor α in liver tissues from patients diagnosed with hepatic steatosis. We reviewed the clinical and histomorphological features of 29 patients diagnosed with hepatic steatosis: 18 with non-alcoholic fatty liver disease (NAFLD), 11 with alcoholic fatty acid disease (AFLD), and a control group (16 biliary cysts and 22 hepatolithiasis). Immunohistochemistry was performed on liver tissue using an automated immunostainer. The histologic severity of hepatic steatosis was evaluated by assessing four key histomorphologic parameters common to NAFLD and AFLD: steatosis, lobular inflammation, ballooning degeneration and fibrosis. mTOR, phosphorylated STAT3, phosphorylated pERK, estrogen-receptor α were found to be more frequently expressed in the hepatic steatosis group than in the control group. Specifically, mTOR was expressed in 78% of hepatocytes, and ERK in 100% of hepatic stellate cells, respectively, in patients with NAFLD. Interestingly, estrogen-receptor α was diffusely expressed in hepatocytes in all NALFD cases. Phosphorylated (active) STAT3 was expressed in 73% of hepatocytes and 45% of hepatic stellate cells in patients with AFLD, and phosphorylated (active) ERK was expressed in hepatic stellate cells in all AFLD cases. Estrogen-receptor α was expressed in all AFLD cases (focally in 64% of AFLD cases, and diffusely in 36%). Phosphorylated STAT3 expression in hepatocytes and hepatic stellate cells correlated with severe lobular inflammation, severe ballooning degeneration and advanced fibrosis, whereas diffusely expressed estrogen-receptor α correlated with a mild stage of fibrosis. Our data indicate ERK activation and estrogen-receptor α may be relevant in the development of hepatic steatosis. However, diffuse expression of estrogen-receptor α would appear to impede disease progression, including hepatic fibrosis. Finally, phosphorylated STAT3 may also contribute to disease progression.

Rejection of syngeneic colon carcinoma by CTLs expressing single-chain antibody receptors codelivering CD28 costimulation.

PubMed

Haynes, Nicole M; Trapani, Joseph A; Teng, Michele W L; Jackson, Jacob T; Cerruti, Loretta; Jane, Stephen M; Kershaw, Michael H; Smyth, Mark J; Darcy, Phillip K

2002-11-15

A new strategy to improve the therapeutic utility of redirected T cells for cancer involves the development of novel Ag-specific chimeric receptors capable of stimulating optimal and sustained T cell antitumor activity in vivo. Given that T cells require both primary and costimulatory signals for optimal activation and that many tumors do not express critical costimulatory ligands, modified single-chain Ab receptors have been engineered to codeliver CD28 costimulation. In this study, we have compared the antitumor potency of primary T lymphocytes expressing carcinoembryonic Ag (CEA)-reactive chimeric receptors that incorporate either TCR-zeta or CD28/TCR-zeta signaling. Although both receptor-transduced T cell effector populations demonstrated cytolysis of CEA(+) tumors in vitro, T cells expressing the single-chain variable fragment of Ig (scFv)-CD28-zeta chimera had a far greater capacity to control the growth of CEA(+) xenogeneic and syngeneic colon carcinomas in vivo. The observed enhanced antitumor activity of T cells expressing the scFv-CD28-zeta receptor was critically dependent on perforin and the production of IFN-gamma. Overall, this study has illustrated the ability of a chimeric scFv receptor capable of harnessing the signaling machinery of both TCR-zeta and CD28 to augment T cell immunity against tumors that have lost expression of both MHC/peptide and costimulatory ligands in vivo.

Differential regulation of NMDA receptor-expressing neurons in the rat hippocampus and striatum following bilateral vestibular loss demonstrated using flow cytometry.

PubMed

Benoit, Alice; Besnard, Stephane; Guillamin, Maryline; Philoxene, Bruno; Sola, Brigitte; Le Gall, Anne; Machado, Marie-Laure; Toulouse, Joseph; Hitier, Martin; Smith, Paul F

2018-06-21

There is substantial evidence that loss of vestibular function impairs spatial learning and memory related to hippocampal (HPC) function, as well as increasing evidence that striatal (Str) plasticity is also implicated. Since the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is considered essential to spatial memory, previous studies have investigated whether the expression of HPC NMDA receptors changes following vestibular loss; however, the results have been contradictory. Here we used a novel flow cytometric method to quantify the number of neurons expressing NMDA receptors in the HPC and Str following bilateral vestibular loss (BVL) in rats. At 7 and 30 days post-op., there was a significant increase in the number of HPC neurons expressing NMDA receptors in the BVL animals, compared to sham controls (P ≤ 0.004 and P ≤ 0.0001, respectively). By contrast, in the Str, at 7 days there was a significant reduction in the number of neurons expressing NMDA receptors in the BVL group (P ≤ 0.05); however, this difference had disappeared by 30 days post-op. These results suggest that BVL causes differential changes in the number of neurons expressing NMDA receptors in the HPC and Str, which may be related to its long-term impairment of spatial memory. Copyright © 2018. Published by Elsevier B.V.

Induction of cyclooxygenase-2 expression by prostaglandin E2 stimulation of the prostanoid EP4 receptor via coupling to Gαi and transactivation of the epidermal growth factor receptor in HCA-7 human colon cancer cells.

PubMed

Yoshida, Kenji; Fujino, Hiromichi; Otake, Sho; Seira, Naofumi; Regan, John W; Murayama, Toshihiko

2013-10-15

Increased expressions of cyclooxygenase-2 (COX-2) and its downstream metabolite, prostaglandin E2 (PGE2), are well documented events in the development of colorectal cancer. Interestingly, PGE2 itself can induce the expression of COX-2 thereby creating the potential for positive feedback. Although evidence for such a positive feedback has been previously described, the specific E-type prostanoid (EP) receptor subtype that mediates this response, as well as the relevant signaling pathways, remain unclear. We now report that the PGE2 stimulated induction of COX-2 expression in human colon cancer HCA-7 cells is mediated by activation of the prostanoid EP4 receptor subtype and is followed by coupling of the receptor to Gαi and the activation of phosphatidylinositol 3-kinase. Subsequent activation of metalloproteinases releases membrane bound heparin-binding epidermal growth factor-like growth factor resulting in the transactivation of epidermal growth factor receptors and the activation of the extracellular signal-regulated kinases and induction of COX-2 expression. This induction of COX-2 expression by PGE2 stimulation of the prostanoid EP4 receptor may underlie the upregulation of COX-2 during colorectal cancer and appears to be an early event in the process of tumorigenesis. © 2013 Elsevier B.V. All rights reserved.

Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila

PubMed Central

Benton, Richard; Vannice, Kirsten S.; Gomez-Diaz, Carolina; Vosshall, Leslie B.

2009-01-01

Summary Ionotropic glutamate receptors (iGluRs) mediate neuronal communication at synapses throughout vertebrate and invertebrate nervous systems. We have characterized a novel family of iGluR-related genes in Drosophila, which we name Ionotropic Receptors (IRs). These receptors do not belong to the well-described Kainate, AMPA, or NMDA classes of iGluRs, and have divergent ligand-binding domains that lack their characteristic glutamate-interacting residues. IRs are expressed in a combinatorial fashion in sensory neurons that respond to many distinct odors but do not express either insect odorant receptors (ORs) or gustatory receptors (GRs). IR proteins accumulate in sensory dendrites and not at synapses. Mis-expression of IRs induces novel odor responses in ectopic neurons. Together, these results lead us to propose that the IRs comprise a novel family of chemosensory receptors. Conservation of IR/iGluR-related proteins in bacteria, plants, and animals suggests that this receptor family represents an evolutionarily ancient mechanism for sensing both internal and external chemical cues. PMID:19135896

P2X receptors in the cardiovascular system and their potential as therapeutic targets in disease.

PubMed

Ralevic, Vera

2015-01-01

This review considers the expression and roles of P2X receptors in the cardiovascular system in health and disease and their potential as therapeutic targets. P2X receptors are ligand gated ion channels which are activated by the endogenous ligand ATP. They are formed from the assembly of three P2X subunit proteins from the complement of seven (P2X1-7), which can associate to form homomeric or heteromeric P2X receptors. The P2X1 receptor is widely expressed in the cardiovascular system, being located in the heart, in the smooth muscle of the majority of blood vessels and in platelets. P2X1 receptors expressed in blood vessels can be activated by ATP coreleased with noradrenaline as a sympathetic neurotransmitter, leading to smooth muscle depolarisation and contraction. There is evidence that the purinergic component of sympathetic neurotransmission is increased in hypertension, identifying P2X1 receptors as a possible therapeutic target in this disorder. P2X3 and P2X2/3 receptors are expressed on cardiac sympathetic neurones and may, through positive feedback of neuronal ATP at this prejunctional site, amplify sympathetic neurotransmission. Activation of P2X receptors expressed in the heart increases cardiac myocyte contractility, and an important role of the P2X4 receptor in this has been identified. Deletion of P2X4 receptors in the heart depresses contractile performance in models of heart failure, while overexpression of P2X4 receptors has been shown to be cardioprotective, thus P2X4 receptors may be therapeutic targets in the treatment of heart disease. P2X receptors have been identified on endothelial cells. Although immunoreactivity for all P2X1-7 receptor proteins has been shown on the endothelium, relatively little is known about their function, with the exception of the endothelial P2X4 receptor, which has been shown to mediate endothelium-dependent vasodilatation to ATP released during shear stress. The potential of P2X receptors as therapeutic targets in the treatment of cardiovascular disease is discussed.

Obesity induces functional astrocytic leptin receptors in hypothalamus

PubMed Central

Hsuchou, Hung; He, Yi; Kastin, Abba J.; Tu, Hong; Markadakis, Emily N.; Rogers, Richard C.; Fossier, Paul B.

2009-01-01

The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood–brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT–PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology. PMID:19293246

Estrogen Receptor-Related Receptor α Mediates Up-Regulation of Aromatase Expression by Prostaglandin E2 in Prostate Stromal Cells

PubMed Central

Miao, Lin; Shi, Jiandang; Wang, Chun-Yu; Zhu, Yan; Du, Xiaoling; Jiao, Hongli; Mo, Zengnan; Klocker, Helmut; Lee, Chung; Zhang, Ju

2010-01-01

Estrogen receptor-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily of transcription factors. ERRα is highly expressed in the prostate, especially in prostate stromal cells. However, little is known about the regulation and function of ERRα, which may contribute to the progression of prostatic diseases. We previously found that prostaglandin E2 (PGE2) up-regulated the expression of aromatase in prostate stromal cells. Here we show that PGE2 also up-regulates the expression of ERRα, which, as a transcription factor, further mediates the regulatory effects of PGE2 on the expression of aromatase. ERRα expression was up-regulated by PGE2 in prostate stromal cell line WPMY-1, which was mediated mainly through the protein kinase A signaling pathway by PGE2 receptor EP2. Suppression of ERRα activity by chlordane (an antagonist of ERRα) or small interfering RNA knockdown of ERRα blocked the increase of expression and promoter activity of aromatase induced by PGE2. Overexpression of ERRα significantly increased aromatase expression and promoter activity, which were further augmented by PGE2. Chromatin immunoprecipitation assay demonstrated that ERRα directly bound to the aromatase promoter in vivo, and PGE2 enhanced the recruitment of ERRα and promoted transcriptional regulatory effects on aromatase expression in WPMY-1. 17β-Estradiol concentration in WPMY-1 medium was up-regulated by ERRα expression, and that was further increased by PGE2. Our results provided evidence that ERRα contributed to local estrogen production by up-regulating aromatase expression in response to PGE2 and provided further insights into the potential role of ERRα in estrogen-related prostatic diseases. PMID:20351196

β3-Adrenergic receptors, adipokines and neuroendocrine activation during stress induced by repeated immune challenge in male and female rats.

PubMed

Csanova, Agnesa; Hlavacova, Natasa; Hasiec, Malgorzata; Pokusa, Michal; Prokopova, Barbora; Jezova, Daniela

2017-05-01

The main hypothesis of the study is that stress associated with repeated immune challenge has an impact on β 3 -adrenergic receptor gene expression in the brain. Sprague-Dawley rats were intraperitoneally injected with increasing doses of lipopolysaccharide (LPS) for five consecutive days. LPS treatment was associated with body weight loss and increased anxiety-like behavior. In LPS-treated animals of both sexes, β 3 -receptor gene expression was increased in the prefrontal cortex but not the hippocampus. LPS treatment decreased β 3 -receptor gene expression in white adipose tissue with higher values in males compared to females. In the adipose tissue, LPS reduced peroxisome proliferator-activated receptor-gamma, leptin and adiponectin gene expression, but increased interleukin-6 expression, irrespective of sex. Repeated immune challenge resulted in increased concentrations of plasma aldosterone and corticosterone with higher values of corticosterone in females compared to males. Concentrations of dehydroepiandrosterone (DHEA) in plasma were unaffected by LPS, while DHEA levels in the frontal cortex were lower in the LPS-treated animals compared to the controls. Thus, changes of DHEA levels in the brain take place irrespective of the changes of this neurosteroid in plasma. We have provided the first evidence on stress-induced increase in β 3 -adrenergic receptor gene expression in the brain. Greater reduction of β 3 -adrenergic receptor expression in the adipose tissue and of the body weight gain by repeated immune challenge in male than in female rats suggests sex differences in the role of β 3 -adrenergic receptors in the metabolic functions. LPS-induced changes in adipose tissue regulatory factors and hormone concentrations might be important for coping with chronic infections.

Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

PubMed Central

Nakagawa, Yuko; Nagasawa, Masahiro; Yamada, Satoko; Hara, Akemi; Mogami, Hideo; Nikolaev, Viacheslav O.; Lohse, Martin J.; Shigemura, Noriatsu; Ninomiya, Yuzo; Kojima, Itaru

2009-01-01

Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions Sweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms. PMID:19352508

Directing Energy Transfer in Panchromatic Platinum Complexes for Dual Vis-Near-IR or Dual Visible Emission from σ-Bonded BODIPY Dyes.

PubMed

Geist, Fabian; Jackel, Andrej; Irmler, Peter; Linseis, Michael; Malzkuhn, Sabine; Kuss-Petermann, Martin; Wenger, Oliver S; Winter, Rainer F

2017-01-17

We report on the platinum complexes trans-Pt(BODIPY)(8-ethynyl-BODIPY)(PEt 3 ) 2 (EtBPtB) and trans-Pt(BODIPY)(4-ethynyl-1,8-naphthalimide)(PR 3 ) 2 (R = Et, EtNIPtB-1; R = Ph, EtNIPtB-2), which all contain two different dye ligands that are connected to the platinum atom by a direct σ bond. The molecular structures of all complexes were established by X-ray crystallography and show that the different dye ligands are in either a coplanar or an orthogonal arrangement. π-stacking and several CH···F and short CH···π interactions involving protons at the phosphine substituents lead to interesting packing motifs in the crystal. The complexes feature several strong absorptions (ε = 3.2 × 10 5 -5.5 × 10 5 M -1 cm -1 ) that cover the regime from 350 to 480 nm (EtNIPtB-1 and EtNIPtB-2) or from 350 to 580 nm (EtBPtB). Besides the typical absorption bands of both kinds of attached dyes, they also feature an intense band near 400-420 nm, which is assigned by time-dependent density functional theory calculations to a higher-energy transition within the ethynyl-BODIPY (EtB) ligand or to charge transfer between the BODIPY (B) and naphthalimide (NI) chromophores. All complexes show dual fluorescence and phosphorescence emission from either the B (EtNIPtB-1 and EtNIPtB-2) or EtB (EtBPtB) ligand with a maximum phosphorescence quantum yield of 41% for EtNIPtB-1. The latter seems to be the highest reported value for room temperature phosphorescence from a BODIPY dye. The complete quenching of the emission from the chromophore absorbing at the higher energy and the appearance of the corresponding absorption bands in the fluorescence and phosphorescence excitation spectra indicate complete and rapid energy transfer to the chromophore with the lower-energy excited state, i.e., EtNI → B in EtNIPtB-1 and EtNIPtB-2 and B → EtB in EtBPtB. The latter process was further investigated by transient absorption spectroscopy, indicating that energy transfer is complete within 0.6 ns. EtNIPtB-1 catalyzes the photooxidation of 1,5-dihydroxynaphthalene with photogenerated 1 O 2 to Juglone at a much faster rate than methylene blue but with only modest quantum yields of 37% and with the onset of photodegradation after 60 min.

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Estrogen receptor-α, progesterone receptor, and c-erbB/HER-family receptor mRNA detection and phenotype analysis in spontaneous canine models of breast cancer

PubMed Central

Kabir, Farruk M. Lutful; DeInnocentes, Patricia; Agarwal, Payal; Mill, Christopher P.; Riese, David J.

2017-01-01

Well characterized, stable, p16-defective canine mammary cancer (CMT) cell lines and normal canine mammary epithelial cells were used to investigate expression of the major breast cancer-specific hormone receptors estrogen receptor alpha (ER1) and progesterone receptor (PR) as well as luminal epithelial-specific proto-oncogenes encoding c-erbB-1 (epidermal growth factor receptor/EGFr), c-erbB-2/HER2, c-erbB-3, and c-erbB-4 receptors. The investigation developed and validated quantitative reverse transcriptase polymerase chain reaction assays for each transcript to provide rapid assessment of breast cancer phenotypes for canine cancers, based on ER1, PR, and c-erbB-2/HER2 expressions, similar to those in human disease. Roles for relatively underexplored c-erbB-3 and c-erbB-4 receptor expressions in each of these breast cancer phenotypes were also evaluated. Each quantitative assay was validated by assessment of amplicon size and DNA sequencing following amplification. Differential expression of ER1, PR, and c-erbB-2 in CMT cell lines clearly defined distinct human-like breast cancer phenotypes for a selection of CMT-derived cell lines. Expression profiles for EGFr family genes c-erbB-3 and c-erbB-4 in CMT models also provided an enriched classification of canine breast cancer identifying new extended phenotypes beyond the conventional luminal-basal characterization used in human breast cancer. PMID:27515268

Effects of chronic ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons.

PubMed

McCool, Brian A; Frye, Gerald D; Pulido, Marisa D; Botting, Shaleen K

2003-02-14

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor's response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanol's anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

Brain aromatase (Cyp19A2) and estrogen receptors, in larvae and adult pejerrey fish Odontesthes bonariensis: Neuroanatomical and functional relations

USGS Publications Warehouse

Strobl-Mazzulla, P. H.; Lethimonier, C.; Gueguen, M.M.; Karube, M.; Fernandino, J.I.; Yoshizaki, G.; Patino, R.; Strussmann, C.A.; Kah, O.; Somoza, G.M.

2008-01-01

Although estrogens exert many functions on vertebrate brains, there is little information on the relationship between brain aromatase and estrogen receptors. Here, we report the cloning and characterization of two estrogen receptors, ?? and ??, in pejerrey. Both receptors' mRNAs largely overlap and were predominantly expressed in the brain, pituitary, liver, and gonads. Also brain aromatase and estrogen receptors were up-regulated in the brain of estradiol-treated males. In situ hybridization was performed to study in more detail, the distribution of the two receptors in comparison with brain aromatase mRNA in the brain of adult pejerrey. The estrogen receptors' mRNAs exhibited distinct but partially overlapping patterns of expression in the preoptic area and the mediobasal hypothalamus, as well as in the pituitary gland. Moreover, the estrogen receptor ??, but not ??, were found to be expressed in cells lining the preoptic recess, similarly as observed for brain aromatase. Finally, it was shown that the onset expression of brain aromatase and both estrogen receptors in the head of larvae preceded the morphological differentiation of the gonads. Because pejerrey sex differentiation is strongly influenced by temperature, brain aromatase expression was measured during the temperature-sensitive window and was found to be significantly higher at male-promoting temperature. Taken together these results suggest close neuroanatomical and functional relationships between brain aromatase and estrogen receptors, probably involved in the sexual differentiation of the brain and raising interesting questions on the origin (central or peripheral) of the brain aromatase substrate. ?? 2008 Elsevier Inc.

