New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

PubMed

Barocelli, E; Ballabeni, V; Bertoni, S; Dallanoce, C; De Amici, M; De Micheli, C; Impicciatore, M

2000-06-30

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

Subtype specific internalization of P2Y1 and P2Y2 receptors induced by novel adenosine 5′-O-(1-boranotriphosphate) derivatives

PubMed Central

Tulapurkar, M E; Laubinger, W; Nahum, V; Fischer, B; Reiser, G

2004-01-01

P2Y-nucleotide receptors represent important targets for drug development. The lack of stable and receptor specific agonists, however, has prevented successful therapeutic applications. A novel series of P-boronated ATP derivatives (ATP-α-B) were synthesized by substitution of a nonbridging O at Pα with a BH3 group. This introduces a chiral center, thus resulting in diastereoisomers. In addition, at C2 of the adenine ring a further substitution was made (Cl- or methylthio-). The pairs of diastereoisomers were denoted here as A and B isomers. Here, we tested the receptor subtype specificity of these analogs on HEK 293 cells stably expressing rat P2Y1 and rat P2Y2 receptors, respectively, both attached to the fluorescent marker protein GFP (rP2Y1-GFP, rP2Y2-GFP). We investigated agonist-induced receptor endocytosis, [Ca2+]i rise and arachidonic acid (AA) release. Agonist-induced endocytosis of rP2Y1-GFP was more pronounced for the A isomers than the corresponding B counterparts for all ATP-α-B analogs. Both 2-MeS-substituted diastereoisomers induced a greater degree of agonist-induced receptor endocytosis as compared to the 2-Cl-substituted derivatives. Endocytosis results are in accordance with the potency to induce Ca2+ release by these compounds in HEK 293 cells stably transfected with rP2Y1. In case of rP2Y2-GFP, the borano-nucleotides were very weak agonists in comparison to UTP and ATP in terms of Ca2+ release, AA release and in inducing receptor endocytosis. The different ATP-α-B derivatives and also the diastereoisomers were equally ineffective. Thus, the new agonists may be considered as potent and highly specific agonist drug candidates for P2Y1 receptors. The difference in activity of the ATP analogs at P2Y receptors could be used as a tool to investigate structural differences between P2Y receptor subtypes. PMID:15197109

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.

Opiate-induced motor stimulation is regulated by gamma-aminobutyric acid type B receptors found in the ventral tegmental area in mice.

PubMed

Leite-Morris, Kimberly A; Fukudome, Eugene Y; Kaplan, Gary B

2002-01-14

Recent studies suggest that gamma-aminobutyric acid type B (GABA(B)) receptors located on dopaminergic cells in the ventral tegmental area (VTA) regulate mesolimbic dopaminergic (A10) activity. In the current study, we identified GABA(B) receptor subtypes in the area of the VTA and examined their role in modulating acute opiate actions. We studied the effects of intra-VTA infusions of the selective GABA(B) agonist baclofen on morphine-induced locomotor stimulation and A10 neuronal activation. Drug treatments were followed by ambulatory activity monitoring for 180 min. Intra-VTA baclofen treatment produced a 70% inhibition of morphine-stimulated locomotor activity. Furthermore, functional activation of A10 neurons was assessed by immunohistochemical staining of c-Fos in the nucleus accumbens (NAc), where A10 neurons terminate. We found that morphine treatment increased the levels of Fos-positive nuclei in the NAc, while intra-VTA baclofen treatment reversed morphine's effects. Finally, GABA(B) receptor subtypes and isoforms were identified in the ventromedial mesencephalon using immunoblotting. We demonstrated the presence of GABA(B)R1a (130 kDa), GABA(B)R1b (100 kDa), and GABA(B)R2 (120 kDa) receptor subtypes in this region. These results suggest that GABA(B) receptor isoforms are found in the VTA and their activation results in the blockade of behavioral effects of opiates via inhibition of dopaminergic neurotransmission.

Structural basis for molecular recognition at serotonin receptors.

PubMed

Wang, Chong; Jiang, Yi; Ma, Jinming; Wu, Huixian; Wacker, Daniel; Katritch, Vsevolod; Han, Gye Won; Liu, Wei; Huang, Xi-Ping; Vardy, Eyal; McCorvy, John D; Gao, Xiang; Zhou, X Edward; Melcher, Karsten; Zhang, Chenghai; Bai, Fang; Yang, Huaiyu; Yang, Linlin; Jiang, Hualiang; Roth, Bryan L; Cherezov, Vadim; Stevens, Raymond C; Xu, H Eric

2013-05-03

Serotonin or 5-hydroxytryptamine (5-HT) regulates a wide spectrum of human physiology through the 5-HT receptor family. We report the crystal structures of the human 5-HT1B G protein-coupled receptor bound to the agonist antimigraine medications ergotamine and dihydroergotamine. The structures reveal similar binding modes for these ligands, which occupy the orthosteric pocket and an extended binding pocket close to the extracellular loops. The orthosteric pocket is formed by residues conserved in the 5-HT receptor family, clarifying the family-wide agonist activity of 5-HT. Compared with the structure of the 5-HT2B receptor, the 5-HT1B receptor displays a 3 angstrom outward shift at the extracellular end of helix V, resulting in a more open extended pocket that explains subtype selectivity. Together with docking and mutagenesis studies, these structures provide a comprehensive structural basis for understanding receptor-ligand interactions and designing subtype-selective serotonergic drugs.

Control of glycinergic input to spinal dorsal horn neurons by distinct muscarinic receptor subtypes revealed using knockout mice.

PubMed

Zhang, Hong-Mei; Zhou, Hong-Yi; Chen, Shao-Rui; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2007-12-01

Muscarinic acetylcholine receptors (mAChRs) play an important role in the tonic regulation of nociceptive transmission in the spinal cord. However, how mAChR subtypes contribute to the regulation of synaptic glycine release is unknown. To determine their role, glycinergic spontaneous inhibitory postsynaptic currents (sIPSCs) were recorded in lamina II neurons by using whole-cell recordings in spinal cord slices of wild-type (WT) and mAChR subtype knockout (KO) mice. In WT mice, the mAChR agonist oxotremorine-M dose-dependently decreased the frequency of sIPSCs in most neurons, but it had variable effects in other neurons. In contrast, in M3-KO mice, oxotremorine-M consistently decreased the glycinergic sIPSC frequency in all neurons tested, and in M2/M4 double-KO mice, it always increased the sIPSC frequency. In M2/M4 double-KO mice, the potentiating effect of oxotremorine-M was attenuated by higher concentrations in some neurons through activation of GABA(B) receptors. In pertussis toxin-treated WT mice, oxotremorine-M also consistently increased the sIPSC frequency. In M2-KO and M4-KO mice, the effect of oxotremorine-M on sIPSCs was divergent because of the opposing functions of the M3 subtype and the M2 and M4 subtypes. This study demonstrates that stimulation of the M2 and M4 subtypes inhibits glycinergic inputs to spinal dorsal horn neurons of mice, whereas stimulation of the M3 subtype potentiates synaptic glycine release. Furthermore, GABA(B) receptors are involved in the feedback regulation of glycinergic synaptic transmission in the spinal cord. This study revealed distinct functions of mAChR subtypes in controlling glycinergic input to spinal dorsal horn neurons.