Finasteride Treatment Alters Tissue Specific Androgen Receptor Expression in Prostate Tissues

PubMed Central

Bauman, Tyler M.; Sehgal, Priyanka D.; Johnson, Karen A.; Pier, Thomas; Bruskewitz, Reginald C.; Ricke, William A.; Huang, Wei

2014-01-01

BACKGROUND Normal and pathologic growth of the prostate is dependent on the synthesis of dihydrotestosterone (DHT) from testosterone by 5α-reductase. Finasteride is a selective inhibitor of 5α-reductase 2, one isozyme of 5α-reductase found in abundance in the human prostate. The objective of this study was to investigate the effects of finasteride on androgen receptor expression and tissue morphology in human benign prostatic hyperplasia specimens. METHODS Patients undergoing transurethral resection of the prostate and either treated or not treated with finasteride between 2004 and 2010 at the University of Wisconsin-Hospital were retrospectively identified using an institutional database. Prostate specimens from each patient were triple-stained for androgen receptor, prostate-specific antigen, and basal marker cytokeratin 5. Morphometric analysis was performed using the multispectral imaging, and results were compared between groups of finasteride treated and non-treated patients. RESULTS Epithelial androgen receptor but not stromal androgen receptor expression was significantly lower in patients treated with finasteride than in non-treated patients. Androgen receptor-regulated prostate-specific antigen was not significantly decreased in finasteride-treated patients. Significant luminal epithelial atrophy and basal cell hyperplasia were prevalent in finasteride treated patients. Epithelial androgen receptor expression was highly correlated to the level of luminal epithelial atrophy. CONCLUSIONS In this study, finasteride decreased the expression of epithelial androgen receptor in a tissue specific manner. The correlation between epithelial androgen receptor and the extent of luminal epithelial atrophy suggests that epithelial androgen receptor may be directly regulating the atrophic effects observed with finasteride treatment. PMID:24789081

Nested Expression Domains for Odorant Receptors in Zebrafish Olfactory Epithelium

NASA Astrophysics Data System (ADS)

Weth, Franco; Nadler, Walter; Korsching, Sigrun

1996-11-01

The mapping of high-dimensional olfactory stimuli onto the two-dimensional surface of the nasal sensory epithelium constitutes the first step in the neuronal encoding of olfactory input. We have used zebrafish as a model system to analyze the spatial distribution of odorant receptor molecules in the olfactory epithelium by quantitative in situ hybridization. To this end, we have cloned 10 very divergent zebrafish odorant receptor molecules by PCR. Individual genes are expressed in sparse olfactory receptor neurons. Analysis of the position of labeled cells in a simplified coordinate system revealed three concentric, albeit overlapping, expression domains for the four odorant receptors analyzed in detail. Such regionalized expression should result in a corresponding segregation of functional response properties. This might represent the first step of spatial encoding of olfactory input or be essential for the development of the olfactory system.

Dopamine D2 Antagonist-Induced Striatal Nur77 Expression Requires Activation of mGlu5 Receptors by Cortical Afferents

PubMed Central

Maheux, Jérôme; St-Hilaire, Michel; Voyer, David; Tirotta, Emanuele; Borrelli, Emiliana; Rouillard, Claude; Rompré, Pierre-Paul; Lévesque, Daniel

2012-01-01

Dopamine D2 receptor antagonists modulate gene transcription in the striatum. However, the molecular mechanism underlying this effect remains elusive. Here we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, as readout to explore the role of dopamine, glutamate, and adenosine receptors in the effect of a dopamine D2 antagonist in the striatum. First, we investigated D2 antagonist-induced Nur77 mRNA in D2L receptor knockout mice. Surprisingly, deletion of the D2L receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Next, we tested if an ibotenic acid-induced cortical lesion could block the effect of eticlopride on Nur77 expression. Cortical lesions strongly reduced eticlopride-induced striatal upregulation of Nur77 mRNA. Then, we investigated if glutamatergic neurotransmission could modulate eticlopride-induced Nur77 expression. A combination of a metabotropic glutamate type 5 (mGlu5) and adenosine A2A receptor antagonists abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of Nur77 expression by striatal glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that blockade of postsynaptic D2 receptors is not sufficient to trigger striatal transcriptional activity and that interaction with corticostriatal presynaptic D2 receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum is required. Thus, these results uncover an unappreciated role of presynaptic D2 heteroreceptors and support a prominent role of glutamate in the effect of D2 antagonists. PMID:22912617

Role of hypothalamic cannabinoid receptors in post-stroke depression in rats.

PubMed

Wang, Shanshan; Sun, Hong; Liu, Sainan; Wang, Ting; Guan, Jinqun; Jia, Jianjun

2016-03-01

One of the most common psychological consequences of stroke is post-stroke depression (PSD). While more than 30 percent of stroke patients eventually develop PSD, the neurobiological mechanisms underlying such a phenomenon have not been well investigated. Given the critical involvement of hypothalamic-pituitary-adrenal axis and endocannabinoid system in response to stressful stimuli, we evaluated the hypothesis that cannabinoid receptors in the hypothalamus are critical for modulation of post-stroke depression-like behaviors in rats. To this end, rats were treated with middle cerebral artery occlusion (MCAO) followed by chronic unpredictable mild stress (CUMS) treatment procedure. We then assessed the expression of CB1 and CB2 receptors in the hypothalamus, and evaluated the effects of pharmacological stimulations of CB1 or CB2 receptors on the expression and development of depression-like behaviors in PSD rats. We found that PSD rats exhibited decreased the expression of CB1 receptor, but not CB2 receptor, in the ventral medial hypothalamus (VMH). Such an effect was not observed in the dorsally adjacent brain regions. Furthermore, intra-VMH injections of CB2 receptor agonist, but not CB1 receptor agonist, attenuated the expression of depression-like behaviors in PSD rats. Finally, repeated intraperitoneal injections of CB1 or CB2 receptor agonists during CUMS treatment inhibited the development of depression-like behaviors in PSD rats. Taken together, these results suggest that decreased CB1 receptor expression is likely associated with the development of post-stroke depression, and CB2 receptor may be a potential therapeutic target for the treatment post-stroke depressive disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

Attenuation of Cocaine-Induced Conditioned Place Preference and Motor Activity via Cannabinoid CB2 Receptor Agonism and CB1 Receptor Antagonism in Rats

PubMed Central

Delis, Foteini; Polissidis, Alexia; Poulia, Nafsika; Justinova, Zuzana; Nomikos, George G.; Goldberg, Steven R.

2017-01-01

Abstract Background: Studies have shown the involvement of cannabinoid (CB) receptors in the behavioral and neurobiological effects of psychostimulants. Most of these studies have focused on the role of CB1 receptors in the psychostimulant effects of cocaine, while very few have investigated the respective role of CB2 receptors. Further studies are warranted to elucidate the extent of CB receptor involvement in the expression of cocaine-induced effects. Methods: The role of CB1 and CB2 receptors in the rewarding and motor properties of cocaine was assessed in conditioned place preference, conditioned motor activity, and open field activity in rats. Results: The CB1 receptor antagonist rimonabant (3 mg/kg) decreased the acquisition and the expression of conditioned place preference induced by cocaine (20 mg/kg). Rimonabant inhibited cocaine-elicited conditioned motor activity when administered during the expression of cocaine-induced conditioned place preference. Rimonabant decreased ambulatory and vertical activity induced by cocaine. The CB2 receptor agonist JWH-133 (10 mg/kg) decreased the acquisition and the expression of cocaine-induced conditioned place preference. JWH-133 inhibited cocaine-elicited conditioned motor activity when administered during the acquisition and the expression of cocaine-induced conditioned place preference. JWH-133 decreased ambulatory activity and abolished vertical activity induced by cocaine. The effects of JWH-133 on cocaine conditioned and stimulated responses were abolished when the CB2 receptor antagonist/inverse agonist AM630 (5 mg/kg) was preadministered. Conclusions: Cannabinoid CB1 and CB2 receptors modulate cocaine-induced rewarding behavior and appear to have opposite roles in the regulation of cocaine’s reinforcing and psychomotor effects. PMID:27994006

Vitamin D receptor FokI genotype may modify the susceptibility to schizophrenia and bipolar mood disorder by regulation of dopamine D1 receptor gene expression.

PubMed

Ahmadi, S; Mirzaei, K; Hossein-Nezhad, A; Shariati, G

2012-10-01

This study is designed to test association of FOKI polymorphism in Vitamin D receptor (VDR) gene and its potential effect on expression of dopamine D1 receptor in schizophrenia and bipolar mood disorder as well as in healthy individuals. In this case-control study 196 patient with schizophrenia, 119 patients with bipolar mood disorder and 192 healthy individuals as the control group were recruited. All psychiatric disorders were diagnosed according to DSM IV criteria. Healthy control group denied any family history of such disorders. FOKI was genotyped by means of PCR-RFLP method. The mRNA was extracted from the peripheral blood mononuclear cells (PBMC) and the cDNA was synthesized. Frequency of ff genotype was more common in patients with bipolar disorders compared to the healthy control group (Odds ratio=1.84, 95% CI; 0.81 to 4.17) with increased relative risk (Relative risk=1.31, CI 95%; 0.86 to 1.99). There were significant differences between relative expressions of dopamine D1 receptor gene in various genotypes. Our results indicated that the ff genotype was associated with lower expression of dopamine D1 receptor gene. VDR as a nuclear receptor may contribute to bipolar disorders via modification of the expression of the neurotransmitters receptor such as dopamine.

Suppression of transient receptor potential melastatin 4 expression promotes conversion of endothelial cells into fibroblasts via transforming growth factor/activin receptor-like kinase 5 pathway.

PubMed

Echeverría, Cesar; Montorfano, Ignacio; Cabello-Verrugio, Claudio; Armisén, Ricardo; Varela, Diego; Simon, Felipe

2015-05-01

To study whether transient receptor potential melastatin 4 (TRPM4) participates in endothelial fibrosis and to investigate the underlying mechanism. Primary human endothelial cells were used and pharmacological and short interfering RNA-based approaches were used to test the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) pathway participation and contribution of TRPM7 ion channel. Suppression of TRPM4 expression leads to decreased endothelial protein expression and increased expression of fibrotic and extracellular matrix markers. Furthermore, TRPM4 downregulation increases intracellular Ca levels as a potential condition for fibrosis. The underlying mechanism of endothelial fibrosis shows that inhibition of TRPM4 expression induces TGF-β1 and TGF-β2 expression, which act through their receptor, ALK5, and the nuclear translocation of the profibrotic transcription factor smad4. TRPM4 acts to maintain endothelial features and its loss promotes fibrotic conversion via TGF-β production. The regulation of TRPM4 levels could be a target for preserving endothelial function during inflammatory diseases.

Genetic tracing of the gustatory and trigeminal neural pathways originating from T1R3-expressing taste receptor cells and solitary chemoreceptor cells.

PubMed

Ohmoto, Makoto; Matsumoto, Ichiro; Yasuoka, Akihito; Yoshihara, Yoshihiro; Abe, Keiko

2008-08-01

We established transgenic mouse lines expressing a transneuronal tracer, wheat germ agglutinin (WGA), under the control of mouse T1R3 gene promoter/enhancer. In the taste buds, WGA transgene was faithfully expressed in T1R3-positive sweet/umami taste receptor cells. WGA protein was transferred not laterally to the synapse-bearing, sour-responsive type III cells in the taste buds but directly to a subset of neurons in the geniculate and nodose/petrosal ganglia, and further conveyed to a rostro-central region of the nucleus of solitary tract. In addition, WGA was expressed in solitary chemoreceptor cells in the nasal epithelium and transferred along the trigeminal sensory pathway to the brainstem neurons. The solitary chemoreceptor cells endogenously expressed T1R3 together with bitter taste receptors T2Rs. This result shows an exceptional signature of receptor expression. Thus, the t1r3-WGA transgenic mice revealed the sweet/umami gustatory pathways from taste receptor cells and the trigeminal neural pathway from solitary chemoreceptor cells.

Neurotrophins and Neurotrophin Receptors in Proliferative Diabetic Retinopathy

PubMed Central

Abu El-Asrar, Ahmed M.; Mohammad, Ghulam; De Hertogh, Gert; Nawaz, Mohd Imtiaz; Van Den Eynde, Kathleen; Siddiquei, Mohammad Mairaj; Struyf, Sofie; Opdenakker, Ghislain; Geboes, Karel

2013-01-01

Neurotrophins (NTs) are emerging as important mediators of angiogenesis and fibrosis. We investigated the expression of the NTs nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) and their receptors TrkA, TrkB, and TrkC in proliferative diabetic retinopathy (PDR). As a comparison, we examined the expression of NTs and their receptors in the retinas of diabetic rats. Vitreous samples from 16 PDR and 15 nondiabetic patients were studied by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Epiretinal membranes from 17 patients with PDR were studied by immunohistochemistry. Rats were made diabetic with a single high dose of streptozotocin and retinas of rats were examined by Western blot analysis. Western blot analysis revealed a significant increase in the expression of NT-3 and NT-4 and the shedding of receptors TrkA and TrkB in vitreous samples from PDR patients compared to nondiabetic controls, whereas NGF and BDNF and the receptor TrkC were not detected with the use of Western blot analysis and ELISA. In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed NT-3 and the receptors TrkA, TrkB and TrkC in situ, whereas NT-4 was not detected. The expression levels of NT-3 and NT-4 and the receptors TrkA and TrkB, both in intact and solubilized forms, were upregulated in the retinas of diabetic rats, whereas the receptor TrkC was not detected. Co-immunoprecipitation studies revealed binding between NT-3 and the receptors TrkA and TrkB in the retinas of diabetic rats. Our findings in diabetic eyes from humans and rats suggest that the increased expression levels within the NT-3 and NT-4/Trk axis are associated with the progression of PDR. PMID:23762379

Profiling neurotransmitter receptor expression in the Ambystoma mexicanum brain.

PubMed

Reyes-Ruiz, Jorge Mauricio; Limon, Agenor; Korn, Matthew J; Nakamura, Paul A; Shirkey, Nicole J; Wong, Jamie K; Miledi, Ricardo

2013-03-22

Ability to regenerate limbs and central nervous system (CNS) is unique to few vertebrates, most notably the axolotl (Ambystoma sp.). However, despite the fact the neurotransmitter receptors are involved in axonal regeneration, little is known regarding its expression profile. In this project, RT-PCR and qPCR were performed to gain insight into the neurotransmitter receptors present in Ambystoma. Its functional ability was studied by expressing axolotl receptors in Xenopus laevis oocytes by either injection of mRNA or by direct microtransplantation of brain membranes. Oocytes injected with axolotl mRNA expressed ionotropic receptors activated by GABA, aspartate+glycine and kainate, as well as metabotropic receptors activated by acetylcholine and glutamate. Interestingly, we did not see responses following the application of serotonin. Membranes from the axolotl brain were efficiently microtransplanted into Xenopus oocytes and two types of native GABA receptors that differed in the temporal course of their responses and affinities to GABA were observed. Results of this study are necessary for further characterization of axolotl neurotransmitter receptors and may be useful for guiding experiments aimed at understanding activity-dependant limb and CNS regeneration. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cocaine-induced adaptation of dopamine D2S, but not D2L autoreceptors

PubMed Central

Radl, Daniela; Borrelli, Emiliana; Williams, John T; Neve, Kim A

2017-01-01

The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants. PMID:29154756

Cocaine-induced adaptation of dopamine D2S, but not D2L autoreceptors.

PubMed

Robinson, Brooks G; Condon, Alec F; Radl, Daniela; Borrelli, Emiliana; Williams, John T; Neve, Kim A

2017-11-20

The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants.

A candidate pheromone receptor and two odorant receptors of the hawkmoth Manduca sexta.

PubMed

Patch, Harland M; Velarde, Rodrigo A; Walden, Kimberly K O; Robertson, Hugh M

2009-05-01

In this study, we cloned and characterized three Manduca sexta odorant receptors (ORs). One receptor is a putative pheromone receptor expressed exclusively in a cell associated with male-specific type-I trichoid sensilla. We describe the results of real-time PCR (RT-PCR) and quantitative real-time PCR (qRT-PCR) experiments that show MsextaOR1 is expressed only in male antennae. In situ hybridization labels a single cell associated with type-1 trichoid sensilla, which houses two neurons that have been previously determined to respond to the major components of the pheromone blend. The second receptor, MsextaOR2, was discovered using degenerate primers designed to conserved motifs of a unique group ORs that share as much as 88% identity. Comparison of RT-PCR, qRT-PCR, and in situ hybridization results with those of ORs in the Drosophila melanogaster Or83b subfamily shows a strong sequence and expression pattern similarity. The third receptor, MsextaOR3, was found by 5'-end sequencing of a normalized and subtracted cDNA library from male M. sexta antennae. RT-PCR and qRT-PCR show that this receptor is expressed only in male and female antennae. These are the first ORs, including a putative pheromone receptor, to be described from M. sexta.

The Peripheral Olfactory Repertoire of the Lightbrown Apple Moth, Epiphyas postvittana

PubMed Central

Thrimawithana, Amali H.; Crowhurst, Ross N.; Newcomb, Richard D.

2015-01-01

The lightbrown apple moth, Epiphyas postvittana is an increasingly global pest of horticultural crops. Like other moths, E. postvittana relies on olfactory cues to locate mates and oviposition sites. To detect these cues, moths have evolved families of genes encoding elements of the peripheral olfactory reception system, including odor carriers, receptors and degrading enzymes. Here we undertake a transcriptomic approach to identify members of these families expressed in the adult antennae of E. postvittana, describing open reading frames encoding 34 odorant binding proteins, 13 chemosensory proteins, 70 odorant receptors, 19 ionotropic receptors, nine gustatory receptors, two sensory neuron membrane proteins, 27 carboxylesterases, 20 glutathione-S-transferases, 49 cytochrome p450s and 18 takeout proteins. For the odorant receptors, quantitative RT-PCR corroborated RNAseq count data on steady state transcript levels. Of the eight odorant receptors that group phylogenetically with pheromone receptors from other moths, two displayed significant male-biased expression patterns, one displayed significant female-biased expression pattern and five were expressed equally in the antennae of both sexes. In addition, we found two male-biased odorant receptors that did not group with previously described pheromone receptors. This suite of olfaction-related genes provides a substantial resource for the functional characterization of this signal transduction system and the development of odor-mediated control strategies for horticultural pests. PMID:26017144

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed Central

2014-01-01

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis. PMID:25011628

How much do we know about the coupling of G-proteins to serotonin receptors?

PubMed

Giulietti, Matteo; Vivenzio, Viviana; Piva, Francesco; Principato, Giovanni; Bellantuono, Cesario; Nardi, Bernardo

2014-07-10

Serotonin receptors are G-protein-coupled receptors (GPCRs) involved in a variety of psychiatric disorders. G-proteins, heterotrimeric complexes that couple to multiple receptors, are activated when their receptor is bound by the appropriate ligand. Activation triggers a cascade of further signalling events that ultimately result in cell function changes. Each of the several known G-protein types can activate multiple pathways. Interestingly, since several G-proteins can couple to the same serotonin receptor type, receptor activation can result in induction of different pathways. To reach a better understanding of the role, interactions and expression of G-proteins a literature search was performed in order to list all the known heterotrimeric combinations and serotonin receptor complexes. Public databases were analysed to collect transcript and protein expression data relating to G-proteins in neural tissues. Only a very small number of heterotrimeric combinations and G-protein-receptor complexes out of the possible thousands suggested by expression data analysis have been examined experimentally. In addition this has mostly been obtained using insect, hamster, rat and, to a lesser extent, human cell lines. Besides highlighting which interactions have not been explored, our findings suggest additional possible interactions that should be examined based on our expression data analysis.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens

USGS Publications Warehouse

Iwanowicz, Luke R.; Stafford, James L.; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W.; Blazer, Vicki

2014-01-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines.

Tumor necrosis factor-α inhibits angiotensin II receptor type 1 expression in dorsal root ganglion neurons via β-catenin signaling.

PubMed

Yang, Y; Wu, H; Yan, J-Q; Song, Z-B; Guo, Q-L

2013-09-17

Both tumor necrosis factor (TNF)-α and the angiotensin (Ang) II/angiotensin II receptor type 1 (AT1) axis play important roles in neuropathic pain and nociception. In the present study, we explored the interaction between the two systems by examining the mutual effects between TNF-α and the Ang II/AT1 receptor axis in dorsal root ganglion (DRG) neurons. Rat DRG neurons were treated with TNF-α in different concentrations for different lengths of time in the presence or absence of transcription inhibitor actinomycin D, TNF receptor 1 (TNFR1) inhibitor SPD304, β-catenin signaling inhibitor CCT031374, or different kinase inhibitors. TNF-α decreased the AT1 receptor mRNA level as well as the AT1a receptor promoter activity in a dose-dependent manner within 30 h, which led to dose-dependent inhibition of Ang II-binding AT1 receptor level on the cell membrane. Actinomycin D (1 mg/ml), SPD304 (50 μM), p38 mitogen-activated protein kinase (MAPK) inhibitor PD169316 (25 μM), and CCT031374 (50 μM) completely abolished the inhibitory effect of TNF-α on AT1 receptor expression. TNF-α dose-dependently increased soluble β-catenin and phosphorylated GSK-3β levels, which was blocked by SPD304 and PD169316. In DRG neurons treated with AT2 receptor agonist CGP421140, or Ang II with or without AT1 receptor antagonist losartan or AT2 receptor antagonist PD123319 for 30 h, we found that Ang II and Ang II+PD123319 significantly decreased TNF-α expression, whereas CPG421140 and Ang II+losartan increased TNF-α expression. In conclusion, we demonstrate that TNF-α inhibits AT1 receptor expression at the transcription level via TNFR1 in rat DRG neurons by increasing the soluble β-catenin level through the p38 MAPK/GSK-3β pathway. In addition, Ang II appears to inhibit and induce TNF-α expression via the AT1 receptor and the AT2 receptor in DRG neurons, respectively. This is the first evidence of crosstalk between TNF-α and the Ang II/AT receptor axis in DRG neurons. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

Flow cytometric monitoring of hormone receptor expression in human solid tumors

NASA Astrophysics Data System (ADS)

Krishan, Awtar

2002-05-01

Hormone receptor expression in human breast and prostate tumors is of diagnostic and therapeutic importance. With the availability of anti-estrogen, androgen and progesterone antibodies, immunohistochemistry has become a standard tool for determination of receptor expression in human tumor biopsies. However, this method is dependent on examination of a small number of cells under a microscope and the data obtained in most cases is not quantitative. As most of the commercially used anti-hormone antibodies have nuclear specificity, we have developed methods for isolation and antigen unmasking of nuclei from formalin fixed/paraffin embedded archival human tumors. After immunostaining with the antibodies and propidium iodide (for DNA content and cell cycle analysis), nuclei are analyzed by multiparametric laser flow cytometry for hormone receptor expression, DNA content, aneuploidy and cell cycle determination. These multiparametric methods are especially important for retrospective studies seeking to correlate hormone receptor expression with clinical response to anti-hormonal therapy of human breast and prostate tumors.