Structure and genomic organization of the human B1 receptor gene for kinins (BDKRB1).

PubMed

Bachvarov, D R; Hess, J F; Menke, J G; Larrivée, J F; Marceau, F

1996-05-01

Two subtypes of mammalian bradykinin receptors, B1 and B2 (BDKRB1 and BDKRB2), have been defined based on their pharmacological properties. The B1 type kinin receptors have weak affinity for intact BK or Lys-BK but strong affinity for kinin metabolites without the C-terminal arginine (e.g., des-Arg9-BK and Lys-des-Arg9-BK, also called des-Arg10-kallidin), which are generated by kininase I. The B1 receptor expression is up-regulated following tissue injury and inflammation (hyperemia, exudation, hyperalgesia, etc.). In the present study, we have cloned and sequenced the gene encoding human B1 receptor from a human genomic library. The human B1 receptor gene contains three exons separated by two introns. The first and the second exon are noncoding, while the coding region and the 3'-flanking region are located entirely on the third exon. The exon-intron arrangement of the human B1 receptor gene shows significant similarity with the genes encoding the B2 receptor subtype in human, mouse, and rat. Sequence analysis of the 5'-flanking region revealed the presence of a consensus TATA box and of numerous candidate transcription factor binding sequences. Primer extension experiments have shown the existence of multiple transcription initiation sites situated downstream and upstream from the consensus TATA box. Genomic Southern blot analysis indicated that the human B1 receptor is encoded by a single-copy gene.

Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors.

PubMed

Disingrini, Teresa; Muth, Mathias; Dallanoce, Clelia; Barocelli, Elisabetta; Bertoni, Simona; Kellershohn, Kerstin; Mohr, Klaus; De Amici, Marco; Holzgrabe, Ulrike

2006-01-12

A novel series of muscarinic receptor ligands of the hexamethonio-type was prepared which contained, on one side, the phthalimidopropane or 1,8-naphthalimido-2,2-dimethylpropane moiety typical for subtype selective allosteric antagonists and, on the other, the acetylenic fragment typical for the nonselective orthosteric muscarinic agonists oxotremorine, oxotremorine-M, and related muscarinic agonists. Binding experiments in M(2) receptors using [(3)H]N-methylscopolamine as an orthosteric probe proved an allosteric action of both groups of hybrids, 7a-10a and 8b-10b. The difference in activity between a-group and b-group hybrids corresponded with the activity difference between the allosteric parent compounds. In M(1)-M(3) muscarinic isolated organ preparations, most of the hybrids behaved as subtype selective antagonists. [(35)S]GTPgammaS binding assays using human M(2) receptors overexpressed in CHO cells revealed that a weak intrinsic efficacy was preserved in 8b-10b. Thus, attaching muscarinic allosteric antagonist moieties to orthosteric muscarinic agonists may lead to hybrid compounds in which functions of both components are mixed.

Differential regulation of the cell cycle by alpha1-adrenergic receptor subtypes.

PubMed

Gonzalez-Cabrera, Pedro J; Shi, Ting; Yun, June; McCune, Dan F; Rorabaugh, Boyd R; Perez, Dianne M

2004-11-01

Alpha(1)-Adrenergic receptors have been implicated in growth-promoting pathways. A microarray study of individual alpha(1)-adrenergic receptor subtypes (alpha(1A), alpha(1B), and alpha(1D)) expressed in Rat-1 fibroblasts revealed that epinephrine altered the transcription of several cell cycle regulatory genes in a direction consistent with the alpha(1A)- and alpha(1D)-adrenergic receptors mediating G(1)-S cell cycle arrest and the alpha(1B-)mediating cell-cycle progression. A time course indicated that in alpha(1A) cells, epinephrine stimulated a G(1)-S arrest, which began after 8 h of stimulation and maximized at 16 h, at which point was completely blocked with cycloheximide. The alpha(1B)-adrenergic receptor profile also showed unchecked cell cycle progression, even under low serum conditions and induced foci formation. The G(1)-S arrest induced by alpha(1A)- and alpha(1D)-adrenergic receptors was associated with decreased cyclin-dependent kinase-6 and cyclin E-associated kinase activities and increased expression of the cyclin-dependent kinase inhibitor p27(Kip1), all of which were blocked by prazosin. There were no differences in kinase activities and/or expression of p27(Kip1) in epinephrine alpha(1B)-AR fibroblasts, although the microarray did indicate differences in p27(Kip1) RNA levels. Cell counts proved the antimitotic effect of epinephrine in alpha(1A) and alpha(1D) cells and indicated that alpha(1B)-adrenergic receptor subtype expression was sufficient to cause proliferation of Rat-1 fibroblasts independent of agonist stimulation. Analysis in transfected PC12 cells also confirmed the alpha(1A)- and alpha(1B)-adrenergic receptor effect. The alpha(1B)-subtype native to DDT1-MF2 cells, a smooth muscle cell line, caused progression of the cell cycle. These results indicate that the alpha(1A)- and alpha(1D)-adrenergic receptors mediate G(1)-S cell-cycle arrest, whereas alpha(1B)-adrenergic receptor expression causes a cell cycle progression and may induce transformation in sensitive cell lines.

Novel oxotremorine-related heterocyclic derivatives: Synthesis and in vitro pharmacology at the muscarinic receptor subtypes.

PubMed

Dallanoce, Clelia; De Amici, Marco; Barocelli, Elisabetta; Bertoni, Simona; Roth, Bryan L; Ernsberger, Paul; De Micheli, Carlo

2007-12-15

A set of novel heterocyclic ligands (6-27) structurally related to Oxotremorine 2 was designed, synthesized and tested at muscarinic receptor subtypes (mAChRs). In the binding experiments at cloned human receptors (hm1-5), compounds 7 and 15 evidenced a remarkable affinity and selectivity for the hm2 subtype. The in vitro functional assays, performed on a selected group of derivatives at M(1), M(2), and M(3) tissue preparations, singled out the 3-butynyloxy-5-methylisoxazole trimethylammonium salt 7 as a potent unselective muscarinic agonist [pEC(50): 7.40 (M(1)), 8.18 (M(2)), and 8.14 (M(3))], whereas its 5-phenyl analogue 12 behaved as a muscarinic antagonist, slightly selective for the M(1) subtype [pK(B): 6.88 (M(1)), 5.95 (M(2)), 5.53 (M(3))]. Moreover, the functional data put in evidence that the presence of the piperidine ring may generate a functional selectivity, e.g., an M(1) antagonist/M(2) partial agonist/M(3) full agonist profile (compound 21), at variance with the corresponding quaternary ammonium salt (compound 22) which behaved as a muscarinic agonist at all M(1-3) receptors, with an appreciable selectivity for the cardiac M(2) receptors.

The three α1-adrenoceptor subtypes show different spatio-temporal mechanisms of internalization and ERK1/2 phosphorylation.

PubMed

Perez-Aso, M; Segura, V; Montó, F; Barettino, D; Noguera, M A; Milligan, G; D'Ocon, P

2013-10-01

We analyzed the kinetic and spatial patterns characterizing activation of the MAP kinases ERK 1 and 2 (ERK1/2) by the three α1-adrenoceptor (α1-AR) subtypes in HEK293 cells and the contribution of two different pathways to ERK1/2 phosphorylation: protein kinase C (PKC)-dependent ERK1/2 activation and internalization-dependent ERK1/2 activation. The different pathways of phenylephrine induced ERK phosphorylation were determined by western blot, using the PKC inhibitor Ro 31-8425, the receptor internalization inhibitor concanavalin A and the siRNA targeting β-arrestin 2. Receptor internalization properties were studied using CypHer5 technology and VSV-G epitope-tagged receptors. Activation of α1A- and α1B-ARs by phenylephrine elicited rapid ERK1/2 phosphorylation that was directed to the nucleus and inhibited by Ro 31-8425. Concomitant with phenylephrine induced receptor internalization α1A-AR, but not α1B-AR, produced a maintained and PKC-independent ERK phosphorylation, which was restricted to the cytosol and inhibited by β-arrestin 2 knockdown or concanavalin A treatment. α1D-AR displayed constitutive ERK phosphorylation, which was reduced by incubation with prazosin or the selective α1D antagonist BMY7378. Following activation by phenylephrine, α1D-AR elicited rapid, transient ERK1/2 phosphorylation that was restricted to the cytosol and not inhibited by Ro 31-8425. Internalization of the α1D-AR subtype was not observed via CypHer5 technology. The three α1-AR subtypes present different spatio-temporal patterns of receptor internalization, and only α1A-AR stimulation translates to a late, sustained ERK1/2 phosphorylation that is restricted to the cytosol and dependent on β-arrestin 2 mediated internalization. Copyright © 2013 Elsevier B.V. All rights reserved.

PAM50 Breast Cancer Subtyping by RT-qPCR and Concordance with Standard Clinical Molecular Markers

PubMed Central

2012-01-01

Background Many methodologies have been used in research to identify the “intrinsic” subtypes of breast cancer commonly known as Luminal A, Luminal B, HER2-Enriched (HER2-E) and Basal-like. The PAM50 gene set is often used for gene expression-based subtyping; however, surrogate subtyping using panels of immunohistochemical (IHC) markers are still widely used clinically. Discrepancies between these methods may lead to different treatment decisions. Methods We used the PAM50 RT-qPCR assay to expression profile 814 tumors from the GEICAM/9906 phase III clinical trial that enrolled women with locally advanced primary invasive breast cancer. All samples were scored at a single site by IHC for estrogen receptor (ER), progesterone receptor (PR), and Her2/neu (HER2) protein expression. Equivocal HER2 cases were confirmed by chromogenic in situ hybridization (CISH). Single gene scores by IHC/CISH were compared with RT-qPCR continuous gene expression values and “intrinsic” subtype assignment by the PAM50. High, medium, and low expression for ESR1, PGR, ERBB2, and proliferation were selected using quartile cut-points from the continuous RT-qPCR data across the PAM50 subtype assignments. Results ESR1, PGR, and ERBB2 gene expression had high agreement with established binary IHC cut-points (area under the curve (AUC) ≥ 0.9). Estrogen receptor positivity by IHC was strongly associated with Luminal (A and B) subtypes (92%), but only 75% of ER negative tumors were classified into the HER2-E and Basal-like subtypes. Luminal A tumors more frequently expressed PR than Luminal B (94% vs 74%) and Luminal A tumors were less likely to have high proliferation (11% vs 77%). Seventy-seven percent (30/39) of ER-/HER2+ tumors by IHC were classified as the HER2-E subtype. Triple negative tumors were mainly comprised of Basal-like (57%) and HER2-E (30%) subtypes. Single gene scoring for ESR1, PGR, and ERBB2 was more prognostic than the corresponding IHC markers as shown in a multivariate analysis. Conclusions The standard immunohistochemical panel for breast cancer (ER, PR, and HER2) does not adequately identify the PAM50 gene expression subtypes. Although there is high agreement between biomarker scoring by protein immunohistochemistry and gene expression, the gene expression determinations for ESR1 and ERBB2 status was more prognostic. PMID:23035882

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

PubMed

Bianchi, B R; Lynch, K J; Touma, E; Niforatos, W; Burgard, E C; Alexander, K M; Park, H S; Yu, H; Metzger, R; Kowaluk, E; Jarvis, M F; van Biesen, T

1999-07-02

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Amygdala Infusions of an NR2B-Selective or an NR2A-Preferring NMDA Receptor Antagonist Differentially Influence Fear Conditioning and Expression in the Fear-Potentiated Startle Test

ERIC Educational Resources Information Center

Walker, David L.; Davis, Michael

2008-01-01

Within the amygdala, most N-methyl-D-aspartic acid (NMDA) receptors consist of NR1 subunits in combination with either NR2A or NR2B subunits. Because the particular subunit composition greatly influences the receptors' properties, we investigated the contribution of both subtypes to fear conditioning and expression. To do so, we infused the…

In vitro study on the effects of some selected agonists and antagonists of alpha(1)-adrenergic receptors on the contractility of the aneurysmally-changed aortic smooth muscle in humans.