Purinergic receptor ligands stimulate pro-opiomelanocortin gene expression in AtT-20 pituitary corticotroph cells.

PubMed

Zhao, L-F; Iwasaki, Y; Oki, Y; Tsugita, M; Taguchi, T; Nishiyama, M; Takao, T; Kambayashi, M; Hashimoto, K

2006-04-01

Although recent studies have suggested that purinergic receptors are expressed in the anterior pituitary gland, their involvement in the regulation of pituitary hormone gene expression is not completely understood. In the present study, we examined the expression of purinergic receptors and the effects of purinergic receptor ligands on pro-opiomelanocortin (POMC) gene expression, in AtT20 mouse corticotroph cells. We identified the expression of most of the purinergic receptor subtypes (A1, A2, P2X1, 3-7, P2Y1, 2, 4) mRNAs, analysed by the reverse transcriptase-polymerase chain reaction. We also found that adenosine and ATP, two representative and endogenous agonists of A1-3 and P2X/P2Y receptors, respectively, stimulated the 5'-promoter activity of the POMC gene in a dose- and time-related manner. When these ligands were simultaneously used with corticotrophin-releasing hormone (CRH), effects that were more than additive were observed, suggesting an enhancing role of these compounds in CRH-mediated adrenocorticotrophic hormone (ACTH) synthesis. These ligands also stimulated the expression of transcription factors involved in the regulation of the POMC gene, but did not enhance ACTH secretion. Finally, the positive effect of adenosine as well as CRH was completely inhibited by the protein kinase A inhibitor H89, whereas that of ATP was not influenced, indicating that different intracellular signalling pathways mediate these effects. Altogether, our results suggest a stimulatory role for these purinergic receptor ligands in the regulation of POMC gene expression in corticotroph cells. Because adenosine and ATP are known to be produced within the pituitary gland, it is possible they may be acting in an autocrine/paracrine fashion.

A role for tachykinins in female mouse and rat reproductive function.

PubMed

Pintado, C Oscar; Pinto, Francisco M; Pennefather, Jocelyn N; Hidalgo, Agustin; Baamonde, Ana; Sanchez, Teresa; Candenas, M Luz

2003-09-01

Tachykinins may be involved in reproduction. A reverse transcription-polymerase chain reaction assay was used to analyze the expression of tachykinins and tachykinin receptors in different types of reproductive cells from mice. The preprotachykinin (PPT) genes, PPT-A, PPT-B and PPT-C, that encode substance P/neurokinin A, neurokinin B, and hemokinin-1, respectively, and the genes that encode the tachykinin NK1, NK2, and NK3 receptors were all expressed, at different levels, in the uterus of superovulated, unfertilized mice. The mRNA of neprilysin (NEP), the main enzyme involved in tachykinin metabolism, was also expressed in the uterus. Isolated cumulus granulosa cells expressed PPT-A, PPT-B, PPT-C, and NEP and low levels of the tachykinin NK1 and NK2 receptors. Mouse oocytes expressed PPT-A and -B mRNA transcripts. A low expression of the three tachykinin receptors was observed but PPT-C and NEP were undetectable. Two- and 8- to 16-cell mouse embryos expressed only a low-abundance transcript corresponding to the NK1 receptor. However, the mRNAs of PPT-B, PPT-C and NEP appeared in blastocyst-stage embryos. A low-abundance transcript corresponding to the NK2 receptor was the only target gene detected in mice sperm. Female mice or rats treated neonatally with capsaicin showed a reduced fertility. A reduction in litter size was observed in female rats treated in vivo with the tachykinin NK3 receptor antagonist SR 142801. These data show that tachykinins of both neuronal and nonneuronal origin are differentially expressed in various types of reproductive cells and may play a role in female reproductive function.

The orphan receptor ERRα interferes with steroid signaling

PubMed Central

Teyssier, Catherine; Bianco, Stéphanie; Lanvin, Olivia; Vanacker, Jean-Marc

2008-01-01

The estrogen receptor-related receptor α (ERRα) is an orphan member of the nuclear receptor superfamily that has been shown to interfere with the estrogen-signaling pathway. In this report, we demonstrate that ERRα also cross-talks with signaling driven by other steroid hormones. Treatment of human prostatic cells with a specific ERRα inverse agonist reduces the expression of several androgen-responsive genes, in a manner that does not involve perturbation of androgen receptor expression or activity. Furthermore, ERRα activates the expression of androgen response elements (ARE)-containing promoters, such as that of the prostate cancer marker PSA, in an ARE-dependent manner. In addition, promoters containing a steroid response element can be activated by all members of the ERR orphan receptor subfamily, and this, even in the presence of antisteroid compounds. PMID:18697814

Epigenetic Reprogramming of the Type III Interferon Response Potentiates Antiviral Activity and Suppresses Tumor Growth

PubMed Central

Ding, Siyuan; Khoury-Hanold, William; Iwasaki, Akiko; Robek, Michael D.

2014-01-01

Type III interferon (IFN-λ) exhibits potent antiviral activity similar to IFN-α/β, but in contrast to the ubiquitous expression of the IFN-α/β receptor, the IFN-λ receptor is restricted to cells of epithelial origin. Despite the importance of IFN-λ in tissue-specific antiviral immunity, the molecular mechanisms responsible for this confined receptor expression remain elusive. Here, we demonstrate that the histone deacetylase (HDAC) repression machinery mediates transcriptional silencing of the unique IFN-λ receptor subunit (IFNLR1) in a cell-type-specific manner. Importantly, HDAC inhibitors elevate receptor expression and restore sensitivity to IFN-λ in previously nonresponsive cells, thereby enhancing protection against viral pathogens. In addition, blocking HDAC activity renders nonresponsive cell types susceptible to the pro-apoptotic activity of IFN-λ, revealing the combination of HDAC inhibitors and IFN-λ to be a potential antitumor strategy. These results demonstrate that the type III IFN response may be therapeutically harnessed by epigenetic rewiring of the IFN-λ receptor expression program. PMID:24409098

Conventional light chains inhibit the autonomous signaling capacity of the B cell receptor.

PubMed

Meixlsperger, Sonja; Köhler, Fabian; Wossning, Thomas; Reppel, Michael; Müschen, Markus; Jumaa, Hassan

2007-03-01

Signals from the B cell antigen receptor (BCR), consisting of mu heavy chain (muHC) and conventional light chain (LC), and its precursor the pre-BCR, consisting of muHC and surrogate light chain (SLC), via the adaptor protein SLP-65 regulate the development and function of B cells. Here, we compare the effect of SLC and conventional LC expression on receptor-induced Ca(2+) flux in B cells expressing an inducible form of SLP-65. We found that SLC expression strongly enhanced an autonomous ability of muHC to induce Ca(2+) flux irrespective of additional receptor crosslinking. In contrast, LC expression reduced this autonomous muHC ability and resulted in antigen-dependent Ca(2+) flux. These data indicate that autonomous ligand-independent signaling can be induced by receptor forms other than the pre-BCR. In addition, our data suggest that conventional LCs play an important role in the inhibition of autonomous receptor signaling, thereby allowing further B cell differentiation.

Early expression of triggering receptors and regulatory role of 2B4 in human natural killer cell precursors undergoing in vitro differentiation

PubMed Central

Sivori, Simona; Falco, Michela; Marcenaro, Emanuela; Parolini, Silvia; Biassoni, Roberto; Bottino, Cristina; Moretta, Lorenzo; Moretta, Alessandro

2002-01-01

In this study we analyzed the progression of cell surface receptor expression during the in vitro-induced human natural killer (NK) cell maturation from CD34+ Lin− cell precursors. NKp46 and NKp30, two major triggering receptors that play a central role in natural cytotoxicity, were expressed before the HLA class I-specific inhibitory receptors. Moreover, their appearance at the cell surface correlated with the acquisition of cytolytic activity by developing NK cells. Although the early expression of triggering receptors may provide activating signals required for inducing further cell differentiation, it may also affect the self-tolerance of developing NK cells. Our data show that a fail-safe mechanism preventing killing of normal autologous cells may be provided by the 2B4 surface molecule, which, at early stages of NK cell differentiation, functions as an inhibitory rather than as an activating receptor. PMID:11917118

The phosphorylated C-terminus of cAR1 plays a role in cell-type-specific gene expression and STATa tyrosine phosphorylation.

PubMed

Briscoe, C; Moniakis, J; Kim, J Y; Brown, J M; Hereld, D; Devreotes, P N; Firtel, R A

2001-05-01

cAMP receptors mediate some signaling pathways via coupled heterotrimeric G proteins, while others are G-protein-independent. This latter class includes the activation of the transcription factors GBF and STATa. Within the cellular mounds formed by aggregation of Dictyostelium, micromolar levels of cAMP activate GBF function, thereby inducing the transcription of postaggregative genes and initiating multicellular differentiation. Activation of STATa, a regulator of culmination and ecmB expression, results from cAMP receptor-dependent tyrosine phosphorylation and nuclear localization, also in mound-stage cells. During mound development, the cAMP receptor cAR1 is in a low-affinity state and is phosphorylated on multiple serine residues in its C-terminus. This paper addresses possible roles of cAMP receptor phosphorylation in the cAMP-mediated stimulation of GBF activity, STATa tyrosine phosphorylation, and cell-type-specific gene expression. To accomplish this, we have expressed cAR1 mutants in a strain in which the endogenous cAMP receptors that mediate postaggregative gene expression in vivo are deleted. We then examined the ability of these cells to undergo morphogenesis and induce postaggregative and cell-type-specific gene expression and STATa tyrosine phosphorylation. Analysis of cAR1 mutants in which the C-terminal tail is deleted or the ligand-mediated phosphorylation sites are mutated suggests that the cAR1 C-terminus is not essential for GBF-mediated postaggregative gene expression or STATa tyrosine phosphorylation, but may play a role in regulating cell-type-specific gene expression and morphogenesis. A mutant receptor, in which the C-terminal tail is constitutively phosphorylated, exhibits constitutive activation of STATa tyrosine phosphorylation in pulsed cells in suspension and a significantly impaired ability to induce cell-type-specific gene expression. The constitutively phosphorylated receptor also exerts a partial dominant negative effect on multicellular development when expressed in wild-type cells. These findings suggest that the phosphorylated C-terminus of cAR1 may be involved in regulating aspects of receptor-mediated processes, is not essential for GBF function, and may play a role in mediating subsequent development. Copyright 2001 Academic Press.

Effects of chronic scopolamine administration on spatial working memory and hippocampal receptors related to learning.

PubMed

Doguc, Duygu K; Delibas, Namik; Vural, Huseyin; Altuntas, Irfan; Sutcu, Recep; Sonmez, Yonca

2012-12-01

Scopolamine has been used in neuropsychopharmacology as a standard drug that leads to symptoms mimicking cognitive deficits seen during the aging process in healthy humans and animals. Scopolamine is known to be a nonselective muscarinic receptor blocker, but its chronic effect on the expression of certain hippocampal receptors is not clear. The aim of the present study was to determine the effect of chronic scopolamine administration on hippocampal receptor expression and spatial working memory in two different learning tasks, the water maze and the eight-arm radial maze. Male rats (8-12 months) were trained in both tasks. Subsequently, different groups received physiological saline or 0.1, 0.8, or 2 mg/kg scopolamine hydrobromide, respectively, for 15 days. After drug administration, the rats were retested for both tasks, and hippocampal expressions of NR2A, NR2B, nAChRα7, and mAChRM1 receptors were assessed by western blotting analysis. In both tasks, the spatial working memory was decreased dose dependently in all groups compared with the control group. In terms of receptor expressions, 0.8 and 2 mg/kg scopolamine administration significantly decreased NR2A protein expression, which corroborates suggestions of an interaction between cholinergic and glutamatergic receptors in the hippocampus.

Brain distribution and molecular cloning of the bovine GABA rho1 receptor.

PubMed

Rosas-Arellano, Abraham; Ochoa-de la Paz, Lenin David; Miledi, Ricardo; Martínez-Torres, Ataúlfo

2007-03-01

GABA(C) receptors were originally found in the mammalian retina and recent evidence shows that they are also expressed in several areas of the brain, including caudate nucleus, brain stem, pons and corpus callosum. In this study, plasma membranes from the caudate nucleus were microinjected into X. laevis oocytes. This led the oocyte plasma membrane to incorporate functional bicuculline-resistant, Cl(-) conducting bovine GABA receptors, similar to those of the retina. Immunolocalization of the GABA rho1 subunit revealed its expression in bovine neurons in the head of the caudate as well as in the olive, cuneiform and reticular nuclei of the brain stem. The same antibodies failed to show expression in the callosum and pons, where the GABA rho1 mRNA was previously detected. The cloned GABA rho1 sequence predicts a protein with 473 amino acids and 74-93% similarity to other GABA rho1 subunits. Oocytes injected with the cDNA express a non-desensitizing, homomeric receptor with a GABA EC(50)=6.0 microM and a Hill coefficient of 1.8. The results confirm the presence of GABA(C) receptor mRNAs in several areas of the mammalian brain and show that some of these areas express functional GABA rho1 receptors that have the classic GABA(C) receptor characteristics.

Neurotrophins and tonsillar hypertrophy in children with obstructive sleep apnea.

PubMed

Goldbart, Aviv D; Mager, Edward; Veling, Maria C; Goldman, Julie L; Kheirandish-Gozal, Leila; Serpero, Laura D; Piedimonte, Giovanni; Gozal, David

2007-10-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p<0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p<0.01), and substance P concentrations in OSA patients were higher than in RI (p<0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA.

Neurotrophins and Tonsillar Hypertrophy in Children With Obstructive Sleep Apnea

PubMed Central

GOLDBART, AVIV D.; MAGER, EDWARD; VELING, MARIA C.; GOLDMAN, JULIE L.; KHEIRANDISH-GOZAL, LEILA; SERPERO, LAURA D.; PIEDIMONTE, GIOVANNI; GOZAL, DAVID

2013-01-01

Enlarged adenotonsillar tissue (AT) is a major determinant of obstructive sleep apnea (OSA) severity in children; however, mechanisms of AT proliferation are poorly understood. We hypothesized that early exposure to respiratory syncytial virus (RSV) may modify AT proliferation through up-regulation of nerve growth factor (NGF)-neurokinin 1 (NK1) receptor dependent pathways. AT harvested from 34 children with OSA and 25 children with recurrent tonsillitis (RI) were examined for mRNA expression of multiple growth factors and their receptors. In addition, NK1 receptor expression and location, and substance P tissue concentrations were compared in AT from OSA and RI children. NGF mRNA and its high-affinity tyrosine kinase receptor (trkA) expression were selectively increased in OSA (p < 0.001). NK1 receptor mRNA and protein expression were also enhanced in OSA (p < 0.01), and substance P concentrations in OSA patients were higher than in RI (p < 0.0001). AT from OSA children exhibit distinct differences in the expression of NGF and trkA receptors, NK1 receptors, and substance P. The homology between these changes and those observed in the lower airways following RSV infection suggests that RSV may have induced neuro-immunomodulatory changes within AT, predisposing them to increased proliferation, and ultimately contribute to emergence of OSA. PMID:17667845

Gene and protein expression of oestrogen-β and progesterone receptors in facial melasma and adjacent healthy skin in women.

PubMed

Tamega, A de A; Miot, H A; Moço, N P; Silva, M G; Marques, M E A; Miot, L D B

2015-04-01

Compare gene and protein expression for oestrogen receptor-β (ER-β) and progesterone receptor (PR) in facial melasma and adjacent healthy skin. A cross-sectional study including 42 women with facial melasma, conducted at the Dermatology Service of Botucatu Medical School of São Paulo State University, Brazil. Biopsies of the melasma skin were performed, together with healthy surrounding skin. The gene expression (real-time PCR) of the hormone receptors in the tissue was evaluated. Subsequently, skin fragments were immunostained for nuclear ER-β and PR, evaluated according to their HSCORE (epidermis) and percentage of staining per microscopic field (dermis). Messenger RNA tissue expression for ER-β and PR showed no difference between melasma-affected skin fragments and the healthy perilesional areas (P > 0.2). Median nuclear epithelial expression for ER-β and PR was higher in lesioned skin (HSCORE 157 and 58) than in the healthy perilesional skin (HSCORE 97 and 19; P < 0.01), with no difference in dermal immunostaining. Nuclear histological expression for ER-β was associated to sun-induced melasma and negative familiar background; PR expression was associated to sun-induced melasma and darker phototypes. No difference was observed in gene expression for oestrogen-β and progesterone receptors in melasma-affected skin compared with adjacent healthy skin. However, the higher protein expression of these receptors in melasma-affected epithelia suggests hormonal participation in the pathogenesis of this disease. © 2014 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

A tissue microarray study of toll-like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

PubMed

Lin, Chih-Kung; Ting, Chun-Chieh; Tsai, Wen-Chiuan; Chen, Yuan-Wu; Hueng, Dueng-Yuan

2016-01-01

Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ERK signaling pathway and ERK is a vital member of this pathway. Toll-like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin-embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

Expression of CCK Receptors in Carcinoma Gallbladder and Cholelithiasis: A Pilot Study.

PubMed

Faridi, Mohammad Shazib; Jaiswal, Mahabir Saran Das; Goel, Sudhir K

2015-07-01

Gastrin and cholecystokinin (CCK) receptors are trophic for various gastrointestinal malignancies. Their role in gallbladder cancer has not been widely studied. To identify expression of CCK-A and CCK-B receptors in the tissue and blood of patients suffering from carcinoma (CA) gallbladder and gallstone disease and to compare expression of CCK A and B receptors in the gall bladder tissue and blood of healthy individuals and patients of CA gallbladder, and gallstone diseases. Forty nine subjects of both genders were recruited, comprising of 22 patients of CA gall bladder, 19 cases of cholelithiasis and, 8 normal gallbladders obtained from patients operated for trauma of the biliary system or Whipple's procedure. RNA extraction and cDNA formation for CCK-A and CCK-B receptors were carried out. Real Time PCR was performed on cDNA and threshold cycle (Ct) value of each sample was obtained and ΔCt was calculated. Chi-square test for comparing two groups and ANOVA test for comparing multiple groups were applied and if p<0.05 then Dunnett-C test was performed. Both CCK-A and CCK-B receptors were expressed irrespective of its origin in all tissues and blood samples studied; be it normal, Cholelithiasis or CA gallbladder and there was no difference among them (p>0.05). This preliminary study showed higher expression of CCK-A receptors in patients of cholelithiasis and decreased expression of CCK-A receptors in patients of CA gallbladder as compared to normal gallbladder although it did not rise to statistical significance.

Expression of CCK Receptors in Carcinoma Gallbladder and Cholelithiasis: A Pilot Study

PubMed Central

Jaiswal, Mahabir Saran Das; Goel, Sudhir K.

2015-01-01

Background: Gastrin and cholecystokinin (CCK) receptors are trophic for various gastrointestinal malignancies. Their role in gallbladder cancer has not been widely studied. Objectives: To identify expression of CCK-A and CCK-B receptors in the tissue and blood of patients suffering from carcinoma (CA) gallbladder and gallstone disease and to compare expression of CCK A and B receptors in the gall bladder tissue and blood of healthy individuals and patients of CA gallbladder, and gallstone diseases. Materials and Methods: Forty nine subjects of both genders were recruited, comprising of 22 patients of CA gall bladder, 19 cases of cholelithiasis and, 8 normal gallbladders obtained from patients operated for trauma of the biliary system or Whipple’s procedure. RNA extraction and cDNA formation for CCK-A and CCK-B receptors were carried out. Real Time PCR was performed on cDNA and threshold cycle (Ct) value of each sample was obtained and ΔCt was calculated. Chi-square test for comparing two groups and ANOVA test for comparing multiple groups were applied and if p<0.05 then Dunnett-C test was performed. Observation and Results: Both CCK-A and CCK-B receptors were expressed irrespective of its origin in all tissues and blood samples studied; be it normal, Cholelithiasis or CA gallbladder and there was no difference among them (p>0.05). Conclusion: This preliminary study showed higher expression of CCK-A receptors in patients of cholelithiasis and decreased expression of CCK-A receptors in patients of CA gallbladder as compared to normal gallbladder although it did not rise to statistical significance. PMID:26393162

Clinical validation of nuclear factor kappa B expression in invasive breast cancer.