PubMed

Gnus, J; Czerski, A; Ferenc, S; Zawadzki, W; Witkiewicz, W; Hauzer, W; Rusiecka, A; Bujok, J

2012-02-01

The study included 18 sections of the aneurysmally-changed abdominal aortas, obtained from patients of the Provincial Specialist Hospital in Wroclaw and 18 sections of normal abdominal aortas obtained from swine. The collected samples were placed horizontally in the incubation chamber. Changes in their transverse section area were registered. They were stretched to a tension of 5 mN. Krebs-Henseleit buffer was used as the incubatory environment. Incubation of the sections was performed at a temperature of 37°C, in the gaseous mixture of oxygen and carbon dioxide used in the following proportion: 95% of O(2) and 5% of CO(2). Contractions of the aorta were registered with isotonic transducers (Letica Scientific Instruments). In the studies, we examined the influence of α(1)-adrenergic receptors (and their subtypes α(1A), α(1B), α(1D)) on the contractility of the aortic muscle in humans and swine by their stimulation or inhibition with some selected agonists or antagonists. This time, it was shown that the stimulation of α(1)-adrenergic receptors leads to contractions of the human and swine aortic muscle; the observed increase in the muscle tone may follow from the stimulation of all subtypes of alpha-1 receptor (α(1A), α(1B), α(1D)). All three subtypes of 1-adrenergic receptor are engaged in vasoconstriction, especially of α(1A) and α(1D) subtypes; the α(1B) subtype is less significant for aortic contractility. The contractile response of the aneurysmally-changed abdominal aorta in humans to agonists of α-adrenergic receptors was significantly less intense than that of the normal porcine aorta. It can be concluded that aneurysms influence the contractile response of the aorta.

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

PubMed

Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

2016-04-01

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

Molecular cloning, molecular evolution and gene expression of cDNAs encoding thyrotropin-releasing hormone receptor subtypes in a teleost, the sockeye salmon (Oncorhynchus nerka).

PubMed

Saito, Yuichi; Mekuchi, Miyuki; Kobayashi, Noriaki; Kimura, Makoto; Aoki, Yasuhiro; Masuda, Tomohiro; Azuma, Teruo; Fukami, Motohiro; Iigo, Masayuki; Yanagisawa, Tadashi

2011-11-01

Molecular cloning of thyrotropin-releasing hormone receptors (TRHR) was performed in a teleost, the sockeye salmon (Oncorhynchus nerka). Four different TRHR cDNAs were cloned and named TRHR1, TRHR2a, TRHR2b and TRHR3 based on their similarity to known TRHR subtypes in vertebrates. Important residues for TRH binding were conserved in deduced amino acid sequences of the three TRHR subtypes except for the TRHR2b. Seven transmembrane domains were predicted for TRHR1, TRHR2a and TRHR3 proteins but only five for TRHR2b which appears to be truncated. In silico database analysis identified putative TRHR sequences including invertebrate TRHR and reptilian, avian and mammalian TRHR3. Phylogenetic analyses predicted the molecular evolution of TRHR in vertebrates: from the common ancestral TRHR (i.e. invertebrate TRHR), the TRHR2 subtype diverged first and then TRHR1 and TRHR3 diverged. Reverse transcription-polymerase chain reaction analyses revealed TRHR1 transcripts in the brain (hypothalamus), retina, pituitary gland and large intestine; TRHR2a in the brain (telencephalon and hypothalamus); and TRHR3 in the brain (olfactory bulbs) and retina. Copyright © 2011 Elsevier Inc. All rights reserved.

Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity and functionality.

PubMed

Osman, Noha A; Ligresti, Alessia; Klein, Christian D; Allarà, Marco; Rabbito, Alessandro; Di Marzo, Vincenzo; Abouzid, Khaled A; Abadi, Ashraf H

2016-10-21

CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki = 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (∼70 and ∼200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

The V1a and V1b, but not V2, vasopressin receptor genes are expressed in the supraoptic nucleus of the rat hypothalamus, and the transcripts are essentially colocalized in the vasopressinergic magnocellular neurons.

PubMed

Hurbin, A; Boissin-Agasse, L; Orcel, H; Rabié, A; Joux, N; Desarménien, M G; Richard, P; Moos, F C

1998-11-01

We have identified and visualized the vasopressin (VP) receptors expressed by hypothalamic magnocellular neurons in supraoptic and paraventricular nuclei. To do this, we used RT-PCR on total RNA extracts from supraoptic nuclei or on single freshly dissociated supraoptic neurons, and in situ hybridization on frontal sections of hypothalamus of Wistar rats. The RT-PCR on supraoptic RNA extracts revealed that mainly V1a, but also V1b, subtypes of VP receptors are expressed from birth to adulthood. No V2 receptor messenger RNA (mRNA) was detected. Furthermore, the single-cell RT-nested PCR indicated that the V1a receptor mRNA is present in vasopressinergic magnocellular neurons. In light of these results, in situ hybridization was performed to visualize the V1a and V1b receptor mRNAs in supraoptic and paraventricular nuclei. Simultaneously, we coupled this approach to: 1) in situ hybridization detection of oxytocin or VP mRNAs; or 2) immunocytochemistry to detect the neuropeptides. This provided a way of identifying the neurons expressing perceptible amounts of V1a or V1b receptor mRNAs as vasopressinergic neurons. Here, we suggest that the autocontrol exerted specifically by VP on vasopressinergic neurons is mediated through, at least, V1a and V1b subtype receptors.

Clinical Characteristics and Prognosis of Pregnancy-Associated Breast Cancer: Poor Survival of Luminal B Subtype.

PubMed

Bae, Soo Youn; Jung, Seung Pil; Jung, Eun Sung; Park, Sung Min; Lee, Se Kyung; Yu, Jong Han; Lee, Jeong Eon; Kim, Seok Won; Nam, Seok Jin

2018-06-18

Pregnancy-associated breast cancer (PABC) is rare and is generally defined as breast cancer diagnosed during pregnancy or within 1 year of delivery. The average ages of marriage and childbearing are increasing, and PABC is expected to also increase. This study is intended to increase understanding of the characteristics of PABC. A database of 2,810 patients with breast cancer diagnosed when they were less than 40 years of age was reviewed. The clinicopathological factors and survival of PABC (40 patients) were compared to those of patients with young breast cancer (YBC, non-pregnant or over 12 months after delivery; 2,770 patients). PABC had significantly lower estrogen receptor (ER) and progesterone receptor (PR) expression (ER-positive 50.0%, PR-positive 45.0%) and higher HER2 overexpression (38.5%) than YBC. The most common subtype of PABC was triple-negative breast cancer (TNBC; 35.9%), and luminal A subtype represented only 7.7% of cases. In univariate analysis, PABC had significantly worse disease-free survival (DFS) and breast cancer-specific survival (BCSS) compared to YBC. In multivariate analysis, PABC was associated with worse BCSS (HR 4.0, 95% CI 1.2-12.9, p = 0.019) and survival, but there was no difference in DFS between PABC and YBC. In subgroup analysis by subtype, luminal B subtype of PABC showed worse DFS (HR 3.5; 95% CI 1.1-11.2, p = 0.039) and BCSS (HR 10.2, 95% CI 1.2-87.1, p = 0.035), especially with high Ki67. However, no differences were demonstrated in other subtypes. In this study, PABC showed lower expression of ER/PR, higher overexpression of HER2, fewer luminal A subtype, and more TNBC subtype compared to YBC. PABC had worse BCSS, especially luminal B subtype, compared to YBC. © 2018 S. Karger AG, Basel.