PubMed

Agrawal, Anil Kumar; Pielka, Ewa; Lipinski, Artur; Jelen, Michal; Kielan, Wojciech; Agrawal, Siddarth

2018-01-01

Breast cancer is the most commonly diagnosed cancer in Polish women. The expression of transcription nuclear factor kappa B, a key inducer of inflammatory response promoting carcinogenesis and cancer progression in breast cancer, is not well-established. We assessed the nuclear factor kappa B expression in a total of 119 invasive breast carcinomas and 25 healthy control samples and correlated this expression pattern with several clinical and pathologic parameters including histologic type and grade, tumor size, lymph node status, estrogen receptor status, and progesterone receptor status. The data used for the analysis were derived from medical records. An immunohistochemical analysis of nuclear factor kappa B, estrogen receptor, and progesterone receptor was carried out and evaluation of stainings was performed. The expression of nuclear factor kappa B was significantly higher than that in the corresponding healthy control samples. No statistical difference was demonstrated in nuclear factor kappa B expression in relation to age, menopausal status, lymph node status, tumor size and location, grade and histologic type of tumor, and hormonal status (estrogen receptor and progesterone receptor). Nuclear factor kappa B is significantly overexpressed in invasive breast cancer tissues. Although nuclear factor kappa B status does not correlate with clinicopathological findings, it might provide important additional information on prognosis and become a promising object for targeted therapy.

Nuclear receptor coactivators: regulators of steroid action in brain and behaviour.

PubMed

Tetel, M J; Acharya, K D

2013-11-01

Steroid hormones act in specific regions of the brain to alter behaviour and physiology. Although it has been well established that the bioavailability of the steroid and the expression of its receptor is critical for understanding steroid action in the brain, the importance of nuclear receptor coactivators in the brain is becoming more apparent. The present review focuses on the function of the p160 family of coactivators, which includes steroid receptor coactivator-1 (SRC-1), SRC-2 and SRC-3, in steroid receptor action in the brain. The expression, regulation and function of these coactivators in steroid-dependent gene expression in both brain and behaviour are discussed. © 2013 British Society for Neuroendocrinology.

Cloning and pharmacological characterization of the rabbit bradykinin B2 receptor.

PubMed

Bachvarov, D R; Saint-Jacques, E; Larrivée, J F; Levesque, L; Rioux, F; Drapeau, G; Marceau, F

1995-12-01

Degenerate primers, corresponding to consensus sequences of third and sixth transmembrane domains of G protein-coupled receptor superfamily, were used for the polymerase chain reaction amplification and consecutive characterization of G protein-coupled receptors present in cultured rabbit aortic smooth muscle cells. One of the isolated resulting fragments was highly homologous to the corresponding region of the bradykinin (BK) B2 receptor cloned in other species. The polymerase chain reaction fragment was used to screen a rabbit genomic library, which allowed the identification of an intronless 1101-nucleotide open reading frame which codes for a 367-amino acid receptor protein. The rabbit B2 receptor sequence is more than 80% identical to the ones determined in three other species and retain putative glycosylation, palmitoylation and phosphorylation sites. In the rabbit genomic sequence, an acceptor splice sequence was found 8 base pairs upstream of the start codon. Northern blot analysis showed a high expression of a major transcript (4.2 kilobases) in the rabbit kidney and duodenum, and a less abundant expression in other tissues. Southern blot experiments suggest that a single copy of this gene exists in the rabbit genome. The cloned rabbit B2 receptor expressed in COS-1 cells binds [3H]BK in a saturable manner (KD 2.1 nM) and this ligand competes with a series of kinin agonists and antagonist with a rank order consistent with the B2 receptor identity. The insurmountable character of the antagonism exerted by Hoe 140 against BK on the rabbit B2 receptor, previously shown in pharmacological experiments, was confirmed in binding experiments with the cloned receptor expressed in a controlled manner. By contrast, Hoe 140 competed with [3H]BK in a surmountable manner for the human B2 receptor expressed in COS-1 cells. The cloning of the rabbit B2 receptor will be useful notably for the study of the structural basis of antagonist binding and for studies on receptor regulation in a relatively large animal.

Immunohistochemical localization of bone morphogenetic proteins and the receptors in epiphyseal growth plate.

PubMed

Yazaki, Y; Matsunaga, S; Onishi, T; Nagamine, T; Origuchi, N; Yamamoto, T; Ishidou, Y; Imamura, T; Sakou, T

1998-01-01

The expression of bone morphogenetic proteins (BMPs) and BMP receptors (BMPRs) in the epiphyseal growth plate has not been clarified. In this study, we studied immunohistochemically the spatial and temporal localization of BMP-2/4, osteogenic protein-1 (OP-1, or BMP-7), and BMP receptors (BMPR-IA, BMPR-IB, and BMPR-II) in the epiphyseal plate of growing rats. The proximal parts of tibia in growing rats were observed. At 12 weeks after birth, BMP-2/4 and OP-1 were expressed markedly in proliferating and maturing chondrocytes. BMPR-IA, IB and II were clearly co-expressed in proliferating and maturing chondrocytes, and the expression was decreased in hypertrophic chondrocytes. At 24 weeks, the expression of BMP-2/4 and OP-1 was decreased, but BMPRs were still well-expressed in proliferating chondrocytes. The temporal and spatial expression of BMP and BMPR suggests that BMP and BMP receptors play roles in the multistep cascade of enchondral ossification in the epiphyseal growth plate.

Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

PubMed

Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

2012-04-01

The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability.

Structure and function of the amygdaloid NPY system: NPY Y2 receptors regulate excitatory and inhibitory synaptic transmission in the centromedial amygdala.

PubMed

Wood, J; Verma, D; Lach, G; Bonaventure, P; Herzog, H; Sperk, G; Tasan, R O

2016-09-01

The amygdala is essential for generating emotional-affective behaviors. It consists of several nuclei with highly selective, elaborate functions. In particular, the central extended amygdala, consisting of the central amygdala (CEA) and the bed nucleus of the stria terminalis (BNST) is an essential component actively controlling efferent connections to downstream effectors like hypothalamus and brain stem. Both, CEA and BNST contain high amounts of different neuropeptides that significantly contribute to synaptic transmission. Among these, neuropeptide Y (NPY) has emerged as an important anxiolytic and fear-reducing neuromodulator. Here, we characterized the expression, connectivity and electrophysiological function of NPY and Y2 receptors within the CEA. We identified several NPY-expressing neuronal populations, including somatostatin- and calretinin-expressing neurons. Furthermore, in the main intercalated nucleus, NPY is expressed primarily in dopamine D1 receptor-expressing neurons but also in interspersed somatostatin-expressing neurons. Interestingly, NPY neurons did not co-localize with the Y2 receptor. Retrograde tract tracing experiments revealed that NPY neurons reciprocally connect the CEA and BNST. Functionally, the Y2 receptor agonist PYY3-36, reduced both, inhibitory as well as excitatory synaptic transmission in the centromedial amygdala (CEm). However, we also provide evidence that lack of NPY or Y2 receptors results in increased GABA release specifically at inhibitory synapses in the CEm. Taken together, our findings suggest that NPY expressed by distinct populations of neurons can modulate afferent and efferent projections of the CEA via presynaptic Y2 receptors located at inhibitory and excitatory synapses.

Expression patterns of lectin-like natural killer receptors, inhibitory CD94/NKG2A, and activating CD94/NKG2C on decidual CD56bright natural killer cells differ from those on peripheral CD56dim natural killer cells.

PubMed

Kusumi, Maki; Yamashita, Takahiro; Fujii, Tomoyuki; Nagamatsu, Takeshi; Kozuma, Shiro; Taketani, Yuji

2006-06-01

The balance of inhibitory and activating natural killer (NK) receptors on maternal decidual NK cells, most of which are CD56bright, is thought to be crucial for the proper growth of trophoblasts in placenta. A lectin-like NK receptor, CD94/NKG2, is the receptor for human leukocyte antigen (HLA)-E, which is expressed on trophoblasts. To clarify the mechanism regulating the activity of decidual NK cells during pregnancy, we investigated the expression patterns of inhibitory NK receptor, CD94/NKG2A, and activating receptor, CD94/NKG2C, on decidual NK cells in an early stage of normal pregnancy and compared them with those on peripheral NK cells, most of which are CD56dim. The rate of NKG2A-positive cells was significantly higher for decidual CD56bright NK cells than for peripheral CD56dim NK cells, but the rates of NKG2C-positive cells were comparable between the two cell types. Interestingly, peripheral CD56dim NK cells reciprocally expressed inhibitory NKG2A and activating NKG2C, but decidual CD56bright NK cells that expressed activating NKG2C simultaneously expressed inhibitory NKG2A. The co-expression of inhibitory and activating NKG2 receptors may fine-tune the immunoregulatory functions of the decidual NK cells to control the trophoblast invasion in constructing placenta.

Angiotensin II AT1 receptor alters ACE2 activity, eNOS expression and CD44-hyaluronan interaction in rats with hypertension and myocardial fibrosis.

PubMed

Bai, Feng; Pang, Xue-Fen; Zhang, Li-Hui; Wang, Ning-Ping; McKallip, Robert J; Garner, Ronald E; Zhao, Zhi-Qing

2016-05-15

This study tested the hypothesis that angiotensin II (Ang II) AT1 receptor is involved in development of hypertension and cardiac fibrosis via modifying ACE2 activity, eNOS expression and CD44-hyaluronan interaction. Male Sprague-Dawley rats were subjected to Ang II infusion (500ng/kg/min) using osmotic minipumps up to 4weeks and the AT1 receptor blocker, telmisartan was administered by gastric gavage (10mg/kg/day) during Ang II infusion. Our results indicated that Ang II enhances AT1 receptor, downregulates AT2 receptor, ACE2 activity and eNOS expression, and increases CD44 expression and hyaluronidase activity, an enzyme for hyaluronan degradation. Further analyses revealed that Ang II increases blood pressure and augments vascular/interstitial fibrosis. Comparison of the Ang II group, treatment with telmisartan significantly increased ACE2 activity and eNOS expression in the intracardiac vessels and intermyocardium. These changes occurred in coincidence with decreased blood pressure. Furthermore, the locally-expressed AT1 receptor was downregulated, as evidenced by an increased ratio of the AT2 over AT1 receptor (1.4±0.4% vs. 0.4±0.1% in Ang II group, P<0.05). Along with these modulations, telmisartan inhibited membrane CD44 expression and hyaluronidase activity, decreased populations of macrophages and myofibroblasts, and reduced expression of TGFβ1 and Smads. Collagen I synthesis and tissue fibrosis were attenuated as demonstrated by the less extensive collagen-rich area. These results suggest that the AT1 receptor is involved in development of hypertension and cardiac fibrosis. Selective activating ACE2/eNOS and inhibiting CD44/HA interaction might be considered as the therapeutic targets for attenuating Ang II induced deleterious cardiovascular effects. Copyright © 2016 Elsevier Inc. All rights reserved.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

PubMed

Kesby, James P; Najera, Julia A; Romoli, Benedetto; Fang, Yiding; Basova, Liana; Birmingham, Amanda; Marcondes, Maria Cecilia G; Dulcis, Davide; Semenova, Svetlana

2017-10-01

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for methamphetamine abuse in individuals with HIV. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

Mizuta, Kentaro; Zhang, Yi; Xu, Dingbang; Mizuta, Fumiko; D'Ovidio, Frank; Masaki, Eiji; Emala, Charles W

2013-09-02

Dopamine signaling is mediated by Gs protein-coupled "D1-like" receptors (D1 and D5) and Gi-coupled "D2-like" receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576. These results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM.

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

PubMed Central

2013-01-01

Background Dopamine signaling is mediated by Gs protein-coupled “D1-like” receptors (D1 and D5) and Gi-coupled “D2-like” receptors (D2-4). In asthmatic patients, inhaled dopamine induces bronchodilation. Although the Gi-coupled dopamine D2 receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle (ASM) cells, the Gs-coupled dopamine D1-like receptor subtypes have never been identified on these cells. Activation of Gs-coupled receptors stimulates cyclic AMP (cAMP) production through the stimulation of adenylyl cyclase, which promotes ASM relaxation. We questioned whether the dopamine D1-like receptor is expressed on ASM, and modulates its function through Gs-coupling. Methods The mRNA and protein expression of dopamine D1-like receptor subtypes in both native human and guinea pig ASM tissue and cultured human ASM (HASM) cells was measured. To characterize the stimulation of cAMP through the dopamine D1 receptor, HASM cells were treated with dopamine or the dopamine D1-like receptor agonists (A68930 or SKF38393) before cAMP measurements. To evaluate whether the activation of dopamine D1 receptor induces ASM relaxation, guinea pig tracheal rings suspended under isometric tension in organ baths were treated with cumulatively increasing concentrations of dopamine or A68930, following an acetylcholine-induced contraction with or without the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMPS, the large-conductance calcium-activated potassium (BKCa) channel blocker iberiotoxin, or the exchange proteins directly activated by cAMP (Epac) antagonist NSC45576. Results Messenger RNA encoding the dopamine D1 and D5 receptors were detected in native human ASM tissue and cultured HASM cells. Immunoblots confirmed the protein expression of the dopamine D1 receptor in both native human and guinea pig ASM tissue and cultured HASM cells. The dopamine D1 receptor was also immunohistochemically localized to both human and guinea pig ASM. The dopamine D1-like receptor agonists stimulated cAMP production in HASM cells, which was reversed by the selective dopamine D1-like receptor antagonists SCH23390 or SCH39166. A68930 relaxed acetylcholine-contracted guinea pig tracheal rings, which was attenuated by Rp-cAMPS but not by iberiotoxin or NSC45576. Conclusions These results demonstrate that the dopamine D1 receptors are expressed on ASM and regulate smooth muscle force via cAMP activation of PKA, and offer a novel target for therapeutic relaxation of ASM. PMID:24004608

Expression of mammalian beta-adrenergic receptors in Xenopus laevis oocytes

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

Bahouth, S.W.; Malbon, C.C.

1987-05-01

Xenopus laevis oocytes are a useful transcription and expression system for DNA and RNA, respectively. Total cellular RNA was extracted from mouse lymphoma S49 cells and poly(A)/sup +/mRNA prepared by affinity chromatography of RNA on oligo(dT) cellulose. The membranes of S49 cells contain beta-adrenergic receptors that display pharmacological characteristics of beta/sub 2/-subtype. Xenopus laevis oocytes were injected with 50 ng of mRNA/oocyte. Expression of beta-adrenergic receptors in oocytes incubated for 30 hr after microinjection was assessed in membranes by radioligand binding using (/sup 3/H) dihydroalprenolol. The injected oocytes displayed 0.34 fmol receptor/oocyte as compared to 0.02 fmol receptor/oocyte in themore » control oocytes. The affinity of beta-adrenergic receptors in injected oocytes for this radioligand was 2 nM, a value similar to the affinity of beta-adrenergic receptors for DHA in S49 cell membranes. The potency of beta-adrenergic agonists in competing for DHA binding to oocytes membranes was isoproterenol > epinephrine > norepineprine, indicating that the expressed beta-adrenergic receptors were of the beta/sub 2/-subtype. The K/sub I/ of these agonists for the beta-adrenergic receptor in oocyte membranes was 0.03, 0.15 and 1.2 ..mu..M, respectively. The role of post-translational modification in dictating receptor subtype is analyzed using mRNA of beta/sub 1/- as well as beta/sub 2/-adrenergic receptors.« less

Exendin-4 Upregulates Adiponectin Level in Adipocytes via Sirt1/Foxo-1 Signaling Pathway

PubMed Central

Wang, Anping; Li, Ting; An, Ping; Yan, Wenhua; Zheng, Hua; Wang, Baoan; Mu, Yiming

2017-01-01

Glucagon-like peptide-1 (GLP-1) receptor plays an essential role in regulating glucose metabolism. GLP-1 receptor agonists have been widely used for treating diabetes and other insulin resistance-related diseases. However, mechanisms underlying the anti-diabetic effects of GLP-1 receptor agonists remain largely unknown. In this study, we investigated the effects of GLP-1 agonist exendin-4 on the expression of adiponectin, an insulin sensitizing hormone. We found that exendin-4 increased the expression and secretion of adiponectin both in vitro and in vivo. Our data showed that exendin-4 upregulated adiponectin expression at both mRNA and protein levels in adipocytes and adipose tissues. The effects of exendin-4 on adiponectin expression were dependent on the GLP-1 receptor. We further demonstrated important roles of Sirt1 and transcriptional factor Foxo-1 in mediating the function of exendin-4 in regulating adiponectin expression. Suppression of Sirt1 or Foxo-1 expression significantly impaired exendin-4-induced adiponectin expression. Consistently, exendin-4 up-regulated Sirt1 and Foxo-1 expression in vivo. Our work is the first study demonstrating the role of Sirt1/Foxo-1 in regulating the regulatory function of a GLP-1 receptor agonist in adiponectin expression both in vitro and in vivo. The results provide important information for the mechanism underlying the function of GLP-1R on improving insulin resistance and related diseases. PMID:28122026

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

PubMed

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

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

USDA-ARS?s Scientific Manuscript database

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

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

USDA-ARS?s Scientific Manuscript database

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

Distribution of metabotropic receptors of serotonin, dopamine, GABA, glutamate, and short neuropeptide F in the central complex of Drosophila.

PubMed

Kahsai, L; Carlsson, M A; Winther, A M E; Nässel, D R

2012-04-19

The central complex is a prominent set of midline neuropils in the insect brain, known to be a higher locomotor control center that integrates visual inputs and modulates motor outputs. It is composed of four major neuropil structures, the ellipsoid body (EB), fan-shaped body (FB), noduli (NO), and protocerebral bridge (PB). In Drosophila different types of central complex neurons have been shown to express multiple neuropeptides and neurotransmitters; however, the distribution of corresponding receptors is not known. Here, we have mapped metabotropic, G-protein-coupled receptors (GPCRs) of several neurotransmitters to neurons of the central complex. By combining immunocytochemistry with GAL4 driven green fluorescent protein, we examined the distribution patterns of six different GPCRs: two serotonin receptor subtypes (5-HT(1B) and 5-HT(7)), a dopamine receptor (DopR), the metabotropic GABA(B) receptor (GABA(B)R), the metabotropic glutamate receptor (DmGluR(A)) and a short neuropeptide F receptor (sNPFR1). Five of the six GPCRs were mapped to different neurons in the EB (sNPFR1 was not seen). Different layers of the FB express DopR, GABA(B)R, DmGluR(A,) and sNPFR1, whereas only GABA(B)R and DmGluR(A) were localized to the PB. Finally, strong expression of DopR and DmGluR(A) was detected in the NO. In most cases the distribution patterns of the GPCRs matched the expression of markers for their respective ligands. In some nonmatching regions it is likely that other types of dopamine and serotonin receptors or ionotropic GABA and glutamate receptors are expressed. Our data suggest that chemical signaling and signal modulation are diverse and highly complex in the different compartments and circuits of the Drosophila central complex. The information provided here, on receptor distribution, will be very useful for future analysis of functional circuits in the central complex, based on targeted interference with receptor expression. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

Signal transduction and functional selectivity of F15599, a preferential post-synaptic 5-HT1A receptor agonist

PubMed Central

Newman-Tancredi, A; Martel, J-C; Assié, M-B; Buritova, J; Lauressergues, E; Cosi, C; Heusler, P; Slot, L Bruins; Colpaert, FC; Vacher, B; Cussac, D

2009-01-01

Background and purpose: Activation of post-synaptic 5-HT1A receptors may provide enhanced therapy against depression. We describe the signal transduction profile of F15599, a novel 5-HT1A receptor agonist. Experimental approach: F15599 was compared with a chemical congener, F13714, and with (+)8-OH-DPAT in models of signal transduction in vitro and ex vivo. Key results: F15599 was highly selective for 5-HT1A receptors in binding experiments and in [35S]-GTPγS autoradiography of rat brain, where F15599 increased labelling in regions expressing 5-HT1A receptors. In cell lines expressing h5-HT1A receptors, F15599 more potently stimulated extracellular signal-regulated kinase (ERK1/2) phosphorylation, compared with G-protein activation, internalization of h5-HT1A receptors or inhibition of cAMP accumulation. F13714, (+)8-OH-DPAT and 5-HT displayed a different rank order of potency for these responses. F15599 stimulated [35S]-GTPγS binding more potently in frontal cortex than raphe. F15599, unlike 5-HT, more potently and efficaciously stimulated Gαi than Gαo activation. In rat prefrontal cortex (a region expressing post-synaptic 5-HT1A receptors), F15599 potently activated ERK1/2 phosphorylation and strongly induced c-fos mRNA expression. In contrast, in raphe regions (expressing pre-synaptic 5-HT1A receptors) F15599 only weakly or did not induce c-fos mRNA expression. Finally, despite its more modest affinity in vitro, F15599 bound to 5-HT1A receptors in vivo almost as potently as F13714. Conclusions and implications: F15599 showed a distinctive activation profiles for 5-HT1A receptor-mediated signalling pathways, unlike those of reference agonists and consistent with functional selectivity at 5-HT1A receptors. In rat, F15599 potently activated signalling in prefrontal cortex, a feature likely to underlie its beneficial effects in models of depression and cognition. PMID:19154445

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) in sickle cell disease vasculopathy

PubMed Central

Chen, Mingyi; Qiu, Hong; Lin, Xin; Nam, David; Ogbu-Nwobodo, Lucy; Archibald, Hannah; Joslin, Amelia; Wun, Ted; Sawamura, Tatsuya; Green, Ralph

2017-01-01

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial receptor for oxidized LDL. Increased expression of LOX-1 has been demonstrated in atherosclerotic lesions and diabetic vasculopathy. In this study, we investigate the expression of LOX-1 receptor in sickle cell disease (SCD) vasculopathy. Expression of LOX-1 in brain vascular endothelium is markedly increased and LOX-1 gene expression is upregulated in cultured human brain microvascular endothelial cells by incubation with SCD erythrocytes. Also, the level of circulating soluble LOX-1 concentration is elevated in the plasma of SCD patients. Increased LOX-1 expression in endothelial cells is potentially involved in the pathogenesis of SCD vasculopathy. Soluble LOX-1 concentration in SCD may provide a novel biomarker for risk stratification of sickle cell vascular complications. PMID:27519944

Expression and purification of functional PDGF receptor beta.