Fyn kinase-mediated phosphorylation of NMDA receptor NR2B subunit at Tyr1472 is essential for maintenance of neuropathic pain.

PubMed

Abe, Tetsuya; Matsumura, Shinji; Katano, Tayo; Mabuchi, Tamaki; Takagi, Kunio; Xu, Li; Yamamoto, Akitsugu; Hattori, Kotaro; Yagi, Takeshi; Watanabe, Masahiko; Nakazawa, Takanobu; Yamamoto, Tadashi; Mishina, Masayoshi; Nakai, Yoshihide; Ito, Seiji

2005-09-01

Despite abundant evidence implicating the importance of N-methyl-D-aspartate (NMDA) receptors in the spinal cord for pain transmission, the signal transduction coupled to NMDA receptor activation is largely unknown for the neuropathic pain state that lasts over periods of weeks. To address this, we prepared mice with neuropathic pain by transection of spinal nerve L5. Wild-type, NR2A-deficient, and NR2D-deficient mice developed neuropathic pain; in addition, phosphorylation of NR2B subunits of NMDA receptors at Tyr1472 was observed in the superficial dorsal horn of the spinal cord 1 week after nerve injury. Neuropathic pain and NR2B phosphorylation at Tyr1472 were attenuated by the NR2B-selective antagonist CP-101,606 and disappeared in mice lacking Fyn kinase, a Src-family tyrosine kinase. Concomitant with the NR2B phosphorylation, an increase in neuronal nitric oxide synthase activity was visualized in the superficial dorsal horn of neuropathic pain mice by NADPH diaphorase histochemistry. Electron microscopy showed that the phosphorylated NR2B was localized at the postsynaptic density in the spinal cord of mice with neuropathic pain. Indomethacin, an inhibitor of prostaglandin (PG) synthesis, and PGE receptor subtype EP1-selective antagonist reduced the NR2B phosphorylation in these mice. Conversely, EP1-selective agonist stimulated Fyn kinase-dependent nitric oxide formation in the spinal cord. The present study demonstrates that Tyr1472 phosphorylation of NR2B subunits by Fyn kinase may have dual roles in the retention of NMDA receptors in the postsynaptic density and in activation of nitric oxide synthase, and suggests that PGE2 is involved in the maintenance of neuropathic pain via the EP1 subtype.

A Molecular Determinant of Subtype-Specific Desensitization in Ionotropic Glutamate Receptors.

PubMed

Alsaloum, Matthew; Kazi, Rashek; Gan, Quan; Amin, Johansen; Wollmuth, Lonnie P

2016-03-02

AMPA and NMDA receptors are glutamate-gated ion channels that mediate fast excitatory synaptic transmission throughout the nervous system. In the continual presence of glutamate, AMPA and NMDA receptors containing the GluN2A or GluN2B subunit enter into a nonconducting, desensitized state that can impact synaptic responses and glutamate-mediated excitotoxicity. The process of desensitization is dramatically different between subtypes, but the basis for these differences is unknown. We generated an extensive sequence alignment of ionotropic glutamate receptors (iGluRs) from diverse animal phyla and identified a highly conserved motif, which we termed the "hydrophobic box," located at the extracellular interface of transmembrane helices. A single position in the hydrophobic box differed between mammalian AMPA and NMDA receptors. Surprisingly, we find that an NMDAR-to-AMPAR exchange mutation at this position in the rat GluN2A or GluN2B subunit had a dramatic and highly specific effect on NMDAR desensitization, making it AMPAR-like. In contrast, a reverse exchange mutation in AMPARs had minimal effects on desensitization. These experiments highlight differences in desensitization between iGluR subtypes and the highly specific contribution of the GluN2 subunit to this process. Rapid communication between cells in the nervous system depends on ion channels that are directly activated by neurotransmitter molecules. Here, we studied ionotropic glutamate receptors (iGluRs), which are ion channels activated by the neurotransmitter glutamate. By comparing the sequences of a vast number of iGluR proteins from diverse animal species, assisted by available structural information, we identified a highly conserved motif. We showed that a single amino acid difference in this motif between mammalian iGluR subtypes has dramatic effects on receptor function. These results have implications in both the evolution of synaptic function, as well as the role of iGluRs in health and disease. Copyright © 2016 the authors 0270-6474/16/362617-06$15.00/0.

First report on molecular breast cancer subtypes and their clinico-pathological characteristics in Eastern Morocco: series of 2260 cases.

PubMed

Elidrissi Errahhali, Manal; Elidrissi Errahhali, Mounia; Ouarzane, Meryem; El Harroudi, Tijani; Afqir, Said; Bellaoui, Mohammed

2017-01-09

Breast cancer is the most frequent malignancy among women in Eastern Morocco. In this paper, we provide the first report on molecular breast cancer subtypes in this region. This is the largest population-based study on breast cancer among Moroccan women. We analyzed 2260 breast cancer cases diagnosed at the Hassan II Regional Oncology Center between October 2005 and December 2012. Clinico-pathological and therapeutic features were studied. Molecular subtypes were determined and their associations with the clinico-pathological characteristics of the tumors were examined. The mean age at diagnosis was 48.7 years ±11.4. Invasive ductal carcinoma was the predominant histological type (77.1%), followed by lobular invasive carcinoma (15.3%). The mean size of breast tumors was 3.5 cm ± 1.96, and 84% of our patients are diagnosed with tumors of more than 2 cm. Histological grade II tumors were the most frequent (70.4%), followed by advanced histological grade (18%). Lymph node positive tumors were observed in 64.8% of cases and 29.3% of patients had distant metastasis. Most tumors were hormone receptor-positive (73%) and 28.6% were HER2 positive. 86.1% of patients with hormone receptor-positive breast cancer were given hormone therapy, while 68.9% of patients with HER2+ breast cancer received targeted therapy with Herceptin. Luminal A was the commonest molecular subtype, followed by Luminal B, Triple Negative and HER2. The highest prevalence of premenopausal patients was observed in Triple Negative subtype (72.2%), followed by HER2 (64.1%), Luminal B (62.2%), and Luminal A (55.1%). Luminal B subtype had a poorer prognosis than Luminal A. Compared with Triple Negative, HER2 subtype tend to spread more aggressively and is associated with poorer prognosis. Unlike Western countries, breast cancer occurs at an earlier age and is diagnosed at a more advanced stage in Eastern Morocco. In this region, hormone receptor-positive tumors are predominant and so the majority of breast cancer patients should benefit from hormone therapy. HER2 subtype presents an aggressive tendency, suggesting the importance of anti-HER2 therapy. This study will contribute in developing appropriate screening and cancer management strategies in Eastern Morocco.

Characterization of the functional activity of botulinum neurotoxin subtype B6.

PubMed

Kohda, Tomoko; Nakamura, Keiji; Hosomi, Koji; Torii, Yasushi; Kozaki, Shunji; Mukamoto, Masafumi

2017-11-01

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A-G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (H C ) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. H C /B1 and H C /B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its H c .domain. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Minoxidil-induced hair growth is mediated by adenosine in cultured dermal papilla cells: possible involvement of sulfonylurea receptor 2B as a target of minoxidil.

PubMed

Li, M; Marubayashi, A; Nakaya, Y; Fukui, K; Arase, S

2001-12-01

The mechanism by which minoxidil, an adenosine-triphosphate-sensitive potassium channel opener, induces hypertrichosis remains to be elucidated. Minoxidil has been reported to stimulate the production of vascular endothelial growth factor, a possible promoter of hair growth, in cultured dermal papilla cells. The mechanism of production of vascular endothelial growth factor remains unclear, however. We hypothesize that adenosine serves as a mediator of vascular endothelial growth factor production. Minoxidil-induced increases in levels of intracellular Ca(2+) and vascular endothelial growth factor production in cultured dermal papilla cells were found to be inhibited by 8-sulfophenyl theophylline, a specific antagonist for adenosine receptors, suggesting that dermal papilla cells possess adenosine receptors and sulfonylurea receptors, the latter of which is a well-known target receptor for adenosine-triphosphate-sensitive potassium channel openers. The expression of sulfonylurea receptor 2B and of the adenosine A1, A2A, and A2B receptors was detected in dermal papilla cells by means of reverse transcription polymerase chain reaction analysis. In order to determine which of the adenosine receptor subtypes contribute to minoxidil-induced hair growth, the effects of subtype-specific antagonists for adenosine receptors were investigated. Significant inhibition in increase in intracellular calcium level by minoxidil or adenosine was observed as the result of pretreatment with 8-cyclopentyl-1,3-dipropylxanthine, an antagonist for adenosine A1 receptor, but not by 3,7-dimethyl-1-propargyl-xanthine, an antagonist for adenosine A2 receptor, whereas vascular endothelial growth factor production was blocked by both adenosine A1 and A2 receptor antagonists. These results indicate that the effect of minoxidil is mediated by adenosine, which triggers intracellular signal transduction via both adenosine A1 and A2 receptors, and that the expression of sulfonylurea receptor 2B in dermal papilla cells might play a role in the production of adenosine.

Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography.

PubMed Central

Gupta, N; McAllister, R; Drance, S M; Rootman, J; Cynader, M S

1994-01-01

Muscarinic cholinergic agents are used to lower intraocular pressure in the medical management of glaucoma and subtypes of muscarinic receptors have now been recognised in many tissues including the eye. To localise muscarinic receptors and their M1 and M2 subtypes in the human eye, in vitro ligand binding and autoradiographic techniques with densitometric quantitation on postmortem eye sections were used. As ligands, [3H] quinuclydinyl benzylate (QNB) (non-subtype specific muscarinic antagonist), [3H]pirenzipine (M1 antagonist), [3H]oxotremorine (M2 muscarinic agonist), [3H]AFDX-116(11[(2[diethylaminomethyl]1-piperidinyl)acetyl]5 , 11dihydro-6H-pyrido [2,3b][1,4]benzodiazepine-6-one) (M2 antagonist) were studied. Specific binding sites for QNB, pirenzipine, and AFDX-116 were localised in the entire ciliary muscle, the iris, and ciliary epithelium. [3H]oxotremorine localised only in the longitudinal portion of the ciliary muscle, and additionally, was not localised in the iris or ciliary epithelium. These results suggest that oxotremorine, by binding selectively to receptors on the longitudinal ciliary muscle and inducing its contraction, may modulate outflow facility independently from accommodation and miosis. Images PMID:7918268

Breast Cancer Subtypes and Response to Docetaxel in Node-Positive Breast Cancer: Use of an Immunohistochemical Definition in the BCIRG 001 Trial

PubMed Central

Hugh, Judith; Hanson, John; Cheang, Maggie Chon U.; Nielsen, Torsten O.; Perou, Charles M.; Dumontet, Charles; Reed, John; Krajewska, Maryla; Treilleux, Isabelle; Rupin, Matthieu; Magherini, Emmanuelle; Mackey, John; Martin, Miguel; Vogel, Charles

2009-01-01

Purpose To investigate the prognostic and predictive significance of subtyping node-positive early breast cancer by immunohistochemistry in a clinical trial of a docetaxel-containing regimen. Methods Pathologic data from a central laboratory were available for 1,350 patients (91%) from the BCIRG 001 trial of docetaxel, doxorubicin, and cyclophosphamide (TAC) versus fluorouracil, doxorubicin, and cyclophosphamide (FAC) for operable node-positive breast cancer. Patients were classified by tumor characteristics as (1) triple negative (estrogen receptor [ER]–negative, progesterone receptor [PR]–negative, HER2/neu [HER2]–negative), (2) HER2 (HER2-positive, ER-negative, PR-negative), (3) luminal B (ER-positive and/or PR-positive and either HER2-positive and/or Ki67high), and (4) luminal A (ER-positive and/or PR-positive and not HER2-positive or Ki67high), and assessed for prognostic significance and response to adjuvant chemotherapy. Results Patients were subdivided into triple negative (14.5%), HER2 (8.5%), luminal B (61.1%), and luminal A (15.9%). Three-year disease-free survival (DFS) rates (P values with luminal B as referent) were 67% (P < .0001), 68% (P = .0008), 82% (referent luminal B), and 91% (P = .0027), respectively, with hazard ratios of 2.22, 2.12, and 0.46. Improved 3-year DFS with TAC was found in the luminal B group (P = .025) and a combined ER-positive/HER2-negative group treated with tamoxifen (P = .041), with a marginal trend in the triple negatives (P = .051) and HER2 (P = .068) subtypes. No DFS advantage was seen in the luminal A population. Conclusion A simple immunopanel can divide breast cancers into biologic subtypes with strong prognostic effects. TAC significantly complements endocrine therapy in patients with luminal B subtype and, in the absence of targeted therapy, is effective in the triple-negative population. PMID:19204205

Effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients.