PubMed

Shang, Qingbin; Zhao, Liang; Wang, Xiaojing; Wang, Meimei; Sui, Sen-Fang; Mi, Li-Zhi

2017-07-29

Platelet Derived Growth Factor receptors (PDGFRs), members of receptor tyrosine kinase superfamily, play essential roles in early hematopoiesis, angiogenesis and organ development. Dysregulation of PDGF receptor signaling under pathological conditions associates with cancers, vascular diseases, and fibrotic diseases. Therefore, they are attractive targets in drug development. Like any other membrane proteins with a single-pass transmembrane domain, the high-resolution structural information of the full-length PDGF receptors is still not resolved. It is caused, at least in part, by the technical challenges in the expression and purification of the functional, full-length PDGF receptors. Herein, we reported our experimental details in expression and purification of the full-length PDGFRβ from mammalian cells. We found that purified PDGFRβ remained in two different oligomeric states, presumably the monomer and the dimer, with basal kinase activity in detergent micelles. Addition of PDGF-B promoted dimerization and elevated kinase activity of the receptor, suggesting that purified receptors were functional. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

PubMed Central

Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

2011-01-01

P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

PubMed

Vita, N; Oury-Donat, F; Chalon, P; Guillemot, M; Kaghad, M; Bachy, A; Thurneyssen, O; Garcia, S; Poinot-Chazel, C; Casellas, P; Keane, P; Le Fur, G; Maffrand, J P; Soubrie, P; Caput, D; Ferrara, P

1998-11-06

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

A protein that interacts with members of the nuclear hormone receptor family: identification and cDNA cloning.

PubMed Central

Zeiner, M; Gehring, U

1995-01-01

In search of proteins which interact with activated steroid hormone receptors, we screened a human liver lambda gt11 expression library with the glucocorticoid receptor. We identified and cloned a cDNA sequence of 1322 bp that encodes a protein of 274 aa. This protein consists predominantly of hydrophilic amino acids and contains a putative bipartite nuclear localization signal. The in vitro translated receptor-associating protein runs in SDS/polyacrylamide gels with an apparent molecular mass of 46 kDa. By use of the bacterially expressed fusion protein with glutathione S-transferase we have found that interaction is not limited to the glucocorticoid receptor but included other nuclear receptors--most notably, the estrogen and thyroid receptors. Binding also occurs with the glucocorticoid receptor complexed with the antiglucocorticoid RU 38486, with the estrogen receptor complexed with the antiestrogen 4-hydroxytamoxifen or ICI 164,384, and even with receptors not complexed with ligand. Association with steroid hormone receptors depends on prior receptor activation--i.e., release from heat shock proteins. The sequence identified here appears to be a general partner protein for nuclear hormone receptors, with the gene being expressed in a variety of mammalian tissues. Images Fig. 2 Fig. 3 Fig. 4 PMID:8524784

Prostaglandins and Their Receptors in Eosinophil Function and As Therapeutic Targets

PubMed Central

Peinhaupt, Miriam; Sturm, Eva M.; Heinemann, Akos

2017-01-01

Of the known prostanoid receptors, human eosinophils express the prostaglandin D2 (PGD2) receptors DP1 [also D-type prostanoid (DP)] and DP2 (also chemoattractant receptor homologous molecule, expressed on Th2 cells), the prostaglandin E2 receptors EP2 and EP4, and the prostacyclin (PGI2) receptor IP. Prostanoids can bind to either one or multiple receptors, characteristically have a short half-life in vivo, and are quickly degraded into metabolites with altered affinity and specificity for a given receptor subtype. Prostanoid receptors signal mainly through G proteins and naturally activate signal transduction pathways according to the G protein subtype that they preferentially interact with. This can lead to the activation of sometimes opposing signaling pathways. In addition, prostanoid signaling is often cell-type specific and also the combination of expressed receptors can influence the outcome of the prostanoid impulse. Accordingly, it is assumed that eosinophils and their (patho-)physiological functions are governed by a sensitive prostanoid signaling network. In this review, we specifically focus on the functions of PGD2, PGE2, and PGI2 and their receptors on eosinophils. We discuss their significance in allergic and non-allergic diseases and summarize potential targets for drug intervention. PMID:28770200

G protein-coupled receptor 30 down-regulates cofactor expression and interferes with the transcriptional activity of glucocorticoid.

PubMed

Ylikomi, Timo; Vienonen, Annika; Ahola, Tytti M

2004-11-01

G protein-coupled receptor 30 (GPR30) has previously been described to be important in steroid-mediated growth and to inhibit cell proliferation. Here we investigated whether the effect of GPR30 on cell growth is dependent on steroid hormone receptors. We stably introduced GPR30 in immortalized normal mammary epithelial (HME) cells using retroviruses for gene delivery. GPR30 inhibited the growth and proliferation of the cells. They expressed glucocorticoid receptor, but not estrogen or progesterone receptor. GPR30 down-regulated the expression of cofactor transcription intermediary factor 2 (TIF2) analyzed using quantitative RT-PCR analysis, and also diminished the expression of TIF2 at protein level analyzed by Western blotting using nuclear extracts from mammary epithelial cells. When HME cells were transiently transfected with the glucocorticoid response element MMTV-luc reporter plasmid, stable expression of GPR30 resulted in the abolition of ligand-induced transactivation of the promoter. In COS cells, transient transfection of GPR30 with glucocorticoid receptor alpha resulted in an abrogation of the MMTV-luc and GRE-luc reporter activities induced by dexamethasone. The results suggest a novel mechanism by which membrane-initiated signaling interferes with steroid signaling.

Control of energy balance by hypothalamic gene circuitry involving two nuclear receptors, neuron-derived orphan receptor 1 and glucocorticoid receptor.

PubMed

Kim, Sun-Gyun; Lee, Bora; Kim, Dae-Hwan; Kim, Juhee; Lee, Seunghee; Lee, Soo-Kyung; Lee, Jae W

2013-10-01

Nuclear receptors (NRs) regulate diverse physiological processes, including the central nervous system control of energy balance. However, the molecular mechanisms for the central actions of NRs in energy balance remain relatively poorly defined. Here we report a hypothalamic gene network involving two NRs, neuron-derived orphan receptor 1 (NOR1) and glucocorticoid receptor (GR), which directs the regulated expression of orexigenic neuropeptides agouti-related peptide (AgRP) and neuropeptide Y (NPY) in response to peripheral signals. Our results suggest that the anorexigenic signal leptin induces NOR1 expression likely via the transcription factor cyclic AMP response element-binding protein (CREB), while the orexigenic signal glucocorticoid mobilizes GR to inhibit NOR1 expression by antagonizing the action of CREB. Also, NOR1 suppresses glucocorticoid-dependent expression of AgRP and NPY. Consistently, relative to wild-type mice, NOR1-null mice showed significantly higher levels of AgRP and NPY and were less responsive to leptin in decreasing the expression of AgRP and NPY. These results identify mutual antagonism between NOR1 and GR to be a key rheostat for peripheral metabolic signals to centrally control energy balance.

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

PubMed

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

2015-06-01

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

Expression of gamma-aminobutyric acid rho 1 and rho 1 Delta 450 as gene fusions with the green fluorescent protein.

PubMed

Martinez-Torres, A; Miledi, R

2001-02-13

The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type rho 1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, rho 1 Delta 450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

Tissue Distribution of the 27.8 kDa Receptor and its Dynamic Expression in Response to Lymphocystis Disease Virus Infection in Flounder (Paralichthys olivaceus).

PubMed

Wu, R-H; Tang, X-Q; Sheng, X-Z; Zhan, W-B

2015-11-01

Lymphocystis disease virus (LCDV) enters and infects the gill cells of flounder (Paralichthys olivaceus) via a 27.8 kDa membrane protein receptor. In the present study, immunohistochemistry was performed to locate the tissue distribution of this molecule in healthy flounder and showed that it was widely distributed in the tissues tested. Indirect enzyme-linked immunosorbent assay (ELISA) showed that the expression of the receptor in healthy flounder was highest in the gills and stomach, then in the skin, intestine and liver, followed by the spleen, head kidney, heart, ovary and brain and finally the kidney. On LCDV infection, ELISA indicated that the expression of the receptor, as determined by ELISA, was significantly upregulated in all tissues of LCDV-infected flounder compared with controls, but this expression decreased over the 4 weeks post infection. In contrast, real-time quantitative polymerase chain reaction demonstrated that the copy number of the LCDV gene in the tissues increased with time post infection, and that viral loads were higher in the tissues with higher expressions of the receptor. These results point to a correlation between high expression of the 27.8 kDa receptor and efficient LCDV propagation. The wide tissue distribution of the receptor might be one reason why LCDV can infect various tissues leading to systemic infection. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of Trace Amine-associated Receptor 1 Agonists on the Expression, Reconsolidation, and Extinction of Cocaine Reward Memory.

PubMed

Liu, Jian-Feng; Thorn, David A; Zhang, Yanan; Li, Jun-Xu

2016-07-01

As a modulator of dopaminergic system, trace amine-associated receptor 1 has been shown to play a critical role in regulating the rewarding properties of additive drugs. It has been demonstrated that activation of trace amine-associated receptor 1 decreased the abuse-related behaviors of cocaine in rats. However, the role of trace amine-associated receptor 1 in specific stages of cocaine reward memory is still unclear. Here, using a cocaine-induced conditioned place preference model, we tested the effects of a selective trace amine-associated receptor 1 agonist RO5166017 on the expression, reconsolidation, and extinction of cocaine reward memory. We found that RO5166017 inhibited the expression but not retention of cocaine-induced conditioned place preference. RO5166017 had no effect on the reconsolidation of cocaine reward memory. Pretreatment with RO5166017 before extinction hindered the formation of extinction long-term memory. RO5166017 did not affect the movement during the conditioned place preference test, indicating the inhibitory effect of RO5166017 on the expression of cocaine-induced conditioned place preference was not caused by locomotion inhibition. Using a cocaine i.v. self-administration model, we found that the combined trace amine-associated receptor 1 partial agonist RO5263397 with extinction had no effect on the following cue- and drug-induced reinstatement of cocaine-seeking behavior. Repeated administration of the trace amine-associated receptor 1 agonist during extinction showed a continually inhibitory effect on the expression of cocaine reward memory both in cocaine-induced conditioned place preference and cocaine self-administration models. Taken together, these results indicate that activation of trace amine-associated receptor 1 specifically inhibited the expression of cocaine reward memory. The inhibitory effect of trace amine-associated receptor 1 agonists on cocaine reward memory suggests that trace amine-associated receptor 1 agonists could be a promising agent to prevent cocaine relapse. © The Author 2016. Published by Oxford University Press on behalf of CINP.

Expression and associations of TRAF1, BMI-1, ALDH1, and Lin28B in oral squamous cell carcinoma.

PubMed

Wu, Tian-Fu; Li, Yi-Cun; Ma, Si-Rui; Bing-Liu; Zhang, Wen-Feng; Sun, Zhi-Jun

2017-04-01

Tumor necrosis factor receptor-associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor-associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor-associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor-associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r 2  = 0.109; p < 0.0001, r 2  = 0.64; and p < 0.001, r 2  = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor-associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor-associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor-associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

Histaminergic Receptors Modulate Spinal Cord Injury-Induced Neuronal Nitric Oxide Synthase Upregulation and Cord Pathology: New Roles of Nanowired Drug Delivery for Neuroprotection.

PubMed

Sharma, Hari S; Patnaik, Ranjana; Muresanu, Dafin F; Lafuente, José V; Ozkizilcik, Asya; Tian, Z Ryan; Nozari, Ala; Sharma, Aruna

2017-01-01

The possibility that histamine influences the spinal cord pathophysiology following trauma through specific receptor-mediated upregulation of neuronal nitric oxide synthase (nNOS) was examined in a rat model. A focal spinal cord injury (SCI) was inflicted by a longitudinal incision into the right dorsal horn of the T10-11 segments. The animals were allowed to survive 5h. The SCI significantly induced breakdown of the blood-spinal cord barrier to protein tracers, reduced the spinal cord blood flow at 5h, and increased the edema formation and massive upregulation of nNOS expression. Pretreatment with histamine H1 receptor antagonist mepyramine (1mg, 5mg, and 10mg/kg, i.p., 30min before injury) failed to attenuate nNOS expression and spinal cord pathology following SCI. On the other hand, blockade of histamine H2 receptors with cimetidine or ranitidine (1mg, 5mg, or 10mg/kg) significantly reduced these early pathophysiological events and attenuated nNOS expression in a dose-dependent manner. Interestingly, TiO 2 -naowire delivery of cimetidine or ranitidine (5mg doses) exerted superior neuroprotective effects on SCI-induced nNOS expression and cord pathology. It appears that effects of ranitidine were far superior than cimetidine at identical doses in SCI. On the other hand, pretreatment with histamine H3 receptor agonist α-methylhistamine (1mg, 2mg, or 5mg/kg, i.p.) that inhibits histamine synthesis and release in the central nervous system thwarted the spinal cord pathophysiology and nNOS expression when used in lower doses. Interestingly, histamine H3 receptor antagonist thioperamide (1mg, 2mg, or 5mg/kg, i.p.) exacerbated nNOS expression and cord pathology after SCI. These novel observations suggest that blockade of histamine H2 receptors or stimulation of histamine H3 receptors attenuates nNOS expression and induces neuroprotection in SCI. Taken together, our results are the first to demonstrate that histamine-induced pathophysiology of SCI is mediated via nNOS expression involving specific histamine receptors. © 2017 Elsevier Inc. All rights reserved.

Human dopamine receptor and its uses

DOEpatents

Civelli, Olivier; Van Tol, Hubert Henri-Marie

1999-01-01

The present invention is directed toward the isolation, characterization and pharmacological use of the human D4 dopamine receptor. The nucleotide sequence of the gene corresponding to this receptor and alleleic variant thereof are provided by the invention. The invention also includes recombinant eukaryotic expression constructs capable of expressing the human D4 dopamine receptor in cultures of transformed eukaryotic cells. The invention provides cultures of transformed eukaryotic cells which synthesize the human D4 dopamine receptor, and methods for characterizing novel psychotropic compounds using such cultures.

Analysis of Ethylene Receptors: Ethylene-Binding Assays.

PubMed

Binder, Brad M; Schaller, G Eric

2017-01-01

Plant ethylene receptors bind ethylene with high affinity. Most of the characterization of ethylene binding to the receptors has been carried out using a radioligand-binding assay on functional receptors expressed in yeast. In this chapter, we describe methods for expressing ethylene receptors in yeast and conducting ethylene-binding assays on intact yeast and yeast membranes. The ethylene-binding assays can be modified to analyze ethylene binding to intact plants and other organisms as well as membranes isolated from any biological source.

Long-Term Haloperidol Treatment Prolongs QT Interval and Increases Expression of Sigma 1 and IP3 Receptors in Guinea Pig Hearts.

PubMed

Stracina, Tibor; Slaninova, Iva; Polanska, Hana; Axmanova, Martina; Olejnickova, Veronika; Konecny, Petr; Masarik, Michal; Krizanova, Olga; Novakova, Marie

2015-07-01

Haloperidol is a neuroleptic drug used for a medication of various psychoses and deliria. Its administration is frequently accompanied by cardiovascular side effects, expressed as QT interval prolongation and occurrence of even lethal arrhythmias. Despite these side effects, haloperidol is still prescribed in Europe in clinical practice. Haloperidol binds to sigma receptors that are coupled with inositol 1,4,5-trisphosphate (IP3) receptors. Sigma receptors are expressed in various tissues, including heart muscle, and they modulate potassium channels. Together with IP3 receptors, sigma receptors are also involved in calcium handling in various tissues. Therefore, the present work aimed to study the effects of long-term haloperidol administration on the cardiac function. Haloperidol (2 mg/kg once a day) or vehiculum was administered by intraperitoneal injection to guinea pigs for 21 consecutive days. We measured the responsiveness of the hearts isolated from the haloperidol-treated animals to additional application of haloperidol. Expression of the sigma 1 receptor and IP3 receptors was studied by real time-PCR and immunohistochemical analyses. Haloperidol treatment caused the significant decrease in the relative heart rate and the prolongation of QT interval of the isolated hearts from the haloperidol-treated animals, compared to the hearts isolated from control animals. The expression of sigma 1 and IP3 type 1 and type 2 receptors was increased in both atria of the haloperidol-treated animals but not in ventricles. The modulation of sigma 1 and IP3 receptors may lead to altered calcium handling in cardiomyocytes and thus contribute to changed sensitivity of cardiac cells to arrhythmias.

Effects of Chronic Ethanol Consumption on Rat GABAA and Strychnine-sensitive Glycine Receptors Expressed by Lateral/Basolateral Amygdala Neurons

PubMed Central

McCool, Brian A.; Frye, Gerald D.; Pulido, Marisa D.; Botting, Shaleen K.

2010-01-01

It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABAA and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABAA receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor’s response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABAA receptors composed of unique α subunits were differentially sensitive to acute ethanol. Likewise, the presence of the β subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the α2 subunit. Our results suggest that the facilitation of GABAA receptors during chronic ethanol exposure may help explain the maintenance of ethanol’s anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABAA and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure. PMID:12560122

PDGF-beta receptor expression and ventilatory acclimatization to hypoxia in the rat.

PubMed

Alea, O A; Czapla, M A; Lasky, J A; Simakajornboon, N; Gozal, E; Gozal, D

2000-11-01

Activation of platelet-derived growth factor-beta (PDGF-beta) receptors in the nucleus of the solitary tract (nTS) modulates the late phase of the acute hypoxic ventilatory response (HVR) in the rat. We hypothesized that temporal changes in PDGF-beta receptor expression could underlie the ventilatory acclimatization to hypoxia (VAH). Normoxic ventilation was examined in adult Sprague-Dawley rats chronically exposed to 10% O(2), and at 0, 1, 2, 7, and 14 days, Northern and Western blots of the dorsocaudal brain stem were performed for assessment of PDGF-beta receptor expression. Although no significant changes in PDGF-beta receptor mRNA occurred over time, marked attenuation of PDGF-beta receptor protein became apparent after day 7 of hypoxic exposure. Such changes were significantly correlated with concomitant increases in normoxic ventilation, i.e., with VAH (r: -0.56, P < 0.005). In addition, long-term administration of PDGF-BB in the nTS via osmotic pumps loaded with either PDGF-BB (n = 8) or vehicle (Veh; n = 8) showed that although no significant changes in the magnitude of acute HVR occurred in Veh over time, the typical attenuation of HVR by PDGF-BB decreased over time. Furthermore, PDGF-BB microinjections did not attenuate HVR in acclimatized rats at 7 and 14 days of hypoxia (n = 10). We conclude that decreased expression of PDGF-beta receptors in the dorsocaudal brain stem correlates with the magnitude of VAH. We speculate that the decreased expression of PDGF-beta receptors is mediated via internalization and degradation of the receptor rather than by transcriptional regulation.

γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

PubMed Central

Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

2014-01-01

Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake

PubMed Central

Jeong, Jae Hoon; Lee, Dong Kun; Liu, Shun-Mei; Chua, Streamson C.; Schwartz, Gary J.

2018-01-01

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)–enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake. PMID:29689050

Activation of temperature-sensitive TRPV1-like receptors in ARC POMC neurons reduces food intake.

PubMed

Jeong, Jae Hoon; Lee, Dong Kun; Liu, Shun-Mei; Chua, Streamson C; Schwartz, Gary J; Jo, Young-Hwan

2018-04-01

Proopiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus (ARC) respond to numerous hormonal and neural signals, resulting in changes in food intake. Here, we demonstrate that ARC POMC neurons express capsaicin-sensitive transient receptor potential vanilloid 1 receptor (TRPV1)-like receptors. To show expression of TRPV1-like receptors in ARC POMC neurons, we use single-cell reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry, electrophysiology, TRPV1 knock-out (KO), and TRPV1-Cre knock-in mice. A small elevation of temperature in the physiological range is enough to depolarize ARC POMC neurons. This depolarization is blocked by the TRPV1 receptor antagonist and by Trpv1 gene knockdown. Capsaicin-induced activation reduces food intake that is abolished by a melanocortin receptor antagonist. To selectively stimulate TRPV1-like receptor-expressing ARC POMC neurons in the ARC, we generate an adeno-associated virus serotype 5 (AAV5) carrying a Cre-dependent channelrhodopsin-2 (ChR2)-enhanced yellow fluorescent protein (eYFP) expression cassette under the control of the two neuronal POMC enhancers (nPEs). Optogenetic stimulation of TRPV1-like receptor-expressing POMC neurons decreases food intake. Hypothalamic temperature is rapidly elevated and reaches to approximately 39 °C during treadmill running. This elevation is associated with a reduction in food intake. Knockdown of the Trpv1 gene exclusively in ARC POMC neurons blocks the feeding inhibition produced by increased hypothalamic temperature. Taken together, our findings identify a melanocortinergic circuit that links acute elevations in hypothalamic temperature with acute reductions in food intake.

Gene Transfer and Molecular Cloning of the Human NGF Receptor

NASA Astrophysics Data System (ADS)

Chao, Moses V.; Bothwell, Mark A.; Ross, Alonzo H.; Koprowski, Hilary; Lanahan, Anthony A.; Buck, C. Randall; Sehgal, Amita

1986-04-01

Nerve growth factor (NGF) and its receptor are important in the development of cells derived from the neural crest. Mouse L cell transformants have been generated that stably express the human NGF receptor gene transfer with total human DNA. Affinity cross-linking, metabolic labeling and immunoprecipitation, and equilibrium binding with 125I-labeled NGF revealed that this NGF receptor had the same size and binding characteristics as the receptor from human melanoma cells and rat PC12 cells. The sequences encoding the NGF receptor were molecularly cloned using the human Alu repetitive sequence as a probe. A cosmid clone that contained the human NGF receptor gene allowed efficient transfection and expression of the receptor.

Convergent Pathways for Steroid Hormone-and Neurotransmitter-Induced Rat Sexual Behavior

NASA Astrophysics Data System (ADS)

Mani, S. K.; Allen, J. M. C.; Clark, J. H.; Blaustein, J. D.; O'Malley, B. W.

1994-08-01

Estrogen and progesterone modulate gene expression in rodents by activation of intracellular receptors in the hypothalamus, which regulate neuronal networks that control female sexual behavior. However, the neurotransmitter dopamine has been shown to activate certain steroid receptors in a ligand-independent manner. A dopamine receptor stimulant and a D_1 receptor agonist, but not a D_2 receptor agonist, mimicked the effects of progesterone in facilitating sexual behavior in female rats. The facilitatory effect of the neurotransmitter was blocked by progesterone receptor antagonists, a D_1 receptor antagonist, or antisense oligonucleotides to the progesterone receptor. The results suggest that in rodents neurotransmitters may regulate in vivo gene expression and behavior by means of cross-talk with steroid receptors in the brain.

The CB₁ cannabinoid receptor signals striatal neuroprotection via a PI3K/Akt/mTORC1/BDNF pathway.

PubMed

Blázquez, C; Chiarlone, A; Bellocchio, L; Resel, E; Pruunsild, P; García-Rincón, D; Sendtner, M; Timmusk, T; Lutz, B; Galve-Roperh, I; Guzmán, M

2015-10-01

The CB1 cannabinoid receptor, the main molecular target of endocannabinoids and cannabis active components, is the most abundant G protein-coupled receptor in the mammalian brain. In particular, the CB1 receptor is highly expressed in the basal ganglia, mostly on terminals of medium-sized spiny neurons, where it plays a key neuromodulatory function. The CB1 receptor also confers neuroprotection in various experimental models of striatal damage. However, the assessment of the physiological relevance and therapeutic potential of the CB1 receptor in basal ganglia-related diseases is hampered, at least in part, by the lack of knowledge of the precise mechanism of CB1 receptor neuroprotective activity. Here, by using an array of pharmacological, genetic and pharmacogenetic (designer receptor exclusively activated by designer drug) approaches, we show that (1) CB1 receptor engagement protects striatal cells from excitotoxic death via the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin complex 1 pathway, which, in turn, (2) induces brain-derived neurotrophic factor (BDNF) expression through the selective activation of BDNF gene promoter IV, an effect that is mediated by multiple transcription factors. To assess the possible functional impact of the CB1/BDNF axis in a neurodegenerative-disease context in vivo, we conducted experiments in the R6/2 mouse, a well-established model of Huntington's disease, in which the CB1 receptor and BDNF are known to be severely downregulated in the dorsolateral striatum. Adeno-associated viral vector-enforced re-expression of the CB1 receptor in the dorsolateral striatum of R6/2 mice allowed the re-expression of BDNF and the concerted rescue of the neuropathological deficits in these animals. Collectively, these findings unravel a molecular link between CB1 receptor activation and BDNF expression, and support the relevance of the CB1/BDNF axis in promoting striatal neuron survival.

F104S c-Mpl responds to a transmembrane domain-binding thrombopoietin receptor agonist: proof of concept that selected receptor mutations in congenital amegakaryocytic thrombocytopenia can be stimulated with alternative thrombopoietic agents.

PubMed

Fox, Norma E; Lim, Jihyang; Chen, Rose; Geddis, Amy E

2010-05-01

To determine whether specific c-Mpl mutations might respond to thrombopoietin receptor agonists. We created cell line models of type II c-Mpl mutations identified in congenital amegakaryocytic thrombocytopenia. We selected F104S c-Mpl for further study because it exhibited surface expression of the receptor. We measured proliferation of cell lines expressing wild-type or F104S c-Mpl in response to thrombopoietin receptor agonists targeting the extracellular (m-AMP4) or transmembrane (LGD-4665) domains of the receptor by 1-methyltetrazole-5-thiol assay. We measured thrombopoietin binding to the mutant receptor using an in vitro thrombopoietin uptake assay and identified F104 as a potentially critical residue for the interaction between the receptor and its ligand by aligning thrombopoietin and erythropoietin receptors from multiple species. Cells expressing F104S c-Mpl proliferated in response to LGD-4665, but not thrombopoietin or m-AMP4. Compared to thrombopoietin, LGD-4665 stimulates signaling with delayed kinetics in both wild-type and F104S c-Mpl-expressing cells. Although F104S c-Mpl is expressed on the cell surface in our BaF3 cell line model, the mutant receptor does not bind thrombopoietin. Comparison to the erythropoietin receptor suggests that F104 engages in hydrogen-bonding interactions that are critical for binding to thrombopoietin. These findings suggest that a small subset of patients with congenital amegakaryocytic thrombocytopenia might respond to treatment with thrombopoietin receptor agonists, but that responsiveness will depend on the type of mutation and agonist used. We postulate that F104 is critical for thrombopoietin binding. The kinetics of signaling in response to a transmembrane domain-binding agonist are delayed in comparison to thrombopoietin. 2010 ISEH Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance.

PubMed

Knott, Thomas K; Madany, Pasil A; Faden, Ashley A; Xu, Mei; Strotmann, Jörg; Henion, Timothy R; Schwarting, Gerald A

2012-07-04

The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2(-/-) mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2(-/-) neurons. Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2(-/-) mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2(-/-) olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2(-/-) mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2(-/-) olfactory neurons. Results presented here show that many odorant receptors are under-expressed in β3GnT2(-/-) mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2(-/-) mice exhibit a relatively small deficit in their ability to discriminate divergent odors. Results suggest that despite the fact that β3GnT2(-/-) mice have decreased AC3 activity, decreased expression of many ORs, and display many axon growth and guidance errors, odor-evoked activity in cilia of mutant olfactory neurons remains largely intact.

Expression of fatty acid sensing G-protein coupled receptors in peripartal Holstein cows.

PubMed

Agrawal, Alea; Alharthi, Abdulrahman; Vailati-Riboni, Mario; Zhou, Zheng; Loor, Juan J

2017-01-01

G-protein coupled receptors (GPCR), also referred as Free Fatty Acid Receptors (FFAR), are widely studied within human medicine as drug targets for metabolic disorders. To combat metabolic disorders prevalent in dairy cows during the transition period, which co-occur with negative energy balance and changes to lipid and glucose metabolism, it may be helpful to identify locations and roles of FFAR and other members of the GPCR family in bovine tissues. Quantitative RT-PCR (qPCR) of subcutaneous adipose, liver, and PMNL samples during the transition period (-10, +7, and +20 or +30 d) were used for expression profiling of medium- (MCFA) and long-chain fatty acid (LCFA) receptors GPR120 and GPR40 , MCFA receptor GPR84 , and niacin receptor HCAR2/3 . Adipose samples were obtained from cows with either high (HI; BCS ≥ 3.75) or low (LO; BCS ≤ 3.25) body condition score (BCS) to examine whether FFAR expression is correlated with this indicator of health and body reserves. Supplementation of rumen-protected methionine (MET), which may improve immune function and production postpartum, was also compared with unsupplemented control (CON) cows for liver and blood polymorphonuclear leukocytes (PMNL) samples. In adipose tissue, GPR84 and GPR120 were differentially expressed over time, while GPR40 was not expressed; in PMNL, GPR40 was differentially expressed over time and between MET vs. CON, GPR84 expression differed only between dietary groups, and GPR120 was not expressed; in liver, GPCR were either not expressed or barely detectable. The data indicate that there is likely not a direct role in liver for the selected GPCR during the transition period, but they do play variable roles in adipose and PMN. In future, these receptors may prove useful targets and/or markers for peripartal metabolism and immunity.

Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc.

PubMed

Le Maitre, Christine L; Richardson, Stephen M A; Baird, Pauline; Freemont, Anthony J; Hoyland, Judith A

2005-12-01

Low back pain (LBP) is a common, debilitating and economically important disorder. Current evidence implicates loss of intervertebral disc (IVD) matrix consequent upon 'degeneration' as a major cause of LBP. Degeneration of the IVD involves increases in degradative enzymes and decreases in the extracellular matrix (ECM) component in a process that is controlled by a range of cytokines and growth factors. Studies have suggested using anabolic growth factors to regenerate the normal matrix of the IVD, hence restoring disc height and reversing degenerative disc disease. However, for such therapies to be successful it is vital that the target cells (i.e. the disc cells) express the appropriate receptors. This immunohistochemical study has for the first time investigated the expression and localization of four potentially beneficial growth factor receptors (i.e. TGFbetaRII, BMPRII, FGFR3 and IGFRI) in non-degenerate and degenerate human IVDs. Receptor expression was quantified across regions of the normal and degenerate disc and showed that cells of the nucleus pulposus (NP) and inner annulus fibrosus (IAF) expressed significantly higher levels of the four growth factor receptors investigated. There were no significant differences between the four growth factor expression in non-degenerate and degenerate biopsies. However, expression of TGFbetaRII, FGFR3 and IGFRI, but not BMP RII, were observed in the ingrowing blood vessels that characterize part of the disease aetiology. In conclusion, this study has demonstrated the expression of the four growth factor receptors at similar levels in the chondrocyte-like cells of the NP and IAF in both non-degenerate and degenerate discs, implicating a role in normal disc homeostasis and suggesting that the application of these growth factors to the degenerate human IVD would stimulate matrix production. However, the expression of some of the growth factor receptors on ingrowing blood vessels might be problematic in a therapeutic approach. Copyright 2005 Pathological Society of Great Britain and Ireland.

Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum.

PubMed

Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

2014-01-01

Growing awareness of cerebellar involvement in addiction is based on the cerebellum's intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that lead to addiction.

Effects of acute versus repeated cocaine exposure on the expression of endocannabinoid signaling-related proteins in the mouse cerebellum

PubMed Central

Palomino, Ana; Pavón, Francisco-Javier; Blanco-Calvo, Eduardo; Serrano, Antonia; Arrabal, Sergio; Rivera, Patricia; Alén, Francisco; Vargas, Antonio; Bilbao, Ainhoa; Rubio, Leticia; Rodríguez de Fonseca, Fernando; Suárez, Juan

2014-01-01

Growing awareness of cerebellar involvement in addiction is based on the cerebellum’s intermediary position between motor and reward, potentially acting as an interface between motivational and cognitive functions. Here, we examined the impact of acute and repeated cocaine exposure on the two main signaling systems in the mouse cerebellum: the endocannabinoid (eCB) and glutamate systems. To this end, we investigated whether eCB signaling-related gene and protein expression {cannabinoid receptor type 1 receptors and enzymes that produce [diacylglycerol lipase alpha/beta (DAGLα/β) and N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD)] and degrade [monoacylglycerol lipase (MAGL) and fatty acid amino hydrolase (FAAH)] eCB} were altered. In addition, we analyzed the gene expression of relevant components of the glutamate signaling system [glutamate synthesizing enzymes liver-type glutaminase isoform (LGA) and kidney-type glutaminase isoform (KGA), metabotropic glutamatergic receptor (mGluR3/5), NMDA-ionotropic glutamatergic receptor (NR1/2A/2B/2C) and AMPA-ionotropic receptor subunits (GluR1/2/3/4)] and the gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, because noradrenergic terminals innervate the cerebellar cortex. Results indicated that acute cocaine exposure decreased DAGLα expression, suggesting a down-regulation of 2-arachidonylglycerol (2-AG) production, as well as gene expression of TH, KGA, mGluR3 and all ionotropic receptor subunits analyzed in the cerebellum. The acquisition of conditioned locomotion and sensitization after repeated cocaine exposure were associated with an increased NAPE-PLD/FAAH ratio, suggesting enhanced anandamide production, and a decreased DAGLβ/MAGL ratio, suggesting decreased 2-AG generation. Repeated cocaine also increased LGA gene expression but had no effect on glutamate receptors. These findings indicate that acute cocaine modulates the expression of the eCB and glutamate systems. Repeated cocaine results in normalization of glutamate receptor expression, although sustained changes in eCB is observed. We suggest that cocaine-induced alterations to cerebellar eCB should be considered when analyzing the adaptations imposed by psychostimulants that lead to addiction. PMID:24634647

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

PubMed

Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

2014-09-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

A Single Dose of LSD Does Not Alter Gene Expression of the Serotonin 2A Receptor Gene (HTR2A) or Early Growth Response Genes (EGR1-3) in Healthy Subjects

PubMed Central

Dolder, Patrick C.; Grünblatt, Edna; Müller, Felix; Borgwardt, Stefan J.; Liechti, Matthias E.

2017-01-01

Rationale: Renewed interest has been seen in the use of lysergic acid diethylamide (LSD) in psychiatric research and practice. The repeated use of LSD leads to tolerance that is believed to result from serotonin (5-HT) 5-HT2A receptor downregulation. In rats, daily LSD administration for 4 days decreased frontal cortex 5-HT2A receptor binding. Additionally, a single dose of LSD acutely increased expression of the early growth response genes EGR1 and EGR2 in rat and mouse brains through 5-HT2A receptor stimulation. No human data on the effects of LSD on gene expression has been reported. Therefore, we investigated the effects of single-dose LSD administration on the expression of the 5-HT2A receptor gene (HTR2A) and EGR1-3 genes. Methods: mRNA expression levels were analyzed in whole blood as a peripheral biomarker in 15 healthy subjects before and 1.5 and 24 h after the administration of LSD (100 μg) and placebo in a randomized, double-blind, placebo-controlled, cross-over study. Results: LSD did not alter the expression of the HTR2A or EGR1-3 genes 1.5 and 24 h after administration compared with placebo. Conclusion: No changes were observed in the gene expression of LSD’s primary target receptor gene or genes that are implicated in its downstream effects. Remaining unclear is whether chronic LSD administration alters gene expression in humans. PMID:28701958

Changes in protein and gene expression of angiotensin II receptors (AT1 and AT2) in aorta of diabetic and hypertensive rats.

PubMed

Romero-Nava, R; Rodriguez, J E; Reséndiz-Albor, A A; Sánchez-Muñoz, F; Ruiz-Hernandéz, A; Huang, F; Hong, E; Villafaña, S

2016-01-01

Diabetes and hypertension have been associated with cardiovascular diseases and stroke. Some reports have related the coexistence of hypertension and diabetes with increase in the risk of developing vascular complications. Recently some studies have shown results suggesting that in the early stages of diabetes and hypertension exist a reduced functional response to vasopressor agents like angiotensin II (Ang II), which plays an important role in blood pressure regulation mechanism through the activation of its AT1 and AT2 receptors. For that reason, the aim of this work was to study the gene and protein expression of AT1 and AT2 receptors in aorta of diabetic SHR and WKY rats. Diabetes was induced by the administration of streptozotocin (60 mg/kg i.p.). After 4 weeks of the onset of diabetes, the protein expression was obtained by western blot and the mRNA expression by RT-PCR. Our results showed that the hypertensive rats have a higher mRNA and protein expression of AT1 receptors than normotensive rats while the AT2 expression remained unchanged. On the other hand, the combination of diabetes and hypertension increased the mRNA and protein expression of AT1 and AT2 receptors significantly. In conclusion, our results suggest that diabetes with hypertension modifies the mRNA and protein expression of AT1 and AT2 receptors. However, the overexpression of AT2 could be associated with the reduction in the response to Ang II in the early stage of diabetes.

Alterations in Gene and Protein Expression of Cannabinoid CB2 and GPR55 Receptors in the Dorsolateral Prefrontal Cortex of Suicide Victims.

PubMed

García-Gutiérrez, María S; Navarrete, Francisco; Navarro, Gemma; Reyes-Resina, Irene; Franco, Rafael; Lanciego, Jose Luis; Giner, Salvador; Manzanares, Jorge

2018-02-12

Recent studies point to the cannabinoid CB 2 receptors (CB 2 r) and the non-cannabinoid receptor GPR55 as potential key targets involved in the response to stress, anxiety, and depression. Considering the close relationship between neuropsychiatric disorders and suicide, the purpose of this study was to evaluate the potential alterations of CB 2 r and GPR55 in suicide victims. We analyzed gene and protein expression of both receptors by real-time PCR and western blot, respectively, in the dorsolateral prefrontal cortex (DLPFC) of 18 suicide victims with no clinical psychiatric history or treatment with anxiolytics or antidepressants, and 15 corresponding controls. We used in situ proximity ligation assay to evaluate whether the receptors formed heteromeric complexes and to determine the expression level of these heteromers, also assessing the co-expression of heteromers in neurons, astroglia, or microglia cells. CB 2 r and GPR55 gene expressions were significantly lower (by 33 and 41%, respectively) in the DLPFC of suicide cases. CB 2 r protein expression was higher, as were CB 2 -GPR55 heteroreceptor complexes. The results also revealed the presence of CB 2 -GPR55 receptor heteromers in both neurons and astrocytes, whereas microglial cells showed no expression. We did not observe any significant alterations of GPR55 protein expression. Additional studies will be necessary to evaluate if these alterations are reproducible in suicide victims diagnosed with different psychiatric disorders. Taken together, the results suggest that CB 2 r and GPR55 may play a relevant role in the neurobiology of suicide.

The cannabinoid receptor CB1 modulates the signaling properties of the lysophosphatidylinositol receptor GPR55.

PubMed

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P; Brown, Andrew J; Heinemann, Akos; Waldhoer, Maria

2012-12-28

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors.

The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

PubMed Central

Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

2012-01-01

The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

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

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

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

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

Expression of ionotropic glutamate receptors, AMPA, kainite and NMDA, in the pigeon retina.