PubMed

Hung, Man-Hsin; Liu, Chun-Yu; Shiau, Cheng-Ying; Hsu, Chin-Yi; Tsai, Yi-Fang; Wang, Yu-Ling; Tai, Ling-Chen; King, Kuang-Liang; Chao, Ta-Chung; Chiu, Jen-Hwey; Su, Cheng-Hsi; Lo, Su-Shun; Tzeng, Cheng-Hwai; Shyr, Yi-Ming; Tseng, Ling-Ming

2014-01-01

Brain metastasis is a major complication of breast cancer. This study aimed to analyze the effect of age and biological subtype on the risk and timing of brain metastasis in breast cancer patients. We identified subtypes of invasive ductal carcinoma of the breast by determining estrogen receptor, progesterone receptor and HER2 status. Time to brain metastasis according to age and cancer subtype was analyzed by Cox proportional hazard analysis. Of the 2248 eligible patients, 164 (7.3%) developed brain metastasis over a median follow-up of 54.2 months. Age 35 or younger, HER2-enriched subtype, and triple-negative breast cancer were significant risk factors of brain metastasis. Among patients aged 35 or younger, the risk of brain metastasis was independent of biological subtype (P = 0.507). Among patients aged 36-59 or >60 years, those with triple-negative or HER2-enriched subtypes had consistently increased risk of brain metastasis, as compared with those with luminal A tumors. Patients with luminal B tumors had higher risk of brain metastasis than luminal A only in patients >60 years. Breast cancer subtypes are associated with differing risks of brain metastasis among different age groups. Patients age 35 or younger are particularly at risk of brain metastasis independent of biological subtype.

Temporal profiling of orexin receptor-arrestin-ubiquitin complexes reveals differences between receptor subtypes.

PubMed

Dalrymple, Matthew B; Jaeger, Werner C; Eidne, Karin A; Pfleger, Kevin D G

2011-05-13

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors.

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.

Basal Subtype of Invasive Breast Cancer Is Associated With a Higher Risk of True Recurrence After Conventional Breast-Conserving Therapy

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

Hattangadi-Gluth, Jona A.; Wo, Jennifer Y.; Nguyen, Paul L.

2012-03-01

Purpose: To determine whether breast cancer subtype is associated with patterns of ipsilateral breast tumor recurrence (IBTR), either true recurrence (TR) or elsewhere local recurrence (ELR), among women with pT1-T2 invasive breast cancer (IBC) who receive breast-conserving therapy (BCT). Methods and Materials: From Jan 1998 to Dec 2003, 1,223 women with pT1-T2N0-3 IBC were treated with BCT (lumpectomy plus whole-breast radiation). Ninety percent of patients received adjuvant systemic therapy, but none received trastuzumab. Biologic cancer subtypes were approximated by determining estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), and human epidermal growth factor receptor-2-positive (HER-2+) expression, classified as luminal A (ER+ ormore » PR+ and HER-2 negative [HER-2-]), luminal B (ER+ or PR+ and HER-2+), HER-2 (ER- and PR- and HER-2+), and basal (ER- and PR- and HER-2- ) subtypes. Imaging, pathology, and operative reports were reviewed by two physicians independently, including an attending breast radiologist. Readers were blinded to subtype and outcome. TR was defined as IBTR within the same quadrant and within 3 cm of the primary tumor. All others were defined as ELR. Results: At a median follow-up of 70 months, 24 patients developed IBTR (5-year cumulative incidence of 1.6%), including 15 TR and 9 ELR patients. At 5 years, basal (4.4%) and HER-2 (9%) subtypes had a significantly higher incidence of TR than luminal B (1.2%) and luminal A (0.2%) subtypes (p < 0.0001). On multivariate analysis, basal subtype (hazard ratio [HR], 4.8, p = 0.01), younger age at diagnosis (HR, 0.97; p = 0.05), and increasing tumor size (HR, 2.1; p = 0.04) were independent predictors of TR. Only younger age (HR, 0.95; p = 0.01) significantly predicted for ELR. Conclusions: Basal and HER-2 subtypes are significantly associated with higher rates of TR among women with pT1-T2 IBC after BCT. Younger age predicts for both TR and ELR. Strategies to reduce TR in basal breast cancers, such as increased boost doses, concomitant radiation and chemotherapy, or targeted therapy agents, should be explored.« less

Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. III. Agonist and antagonist properties at serotonin, 5-HT(1) and 5-HT(2), receptor subtypes.

PubMed

Newman-Tancredi, Adrian; Cussac, Didier; Quentric, Yann; Touzard, Manuelle; Verrièle, Laurence; Carpentier, Nathalie; Millan, Mark J

2002-11-01

Although certain antiparkinson agents interact with serotonin (5-HT) receptors, little information is available concerning functional actions. Herein, we characterized efficacies of apomorphine, bromocriptine, cabergoline, lisuride, piribedil, pergolide, roxindole, and terguride at human (h)5-HT(1A), h5-HT(1B), and h5-HT(1D) receptors [guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding], and at h5-HT(2A), h5-HT(2B), and h5-HT(2C) receptors (depletion of membrane-bound [(3)H]phosphatydilinositol). All drugs stimulated h5-HT(1A) receptors with efficacies (compared with 5-HT, 100%) ranging from modest (apomorphine, 35%) to high (cabergoline, 93%). At h5-HT(1B) receptors, efficacies varied from mild (terguride, 37%) to marked (cabergoline, 102%) and potencies were modest (pEC(50) values of 5.8-7.6): h5-HT(1D) sites were activated with a similar range of efficacies and greater potency (7.1-8.5). Piribedil and apomorphine were inactive at h5-HT(1B) and h5-HT(1D) receptors. At h5-HT(2A) receptors, terguride, lisuride, bromocriptine, cabergoline, and pergolide displayed potent (7.6-8.8) agonist properties (49-103%), whereas apomorphine and roxindole were antagonists and piribedil was inactive. Only pergolide (113%/8.2) and cabergoline (123%/8.6) displayed pronounced agonist properties at h5-HT(2B) receptors. At 5-HT(2C) receptors, lisuride, bromocriptine, pergolide, and cabergoline were efficacious (75-96%) agonists, apomorphine and terguride were antagonists, and piribedil was inactive. MDL100,907 and SB242,084, selective antagonists at 5-HT(2A) and 5-HT(2C) receptors, respectively, abolished these actions of pergolide, cabergoline, and bromocriptine. In conclusion, antiparkinson agents display markedly different patterns of agonist and antagonist properties at multiple 5-HT receptor subtypes. Although all show modest (agonist) activity at 5-HT(1A) sites, their contrasting actions at 5-HT(2A) and 5-HT(2C) sites may be of particular significance to their functional profiles in vivo.

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.

Single-Neuron NMDA Receptor Phenotype Influences Neuronal Rewiring and Reintegration following Traumatic Injury

PubMed Central

Patel, Tapan P.; Ventre, Scott C.; Geddes-Klein, Donna; Singh, Pallab K.