PubMed

Atoji, Yasuro

2015-07-01

Glutamate is an excitatory neurotransmitter in the vertebrate retina. A previous study found vesicular glutamate transporter 2 (vGluT2) mRNA in the pigeon retina, suggesting that bipolar and ganglion cells are glutamatergic. The present study examined the localization of ionotropic glutamate receptors to identify receptor cells in the pigeon retina using in situ hybridization histochemistry. Nine subunits of AMPA receptor (GluA1, GluA2, GluA3, and GluA4), kainate receptor (GluK1, GluK2, and GluK4), and NMDA receptor (GluN1 and GluN2A) were found to be expressed in the inner nuclear layer (INL) and ganglion cell layers. GluA1, GluA2, GluA3, and GluA4 were primarily expressed in the inner half of INL, and the signal intensity was strong for GluA2, GluA3, and GluA4. GluK1 was intensely expressed in the outer half of INL, whereas GluK2 and GluK4 were mainly localized in the inner half of INL. GluN1 and GluN2A were moderately expressed in the inner half of INL. Horizontal cells expressed GluA3 and GluA4, and ganglion cells expressed all subunits examined. These results suggest that the glutamatergic neurotransmission in the pigeon retina is similar to that in mammals. Copyright © 2015 Elsevier Ltd. All rights reserved.

Molecular characterization and gene expression patterns of retinoid receptors, in normal and regenerating tissues of the sea cucumber, Holothuria glaberrima.

PubMed

Viera-Vera, Jorge; García-Arrarás, José E

2018-05-15

Retinoic acid receptors (RAR) and retinoid X receptors (RXR) are ligand-mediated transcription factors that synchronize intricate signaling networks in metazoans. Dimer formation between these two nuclear receptors mediates the recruitment of co-regulatory complexes coordinating the progression of signaling cascades during developmental and regenerative events. In the present study we identified and characterized the receptors for retinoic acid in the sea cucumber Holothuria glaberrima; a model system capable of regenerative organogenesis during adulthood. Molecular characterizations revealed the presence of three isoforms of RAR and two of RXR as a consequence of alternative splicing events. Various analyses including: primary structure sequencing, phylogenetic analysis, protein domain prediction, and multiple sequence alignment further confirmed their identity. Semiquantitative reverse transcription PCR analysis of each receptor isoform herein identified showed that the retinoid receptors are expressed in all tissues sampled: the mesenteries, respiratory trees, muscles, gonads, and the digestive tract. During regenerative organogenesis two of the receptors (RAR-L and RXR-T) showed differential expression in the posterior segment while RAR-S is differentially expressed in the anterior segment of the intestine. This work presents the first description of the components relaying the signaling for retinoic acid within this model system. Copyright © 2018 Elsevier B.V. All rights reserved.

Monocyte and plasma expression of TAM ligand and receptor in renal failure: Links to unregulated immunity and chronic inflammation.

PubMed

Lee, Iris J; Hilliard, Brendan A; Ulas, Mehriban; Yu, Daohai; Vangala, Chandan; Rao, Swati; Lee, Jean; Gadegbeku, Crystal A; Cohen, Philip L

2015-06-01

Chronic inflammation is increased in patients with chronic kidney disease (CKD) and contributes to cardiovascular morbidity and mortality. Specific immune mechanisms and pathways that drive and maintain chronic inflammation in CKD are not well described. The TAM ligands (Gas6 and protein S) and receptors (Axl and Mer) have been recently recognized as playing a prominent role in immune regulation. The receptors exist in both soluble and cell-bound forms; the soluble receptors (sAxl and sMer) are believed to compete with the bound receptors and thus inhibit their function. In this study, we determined the expression of cell-bound and soluble TAM proteins in patients with CKD. CKD patients had significantly lower expression of Mer in monocytes, yet increased expression of soluble TAM receptors sAxl and sMer in plasma compared to controls. The metalloproteinase ADAM 17, responsible for cleavage of Mer to its soluble form, was increased in patient monocytes. Elevated levels of soluble TAM receptors were more evident in patients with progressive renal failure. These observations suggest that functional deficiency of TAM receptor-mediated regulation of inflammation may contribute to chronic inflammation in patients with CKD. Copyright © 2015 Elsevier Inc. All rights reserved.

Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata

PubMed Central

Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin

2016-01-01

The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata. Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation. PMID:27421895

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

PubMed

Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

2004-09-01

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

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

PubMed Central

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

2012-01-01

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

Induction of hepatic ABC transporter expression is part of the PPARalpha-mediated fasting response in the mouse.

PubMed

Kok, Tineke; Wolters, Henk; Bloks, Vincent W; Havinga, Rick; Jansen, Peter L M; Staels, Bart; Kuipers, Folkert

2003-01-01

Fatty acids are natural ligands of the peroxisome proliferator-activated receptor alpha (PPARalpha). Synthetic ligands of this nuclear receptor, i.e., fibrates, induce the hepatic expression of the multidrug resistance 2 gene (Mdr2), encoding the canalicular phospholipid translocator, and affect hepatobiliary lipid transport. We tested whether fasting-associated fatty acid release from adipose tissues alters hepatic transporter expression and bile formation in a PPARalpha-dependent manner. A 24-hour fasting/48-hour refeeding schedule was used in wild-type and Pparalpha((-/-)) mice. Expression of genes involved in the control of bile formation was determined and related to secretion rates of biliary components. Expression of Pparalpha, farnesoid X receptor, and liver X receptor alpha genes encoding nuclear receptors that control hepatic bile salt and sterol metabolism was induced on fasting in wild-type mice only. The expression of Mdr2 was 5-fold increased in fasted wild-type mice and increased only marginally in Pparalpha((-/-)) mice, and it normalized on refeeding. Mdr2 protein levels and maximal biliary phospholipid secretion rates were clearly increased in fasted wild-type mice. Hepatic expression of the liver X receptor target genes ATP binding cassette transporter a1 (Abca1), Abcg5, and Abcg8, implicated in hepatobiliary cholesterol transport, was induced in fasted wild-type mice only. However, the maximal biliary cholesterol secretion rate was reduced by approximately 50%. Induction of Mdr2 expression and function is part of the PPARalpha-mediated fasting response in mice. Fasting also induces expression of the putative hepatobiliary cholesterol transport genes Abca1, Abcg5, and Abcg8, but, nonetheless, maximal biliary cholesterol excretion is decreased after fasting.

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

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

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

2006-01-01

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

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

PubMed

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

2016-03-01

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

The effects of abnormalities of glucose homeostasis on the expression and binding of muscarinic receptors in cerebral cortex of rats.

PubMed

Sherin, Antony; Peeyush, Kumar T; Naijil, George; Nandhu, Mohan Sobhana; Jayanarayanan, Sadanandan; Jes, Paul; Paulose, Cheramadathikudiyil Skaria

2011-01-25

Glucose homeostasis in humans is an important factor for the functioning of nervous system. Both hypo and hyperglycemia contributes to neuronal functional deficit. In the present study, effect of insulin induced hypoglycemia and streptozotocin induced diabetes on muscarinic receptor binding, cholinergic enzymes; AChE, ChAT expression and GLUT3 in the cerebral cortex of experimental rats were analysed. Total muscarinic, muscarinic M(1) receptor showed a significant decrease and muscarinic M(3) receptor subtype showed a significant increased binding in the cerebral cortex of hypoglycemic rats compared to diabetic and control. Real-Time PCR analysis of muscarinic M(1), M(3) receptor subtypes confirmed the receptor binding studies. Immunohistochemistry of muscarinic M(1), M(3) receptors using specific antibodies were also carried out. AChE and GLUT3 expression up regulated and ChAT expression down regulated in hypoglycemic rats compared to diabetic and control rats. Our results showed that hypo/hyperglycemia caused impaired glucose transport in neuronal cells as shown by altered expression of GLUT3. Increased AChE and decreased ChAT expression is suggested to alter cortical acetylcholine metabolism in experimental rats along with altered muscarinic receptor binding in hypo/hyperglycemic rats, impair cholinergic transmission, which subsequently lead to cholinergic dysfunction thereby causing learning and memory deficits. We observed a prominent cholinergic functional disturbance in hypoglycemic condition than in hyperglycemia. Hypoglycemia exacerbated the neurochemical changes in cerebral cortex induced by hyperglycemia. These findings have implications for both therapy and identification of causes contributing to neuronal dysfunction in diabetes. Copyright © 2010 Elsevier B.V. All rights reserved.

Immunolocalization of Leptin Receptor and mRNA Expression of Leptin and Estrogen Receptors as well as Caspases in the Chorioallantoic Membrane (CAM) of the Chicken Embryo.

PubMed

Grzegorzewska, Agnieszka K; Lis, Marcin W; Sechman, Andrzej

The chicken chorioallantoic membrane (CAM) is used as a model in tests of angiogenesis, the biocompatibility of materials as well as tumor invasive potential. To assess the properties of CAM tissue, the localization of leptin receptor in the CAM, and the mRNA expression of two leptin receptor isoforms, estrogen receptors (ERα and ERβ) and caspases (-1 and -3) in the CAM on embryonic days 12 (E12), 15 (E15) and 18 (E18) were investigated. The leptin receptor was immunolocalized in each structure of the CAM (chorionic epithelium, allantoic epithelium, mesodermal layer and the walls of blood vessels) and did not change among analyzed stages of embryonic development (E12, E15 and E18) and between sexes. Expression of mRNA of genes encoding leptin and estrogen receptors as well as caspases was detected in the CAM of female and male chicken embryos at all three analysed stages of development. The relative mRNA expression of the long form of leptin receptor exceeded that of its short isoform. The mRNA expression of ERβ was significantly higher than ERα as well as caspase-3 in comparison with caspase-1. There were no differences in mRNA expression of these genes between sexes and among analyzed developmental days. The results indicate that the CAM is a target tissue for leptin as well as for estrogens and that CAM development is partially regulated by caspase-1 and caspase-3 dependent cell death. These results should be taken into consideration in studies in which the CAM is used as an experimental model.

Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

PubMed

VandenBussche, C J; Mulrooney, T J; Frazier, W R; Dakshanamurthy, S; Hurley, C K

2009-03-01

Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

Characterization of interleukin-4-stimulated nasal polyp fibroblasts.

PubMed

Steinke, John W; Crouse, Charles D; Bradley, Dewayne; Hise, Kathleen; Lynch, Kevin; Kountakis, Stilianos E; Borish, Larry

2004-02-01

Chronic hyperplastic eosinophilic sinusitis is an inflammatory disease that results in the accumulation of eosinophils, fibroblasts, mast cells, and goblet cells at the site of injury. A common feature of this disease is the presence of nasal polyposis (NP). The current studies were designed to assess the contribution of interleukin (IL)-4 to fibroblast-mediated inflammation in chronic hyperplastic eosinophilic sinusitis/NP. In addition, we hypothesized that cysteinyl leukotrienes (CysLT) may directly influence fibroblast-mediated fibrotic and remodeling pathways in this disorder. Fibroblasts were isolated from NP tissue. All fibroblast lines expressed the IL-4 receptor. IL-4 induced changes in mRNA and protein expression of fibrotic (transforming growth factor-beta1 and -beta2) and inflammatory cytokines and chemokines (IL-6 and CCL11) by fibroblasts as measured by semiquantitative and quantitative polymerase chain reaction, RNase protection assay, and enzyme-linked immunosorbent assay. The expression of CysLT and other proinflammatory lipid receptors on fibroblasts was evaluated. CysLT1 and CysLT2 receptors were not expressed on fibroblasts; however, LPA(1) receptor was constitutively expressed and LPA(2) receptor expression was upregulated by IL-4. The metabolic cascade involved in CysLT synthesis was not expressed in fibroblasts and could not be induced by IL-4 treatment.

Expression profiling of nuclear receptors in breast cancer identifies TLX as a mediator of growth and invasion in triple-negative breast cancer.

PubMed

Lin, Meng-Lay; Patel, Hetal; Remenyi, Judit; Banerji, Christopher R S; Lai, Chun-Fui; Periyasamy, Manikandan; Lombardo, Ylenia; Busonero, Claudia; Ottaviani, Silvia; Passey, Alun; Quinlan, Philip R; Purdie, Colin A; Jordan, Lee B; Thompson, Alastair M; Finn, Richard S; Rueda, Oscar M; Caldas, Carlos; Gil, Jesus; Coombes, R Charles; Fuller-Pace, Frances V; Teschendorff, Andrew E; Buluwela, Laki; Ali, Simak

2015-08-28

The Nuclear Receptor (NR) superfamily of transcription factors comprises 48 members, several of which have been implicated in breast cancer. Most important is estrogen receptor-α (ERα), which is a key therapeutic target. ERα action is facilitated by co-operativity with other NR and there is evidence that ERα function may be recapitulated by other NRs in ERα-negative breast cancer. In order to examine the inter-relationships between nuclear receptors, and to obtain evidence for previously unsuspected roles for any NRs, we undertook quantitative RT-PCR and bioinformatics analysis to examine their expression in breast cancer. While most NRs were expressed, bioinformatic analyses differentiated tumours into distinct prognostic groups that were validated by analyzing public microarray data sets. Although ERα and progesterone receptor were dominant in distinguishing prognostic groups, other NR strengthened these groups. Clustering analysis identified several family members with potential importance in breast cancer. Specifically, RORγ is identified as being co-expressed with ERα, whilst several NRs are preferentially expressed in ERα-negative disease, with TLX expression being prognostic in this subtype. Functional studies demonstrated the importance of TLX in regulating growth and invasion in ERα-negative breast cancer cells.

Prostaglandin E2 suppresses beta1-integrin expression via E-prostanoid receptor in human monocytes/macrophages.

PubMed

Hasegawa, Shunji; Ichiyama, Takashi; Kohno, Fumitaka; Korenaga, Yuno; Ohsaki, Ayami; Hirano, Reiji; Haneda, Yasuhiro; Fukano, Reiji; Furukawa, Susumu

2010-01-01

Beta1-integrins mediate cell attachment to different extracellular matrix proteins, intracellular proteins, and intercellular adhesions. Recently, it has been reported that prostaglandin E2 (PGE2) has anti-inflammatory properties such as inhibition of the expression of adhesion molecules or production of chemokines. However, the effect of PGE2 on the expression of beta1-integrin remains unknown. In this study, we investigated the effects of PGE2 on the expression of beta1-integrin in the human monocytic cell line THP-1 and in CD14+ monocytes/macrophages in human peripheral blood. For this, we examined the role of four subtypes of PGE2 receptors and E-prostanoid (EP) receptors on PGE2-mediated inhibition. We found that PGE2 significantly inhibited the expression of beta1-integrin, mainly through EP4 receptors in THP-1 cells and CD14+ monocytes/macrophages in human peripheral blood. We suggest that PGE2 has anti-inflammatory effects, leading to the inhibited expression of beta1-integrin in human monocytes/macrophages, and that the EP4 receptor may play an important role in PGE2-mediated inhibition. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Death Receptor Expression on Blasts in AML Is Associated with Unfavorable Prognosis.

PubMed

Schmohl, Joerg Uwe; Nuebling, Tina; Wild, Julia; Jung, Johannes; Kroell, Tanja; Kanz, Lothar; Salih, Helmut R; Schmetzer, Helga

2015-07-01

Tumor necrosis factor (TNF) receptor family members play a key role in the regulation of biological functions such as differentiation, proliferation and apoptosis of various cell types. We studied co-expression profiles of death receptors from the TNF family [TNF-related apoptosis-inducing ligand receptor (TRAILR) 1 to 3, TNF receptor 1 (TNFR1) and FAS receptor (FAS)] on peripheral blood blasts from 46 patients with acute myeloid leukemia (AML) at first diagnosis by flow cytometry and correlated the obtained specific fluorescence indices (SFI) with morphological, cytogenetic and clinical parameters. We found that the expression of TRAILR2 and R3 was significantly increased in unfavorable risk groups, according to the National Comprehensive Cancer Network. Additionally, cut-off analyses for TRAILR2 and TNFR1 showed significantly shorter overall survival, earlier disease onset, higher proportions of cases with unfavorable prognosis and higher probability of relapse when SFIs were above the established cut-off. We demonstrate that high co-expression of death receptors on blasts is an independent predictor of poor prognosis in AML. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Different Hypothalamic Nicotinic α7 Receptor Expression and Response to Low Nicotine Dose in Alcohol-Preferring and Alcohol-Avoiding Rats.

PubMed

Nuutinen, Saara; Panula, Pertti; Salminen, Outi

2016-02-01

The aim of this study was to examine possible differences in nicotinic acetylcholine receptors and responses in rats with genetic preference or avoidance for alcohol. This was done by using 2 rat lines with high alcohol preference (Alko Alcohol [AA]) or alcohol avoidance (Alko Non-Alcohol [ANA]). Locomotor activity was measured following nicotine and histamine H3 receptor (H3R) antagonist treatment. In situ hybridization and receptor ligand binding experiments were used in drug-naïve animals to examine the expression of different α nicotinic receptor subunits. The AA rats were found to be more sensitive to the stimulatory effect of a low dose of nicotine than ANA rats, which were not significantly activated. Combination of histamine H3R antagonist, JNJ-39220675, and nicotine resulted to similar locomotor activation as nicotine alone. To further understand the mechanism underlying the difference in nicotine response in AA and ANA rats, we studied the expression of α5, α6, and α7 nicotinic receptor subunits in specific brain areas of AA and ANA rats. We found no differences in the expression of α5 nicotinic receptor subunits in the medial habenula and hippocampus or in α6 subunit in the ventral tegmental area and substantia nigra. However, the level of α7 nicotinic receptor subunit mRNA was significantly lower in the tuberomamillary nucleus of posterior hypothalamus of alcohol-preferring AA rats than in alcohol-avoiding ANA rats. Also the hypothalamic [125I-α-bungarotoxin binding was lower in AA rats indicating lower levels of α7 nicotinic receptors. The lower expression and receptor binding of α7 nicotinic receptors in the tuberomamillary nucleus of AA rats suggest a difference in the regulation of brain histamine neurons between the rat lines since the α7 nicotinic receptors are located in histaminergic neurons. Stronger nicotine-induced locomotor response, mediated partially via α7 receptors, and previously described high alcohol consumption in AA rats could be explained by the found difference in tuberomamillary α7 receptor levels. Copyright © 2016 by the Research Society on Alcoholism.

Design, synthesis and biological evaluation of a bivalent micro opiate and adenosine A1 receptor antagonist.

PubMed

Mathew, Smitha C; Ghosh, Nandita; By, Youlet; Berthault, Aurélie; Virolleaud, Marie-Alice; Carrega, Louis; Chouraqui, Gaëlle; Commeiras, Laurent; Condo, Jocelyne; Attolini, Mireille; Gaudel-Siri, Anouk; Ruf, Jean; Parrain, Jean-Luc; Rodriguez, Jean; Guieu, Régis

2009-12-01

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A(1) adenosine and mu opiate receptors with a K(i) of 0.8+/-0.05 and 0.7+/-0.03 microM, respectively. This hybrid molecule increases cAMP production in cells that over express the mu receptor as well as those over expressing the A(1) adenosine receptor and reverses the antalgic effects of mu and A(1) adenosine receptor agonists in animals.

NRP-1 Receptor Expression Mismatch in Skin of Subjects with Experimental and Diabetic Small Fiber Neuropathy

PubMed Central

Van Acker, Nathalie; Ragé, Michael; Vermeirsch, Hilde; Schrijvers, Dorien; Nuydens, Rony; Byttebier, Geert; Timmers, Maarten; De Schepper, Stefanie; Streffer, Johannes; Andries, Luc; Plaghki, Léon; Cras, Patrick; Meert, Theo

2016-01-01

The in vivo cutaneous nerve regeneration model using capsaicin is applied extensively to study the regenerative mechanisms and therapeutic efficacy of disease modifying molecules for small fiber neuropathy (SFN). Since mismatches between functional and morphological nerve fiber recovery are described for this model, we aimed at determining the capability of the capsaicin model to truly mimic the morphological manifestations of SFN in diabetes. As nerve and blood vessel growth and regenerative capacities are defective in diabetes, we focused on studying the key regulator of these processes, the neuropilin-1 (NRP-1)/semaphorin pathway. This led us to the evaluation of NRP-1 receptor expression in epidermis and dermis of subjects presenting experimentally induced small fiber neuropathy, diabetic polyneuropathy and of diabetic subjects without clinical signs of small fiber neuropathy. The NRP-1 receptor was co-stained with CD31 vessel-marker using immunofluorescence and analyzed with Definiens® technology. This study indicates that capsaicin application results in significant loss of epidermal NRP-1 receptor expression, whereas diabetic subjects presenting small fiber neuropathy show full epidermal NRP-1 expression in contrast to the basal expression pattern seen in healthy controls. Capsaicin induced a decrease in dermal non-vascular NRP-1 receptor expression which did not appear in diabetic polyneuropathy. We can conclude that the capsaicin model does not mimic diabetic neuropathy related changes for cutaneous NRP-1 receptor expression. In addition, our data suggest that NRP-1 might play an important role in epidermal nerve fiber loss and/or defective regeneration and that NRP-1 receptor could change the epidermal environment to a nerve fiber repellant bed possibly through Sem3A in diabetes. PMID:27598321

Rearrangement and expression of the human {Psi}C{lambda}6 gene segment results in a surface Ig receptor with a truncated light chain constant region

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

Stiernholm, N.B.J.; Verkoczy, L.K.; Berinstein, N.L.

1995-05-01

The constant region of the human Ig{lambda} locus consists of seven tandemly organized J-C gene segments. Although it has been established that the J-C{lambda}1, J-C{lambda}2, J-C{lambda}3, and J-C{lambda}7 gene segments are functional, and code for the four distinct Ig{lambda} isotypes found in human serum, the J-C{lambda}4, J-C{lambda}5, and J-C{lambda}6 gene segments are generally considered to be pseudogenes. Although one example of a functional J-C{lambda}6 gene segment has been documented, in the majority of cases, J-C{lambda}6 is rendered nonfunctional by virtue of a single duplication of four nucleotides, creating a premature translational arrest. We show here that rearrangements to the J-C{lambda}6more » gene segment do occur, and that such a rearrangement encodes an Ig{lambda} protein that lacks the terminal end of the constant region. We also show that this truncated protein is expressed on the surface with the IgH chain, creating an unusual surface Ig (sIg) receptor (sIg{triangle}CL). Cells that express this receptor on the surface do so at significantly reduced levels compared with clonally related variants, which express sIg receptors with conventional Ig{lambda} L chains. However, the effects of sIg cross-linking on tyrosine phosphorylation and surface expression of the CD25 and CD71 Ags are similar in cells that express conventional sIg receptors and in those that express sIg{triangle}CL receptors, suggesting that the latter could possibly function as an Ag receptor. 35 refs., 7 figs.« less

A novel thromboxane A2 receptor N42S variant results in reduced surface expression and platelet dysfunction.

PubMed

Nisar, Shaista P; Lordkipanidzé, Marie; Jones, Matthew L; Dawood, Ban; Murden, Sherina; Cunningham, Margaret R; Mumford, Andrew D; Wilde, Jonathan T; Watson, Steve P; Mundell, Stuart J; Lowe, Gillian C

2014-05-05

A small number of thromboxane receptor variants have been described in patients with a bleeding history that result in platelet dysfunction. We have identified a patient with a history of significant bleeding, who expresses a novel heterozygous thromboxane receptor variant that predicts an asparagine to serine substitution (N42S). This asparagine is conserved across all class A GPCRs, suggesting a vital role for receptor structure and function.We investigated the functional consequences of the TP receptor heterozygous N42S substitution by performing platelet function studies on platelet-rich plasma taken from the patient and healthy controls. We investigated the N42S mutation by expressing the wild-type (WT) and mutant receptor in human embryonic kidney (HEK) cells. Aggregation studies showed an ablation of arachidonic acid responses in the patient, whilst there was right-ward shift of the U46619 concentration response curve (CRC). Thromboxane generation was unaffected. Calcium mobilisation studies in cells lines showed a rightward shift of the U46619 CRC in N42S-expressing cells compared to WT. Radioligand binding studies revealed a reduction in BMax in platelets taken from the patient and in N42S-expressing cells, whilst cell studies confirmed poor surface expression. We have identified a novel thromboxane receptor variant, N42S, which results in platelet dysfunction due to reduced surface expression. It is associated with a significant bleeding history in the patient in whom it was identified. This is the first description of a naturally occurring variant that results in the substitution of this highly conserved residue and confirms the importance of this residue for correct GPCR function.

A Distributed Network for Social Cognition Enriched for Oxytocin Receptors

PubMed Central

Mitre, Mariela; Marlin, Bianca J.; Schiavo, Jennifer K.; Morina, Egzona; Norden, Samantha E.; Hackett, Troy A.; Aoki, Chiye J.

2016-01-01

Oxytocin is a neuropeptide important for social behaviors such as maternal care and parent–infant bonding. It is believed that oxytocin receptor signaling in the brain is critical for these behaviors, but it is unknown precisely when and where oxytocin receptors are expressed or which neural circuits are directly sensitive to oxytocin. To overcome this challenge, we generated specific antibodies to the mouse oxytocin receptor and examined receptor expression throughout the brain. We identified a distributed network of female mouse brain regions for maternal behaviors that are especially enriched for oxytocin receptors, including the piriform cortex, the left auditory cortex, and CA2 of the hippocampus. Electron microscopic analysis of the cerebral cortex revealed that oxytocin receptors were mainly expressed at synapses, as well as on axons and glial processes. Functionally, oxytocin transiently reduced synaptic inhibition in multiple brain regions and enabled long-term synaptic plasticity in the auditory cortex. Thus modulation of inhibition may be a general mechanism by which oxytocin can act throughout the brain to regulate parental behaviors and social cognition. SIGNIFICANCE STATEMENT Oxytocin is an important peptide hormone involved in maternal behavior and social cognition, but it has been unclear what elements of neural circuits express oxytocin receptors due to the paucity of suitable antibodies. Here, we developed new antibodies to the mouse oxytocin receptor. Oxytocin receptors were found in discrete brain regions and at cortical synapses for modulating excitatory-inhibitory balance and plasticity. These antibodies should be useful for future studies of oxytocin and social behavior. PMID:26911697

Pharmacological characterization of a tyramine receptor from the southern cattle tick, Rhipicephalus (Boophilus) microplus.

PubMed

Gross, Aaron D; Temeyer, Kevin B; Day, Tim A; Pérez de León, Adalberto A; Kimber, Michael J; Coats, Joel R

2015-08-01

The southern cattle tick (Rhipicephalus (Boophilus) microplus) is a hematophagous external parasite that vectors the causative agents of bovine babesiosis or cattle tick fever, Babesia bovis and B. bigemina, and anaplasmosis, Anaplasma marginale. The southern cattle tick is a threat to the livestock industry in many locations throughout the world. Control methods include the use of chemical acaricides including amitraz, a formamidine insecticide, which is proposed to activate octopamine receptors. Previous studies have identified a putative octopamine receptor from the southern cattle tick in Australia and the Americas. Furthermore, this putative octopamine receptor could play a role in acaricide resistance to amitraz. Recently, sequence data indicated that this putative octopamine receptor is probably a type-1 tyramine receptor (TAR1). In this study, the putative TAR1 was heterologously expressed in Chinese hamster ovary (CHO-K1) cells, and the expressed receptor resulted in a 39-fold higher potency for tyramine compared to octopamine. Furthermore, the expressed receptor was strongly antagonized by yohimbine and cyproheptadine, and mildly antagonized by mianserin and phentolamine. Tolazoline and naphazoline had agonistic or modulatory activity against the expressed receptor, as did the amitraz metabolite, BTS-27271; however, this was only observed in the presence of tyramine. The southern cattle tick's tyramine receptor may serve as a target for the development of anti-parasitic compounds, in addition to being a likely target of formamidine insecticides. Copyright © 2015 Elsevier Ltd. All rights reserved.

Am5-HT7: molecular and pharmacological characterization of the first serotonin receptor of the honeybee (Apis mellifera).

PubMed

Schlenstedt, Jana; Balfanz, Sabine; Baumann, Arnd; Blenau, Wolfgang

2006-09-01

The biogenic amine serotonin (5-HT) plays a key role in the regulation and modulation of many physiological and behavioural processes in both vertebrates and invertebrates. These functions are mediated through the binding of serotonin to its receptors, of which 13 subtypes have been characterized in vertebrates. We have isolated a cDNA from the honeybee Apis mellifera (Am5-ht7) sharing high similarity to members of the 5-HT(7) receptor family. Expression of the Am5-HT(7) receptor in HEK293 cells results in an increase in basal cAMP levels, suggesting that Am5-HT(7) is expressed as a constitutively active receptor. Serotonin application to Am5-ht7-transfected cells elevates cyclic adenosine 3',5'-monophosphate (cAMP) levels in a dose-dependent manner (EC(50) = 1.1-1.8 nm). The Am5-HT(7) receptor is also activated by 5-carboxamidotryptamine, whereas methiothepin acts as an inverse agonist. Receptor expression has been investigated by RT-PCR, in situ hybridization, and western blotting experiments. Receptor mRNA is expressed in the perikarya of various brain neuropils, including intrinsic mushroom body neurons, and in peripheral organs. This study marks the first comprehensive characterization of a serotonin receptor in the honeybee and should facilitate further analysis of the role(s) of the receptor in mediating the various central and peripheral effects of 5-HT.

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

PubMed Central

Oliveira, Cleida A; Nie, Rong; Carnes, Kay; Franca, Luiz R; Prins, Gail S; Saunders, Philippa TK; Hess, Rex A

2003-01-01

Background The antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown. Methods In the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERα, ERβ and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry. Results ERα, ERβ and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERα is nearly opposite of that for ERβ, as ERα shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERβ shows the reverse. Androgen receptor follows that of ERα. ICI 182,780 caused a gradual but dramatic decrease in ERα expression in the testis and efferent ductules, but no change in ERβ and AR expression. Conclusions The differential response of ERα and ERβ proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract. PMID:14613549

A2BR Adenosine Receptor Modulates Sweet Taste in Circumvallate Taste Buds

PubMed Central

Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C.; Finger, Thomas E.

2012-01-01

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields. PMID:22253866

Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation.

PubMed

Chen, Lei; Meng, Yue; Guo, Xiaoqing; Sheng, Xiaotong; Tai, Guihua; Zhang, Fenglei; Cheng, Hairong; Zhou, Yifa

2016-11-01

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.

A2BR adenosine receptor modulates sweet taste in circumvallate taste buds.

PubMed

Kataoka, Shinji; Baquero, Arian; Yang, Dan; Shultz, Nicole; Vandenbeuch, Aurelie; Ravid, Katya; Kinnamon, Sue C; Finger, Thomas E

2012-01-01

In response to taste stimulation, taste buds release ATP, which activates ionotropic ATP receptors (P2X2/P2X3) on taste nerves as well as metabotropic (P2Y) purinergic receptors on taste bud cells. The action of the extracellular ATP is terminated by ectonucleotidases, ultimately generating adenosine, which itself can activate one or more G-protein coupled adenosine receptors: A1, A2A, A2B, and A3. Here we investigated the expression of adenosine receptors in mouse taste buds at both the nucleotide and protein expression levels. Of the adenosine receptors, only A2B receptor (A2BR) is expressed specifically in taste epithelia. Further, A2BR is expressed abundantly only in a subset of taste bud cells of posterior (circumvallate, foliate), but not anterior (fungiform, palate) taste fields in mice. Analysis of double-labeled tissue indicates that A2BR occurs on Type II taste bud cells that also express Gα14, which is present only in sweet-sensitive taste cells of the foliate and circumvallate papillae. Glossopharyngeal nerve recordings from A2BR knockout mice show significantly reduced responses to both sucrose and synthetic sweeteners, but normal responses to tastants representing other qualities. Thus, our study identified a novel regulator of sweet taste, the A2BR, which functions to potentiate sweet responses in posterior lingual taste fields.

Bromocriptine modulates the expression of PTHrP receptor, Indian hedgehog, and Runx2 proteins in the growth plate of lactating rats.

PubMed

Wongdee, Kannikar; Thonapan, Natchayaporn; Saengamnart, Wasana; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

2013-09-01

In lactating rats, the endochondral bone growth is markedly enhanced, leading to the lengthening of long bone. This lactation-induced bone elongation could be abolished by a dopaminergic D2 receptor agonist bromocriptine, but how bromocriptine altered the expression of major chondroregulatory proteins in the growth plate cartilage was elusive. Here, we performed a quantitative immunohistochemical analysis to determine the expression of various peptides and transcription factors known to control the growth plate chondrocyte proliferation and differentiation [i.e., parathyroid hormone-related protein (PTHrP), PTHrP receptor, Indian hedgehog (Ihh), and runt-related transcription factor 2 (Runx2)], in bromocriptine-treated lactating rats. The results showed that bromocriptine markedly increased Ihh expression in hypertrophic chondrocytes during early and mid-lactation, while the expression of PTHrP receptor, but not its ligand PTHrP, was upregulated in the proliferative and hypertrophic zones during mid and late lactation. In contrast, the expression of Runx2, an important transcription factor for chondrocyte differentiation, was suppressed in the hypertrophic chondrocytes of bromocriptine-treated rats. In conclusion, bromocriptine increased Ihh and PTHrP receptor expressions and decreased Runx2 expression, which might, in turn, enhance chondrocyte proliferation and delay chondrocyte hypertrophy, thereby slowing down endochondral bone growth. This finding could explain how bromocriptine compromised the lactation-induced bone elongation.

Postsynaptic Synaptotagmins Mediate AMPA Receptor Exocytosis During LTP

PubMed Central

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

2017-01-01

Strengthening of synaptic connections by NMDA-receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During NMDA-receptor-dependent LTP induction, Ca2+-influx stimulates recruitment of synaptic AMPA-receptors, thereby strengthening synapses. How Ca2+ induces AMPA-receptor recruitment, however, remains unclear. Here we show that, in pyramidal neurons of the hippocampal CA1-region, blocking postsynaptic expression of both synaptotagmin-1 and synaptotagmin-7, but not of synaptotagmin-1 or synaptotagmin-7 alone, abolished LTP. LTP was rescued by wild-type but not by Ca2+-binding-deficient mutant synaptotagmin-7. Blocking postsynaptic synaptotagmin-1/7 expression did not impair basal synaptic transmission, synaptic or extrasynaptic AMPA-receptor levels, or other AMPA-receptor trafficking events. Moreover, expression of dominant-negative mutant synaptotagmin-1 that inhibited Ca2+-dependent presynaptic vesicle exocytosis also blocked Ca2+-dependent postsynaptic AMPA-receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic synaptotagmin-1 and synaptotagmin-7 act as redundant Ca2+-sensors for Ca2+-dependent exocytosis of AMPA-receptors during LTP, thus delineating a simple mechanism for the recruitment of AMPA-receptors that mediates LTP. PMID:28355182

Reassessing ecdysteroidogenic cells from the cell membrane receptors' perspective.

PubMed

Alexandratos, Alexandros; Moulos, Panagiotis; Nellas, Ioannis; Mavridis, Konstantinos; Dedos, Skarlatos G

2016-02-05

Ecdysteroids secreted by the prothoracic gland (PG) cells of insects control the developmental timing of their immature life stages. These cells have been historically considered as carrying out a single function in insects, namely the biochemical conversion of cholesterol to ecdysteroids and their secretion. A growing body of evidence shows that PG cells receive multiple cues during insect development so we tested the hypothesis that they carry out more than just one function in insects. We characterised the molecular nature and developmental profiles of cell membrane receptors in PG cells of Bombyx mori during the final larval stage and determined what receptors decode nutritional, developmental and physiological signals. Through iterative approaches we identified a complex repertoire of cell membrane receptors that are expressed in intricate patterns and activate previously unidentified signal transduction cascades in PG cells. The expression patterns of some of these receptors explain precisely the mechanisms that are known to control ecdysteroidogenesis. However, the presence of receptors for the notch, hedgehog and wingless signalling pathways and the expression of innate immunity-related receptors such as phagocytosis receptors, receptors for microbial ligands and Toll-like receptors call for a re-evaluation of the role these cells play in insects.

Functional identification and reconstitution of an odorant receptor in single olfactory neurons

PubMed Central

Touhara, Kazushige; Sengoku, Shintaro; Inaki, Koichiro; Tsuboi, Akio; Hirono, Junzo; Sato, Takaaki; Sakano, Hitoshi; Haga, Tatsuya

1999-01-01

The olfactory system is remarkable in its capacity to discriminate a wide range of odorants through a series of transduction events initiated in olfactory receptor neurons. Each olfactory neuron is expected to express only a single odorant receptor gene that belongs to the G protein coupled receptor family. The ligand–receptor interaction, however, has not been clearly characterized. This study demonstrates the functional identification of olfactory receptor(s) for specific odorant(s) from single olfactory neurons by a combination of Ca2+-imaging and reverse transcription–coupled PCR analysis. First, a candidate odorant receptor was cloned from a single tissue-printed olfactory neuron that displayed odorant-induced Ca2+ increase. Next, recombinant adenovirus-mediated expression of the isolated receptor gene was established in the olfactory epithelium by using green fluorescent protein as a marker. The infected neurons elicited external Ca2+ entry when exposed to the odorant that originally was used to identify the receptor gene. Experiments performed to determine ligand specificity revealed that the odorant receptor recognized specific structural motifs within odorant molecules. The odorant receptor-mediated signal transduction appears to be reconstituted by this two-step approach: the receptor screening for given odorant(s) from single neurons and the functional expression of the receptor via recombinant adenovirus. The present approach should enable us to examine not only ligand specificity of an odorant receptor but also receptor specificity and diversity for a particular odorant of interest. PMID:10097159

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

PubMed

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Different expressions and DNA methylation patterns of lysophosphatidic acid receptor genes in mouse tumor cells.

PubMed

Okabe, Kyoko; Hayashi, Mai; Wakabayashi, Naoko; Yamawaki, Yasuna; Teranishi, Miki; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

2010-01-01

Lysophosphatidic acid (LPA) receptors act as several biological effectors through LPA, which is a bioactive phospholipid. Recently, aberrant expressions of LPA receptor genes due to DNA methylation have been detected in several tumor cells. In this study, we measured expression levels and DNA methylation status of LPA receptor genes in mouse tumor cells, LL/2 lung carcinoma, B16F0 melanoma, FM3A mammary carcinoma and L1210 leukemia cells, compared with normal tissues. Total RNAs were extracted and RT-PCR analysis was performed. For DNA methylation status, bisulfite sequencing analysis was carried out, comparing outcomes with other tumor cells and normal tissues. The expressions of LPA1 gene were shown in LL/2, but not in B16F0, FM3A and L1210 cells. While the LPA2 gene was expressed in all 4 tumor cells, the LPA3 gene was unexpressed in them. The LPA1 and LPA3 unexpressed cells were highly methylated, although normal tissues were all unmethylated. The DNA methylation status was correlated with gene expression levels in cancer cells. The present results demonstrate that DNA methylation patterns of LPA receptor genes are dependent on cancer cell types, suggesting that LPA receptors may be new molecular targets for therapeutic approaches and chemoprevention. Copyright © 2011 S. Karger AG, Basel.

Permeability and single channel conductance of human homomeric ρ1 GABAC receptors

PubMed Central

Wotring, Virginia E; Chang, Yongchang; Weiss, David S

1999-01-01

Homomeric human ρ1 GABAC receptors were expressed in Xenopus oocytes and in human embryonic kidney cells (HEK293) in order to examine their conductance and permeability. Reversal potentials of currents elicited by γ-aminobutyric acid (GABA) were measured in extracellular solutions of various ionic composition to determine relative permeability of homomeric ρ1 receptors. The rank order of anionic permeability was: SCN− > I− > NO3− > Br− > Cl− > formate (For−) > HCO3− > acetate (Ac−) ≈ proprionate (Prop−) ≈ isethionate (Ise−) ≈ F−≈ PO4−. In the oocyte expression system, relative permeabilities to SCN−, I−, NO3−, Br− and HCO3− were higher for ρ1 GABAC receptors than α1β2γ2L GABAA receptors. Expression of ρ1 GABAC receptors in Xenopus oocytes and in HEK293 cells gave similar relative permeabilities for selected anions, suggesting that the expression system does not significantly alter permeation properties. The pore diameter of the homomeric ρ1 GABAC receptor expressed in oocytes was estimated to be 0.61 nm, which is somewhat larger than the 0.56 nm pore diameter estimated for α1β2γ2L GABAA receptors. Homomeric ρ1 GABA receptors expressed in oocytes had a single channel chord conductance of 0.65 ± 0.04 pS (mean ±s.e.m.s) when the internal chloride concentration ([Cl−]i) was 20 mm. With a [Cl−]i of 100 mm, the single channel chord conductance was 1.59 ± 0.24 pS. The mean open time directly measured from 43 GABA-induced channel openings in six patches was 3.2 ± 0.8 s. The mean open time in the presence of 100 μm picrotoxin was 0.07 ± 0.01 s (77 openings from 3 patches). The differences observed in ionic permeabilities, pore size, single channel conductance and mean open time suggest that the ρ1 homomeric receptor may not be the native retinal GABAC receptor reported previously. PMID:10581305

Identification and expression analyses of a novel serotonin receptor gene, 5-HT2β, in the field cricket, Gryllus bimaculatus.

PubMed

Watanabe, T; Aonuma, H

2012-01-01

Biogenic amine serotonin (5-HT) modulates various aspects of behaviors such as aggressive behavior and circadian behavior in the cricket. In our previous report, in order to elucidate the molecular basis of the cricket 5-HT system, we identified three genes involved in 5-HT biosynthesis, as well as four 5-HT receptor genes (5-HT1A, 5-HT1B, 5-HT2α, and 5-HT7) expressed in the brain of the field cricket Gryllus bimaculatus DeGeer [7]. In the present study, we identified Gryllus 5-HT2β gene, an additional 5-HT receptor gene expressed in the cricket brain, and examined its tissue-specific distribution and embryonic stage-dependent expression. Gryllus 5-HT2β gene was ubiquitously expressed in the all examined adult tissues, and was expressed during early embryonic development, as well as during later stages. This study suggests functional differences between two 5-HT2 receptors in the cricket.