2014-01-01

Alterations in the activity of neural circuits are a common consequence of traumatic brain injury (TBI), but the relationship between single-neuron properties and the aggregate network behavior is not well understood. We recently reported that the GluN2B-containing NMDA receptors (NMDARs) are key in mediating mechanical forces during TBI, and that TBI produces a complex change in the functional connectivity of neuronal networks. Here, we evaluated whether cell-to-cell heterogeneity in the connectivity and aggregate contribution of GluN2B receptors to [Ca2+]i before injury influenced the functional rewiring, spontaneous activity, and network plasticity following injury using primary rat cortical dissociated neurons. We found that the functional connectivity of a neuron to its neighbors, combined with the relative influx of calcium through distinct NMDAR subtypes, together contributed to the individual neuronal response to trauma. Specifically, individual neurons whose [Ca2+]i oscillations were largely due to GluN2B NMDAR activation lost many of their functional targets 1 h following injury. In comparison, neurons with large GluN2A contribution or neurons with high functional connectivity both independently protected against injury-induced loss in connectivity. Mechanistically, we found that traumatic injury resulted in increased uncorrelated network activity, an effect linked to reduction of the voltage-sensitive Mg2+ block of GluN2B-containing NMDARs. This uncorrelated activation of GluN2B subtypes after injury significantly limited the potential for network remodeling in response to a plasticity stimulus. Together, our data suggest that two single-cell characteristics, the aggregate contribution of NMDAR subtypes and the number of functional connections, influence network structure following traumatic injury. PMID:24647941

Robo2 determines subtype-specific axonal projections of trigeminal sensory neurons

PubMed Central

Pan, Y. Albert; Choy, Margaret; Prober, David A.; Schier, Alexander F.

2012-01-01

How neurons connect to form functional circuits is central to the understanding of the development and function of the nervous system. In the somatosensory system, perception of sensory stimuli to the head requires specific connections between trigeminal sensory neurons and their many target areas in the central nervous system. Different trigeminal subtypes have specialized functions and downstream circuits, but it has remained unclear how subtype-specific axonal projection patterns are formed. Using zebrafish as a model system, we followed the development of two trigeminal sensory neuron subtypes: one that expresses trpa1b, a nociceptive channel important for sensing environmental chemicals; and a distinct subtype labeled by an islet1 reporter (Isl1SS). We found that Trpa1b and Isl1SS neurons have overall similar axon trajectories but different branching morphologies and distributions of presynaptic sites. Compared with Trpa1b neurons, Isl1SS neurons display reduced branch growth and synaptogenesis at the hindbrain-spinal cord junction. The subtype-specific morphogenesis of Isl1SS neurons depends on the guidance receptor Robo2. robo2 is preferentially expressed in the Isl1SS subset and inhibits branch growth and synaptogenesis. In the absence of Robo2, Isl1SS afferents acquire many of the characteristics of Trpa1b afferents. These results reveal that subtype-specific activity of Robo2 regulates subcircuit morphogenesis in the trigeminal sensory system. PMID:22190641

Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.

PubMed

Cousins, Sarah L; Stephenson, F Anne

2012-04-13

N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.

Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95*

PubMed Central

Cousins, Sarah L.; Stephenson, F. Anne

2012-01-01

N-methyl-d-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149–1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins. PMID:22375001

Synthesis and serotonergic activity of 3-[2-(pyrrolidin-1-yl)ethyl]indoles: potent agonists for the h5-HT1D receptor with high selectivity over the h5-HT1B receptor.

PubMed

Sternfeld, F; Guiblin, A R; Jelley, R A; Matassa, V G; Reeve, A J; Hunt, P A; Beer, M S; Heald, A; Stanton, J A; Sohal, B; Watt, A P; Street, L J

1999-02-25

The design, synthesis, and biological evaluation of a novel series of 3-[2-(pyrrolidin-1-yl)ethyl]indoles with excellent selectivity for h5-HT1D (formerly 5-HT1Dalpha) receptors over h5-HT1B (formerly 5-HT1Dbeta) receptors are described. Clinically effective antimigraine drugs such as Sumatriptan show little selectivity between h5-HT1D and h5-HT1B receptors. The differential expression of h5-HT1D and h5-HT1B receptors in neural and vascular tissue prompted an investigation of whether a compound selective for the h5-HT1D subtype would have the same clinical efficacy but with reduced side effects. The pyrrolidine 3b was initially identified as having 9-fold selectivity for h5-HT1D over h5-HT1B receptors. Substitution of the pyrrolidine ring of 3b with methylbenzylamine groups gave compounds with nanomolar affinity for the h5-HT1D receptor and 100-fold selectivity with respect to h5-HT1B receptors. Modification of the indole 5-substituent led to the oxazolidinones 24a,b with up to 163-fold selectivity for the h5-HT1D subtype and improved selectivity over other serotonin receptors. The compounds were shown to be full agonists by measurement of agonist-induced [35S]GTPgammaS binding in CHO cells expressed with h5-HT receptors. This study suggests that the h5-HT1D and h5-HT1B receptors can be differentiated by appropriate substitution of the ligand in the region which binds to the aspartate residue and reveals a large binding pocket in the h5-HT1D receptor domain which is absent for the h5-HT1B receptor. The compounds described herein will be important tools to delineate the role of h5-HT1D receptors in migraine.

P2 receptor subtypes in the cardiovascular system.

PubMed Central

Kunapuli, S P; Daniel, J L

1998-01-01

Extracellular nucleotides have been implicated in a number of physiological functions. Nucleotides act on cell-surface receptors known as P2 receptors, of which several subtypes have been cloned. Both ATP and ADP are stored in platelets and are released upon platelet activation. Furthermore, nucleotides are also released from damaged or broken cells. Thus during vascular injury nucleotides play an important role in haemostasis through activation of platelets, modulation of vascular tone, recruitment of neutrophils and monocytes to the site of injury, and facilitation of adhesion of leucocytes to the endothelium. Nucleotides also moderate these functions by generating nitric oxide and prostaglandin I2 through activation of endothelial cells, and by activating different receptor subtypes on vascular smooth muscle cells. In the heart, P2 receptors regulate contractility through modulation of L-type Ca2+ channels, although the molecular mechanisms involved are still under investigation. Classical pharmacological studies have identified several P2 receptor subtypes in the cardiovascular system. Molecular pharmacological studies have clarified the nature of some of these receptors, but have complicated the picture with others. In platelets, the classical P2T receptor has now been resolved into three P2 receptor subtypes: the P2Y1, P2X1 and P2TAC receptors (the last of these, which is coupled to the inhibition of adenylate cyclase, is yet to be cloned). In peripheral blood leucocytes, endothelial cells, vascular smooth muscle cells and cardiomyocytes, the effects of classical P2X, P2Y and P2U receptors have been found to be mediated by more than one P2 receptor subtype. However, the exact functions of these multiple receptor subtypes remain to be understood, as P2-receptor-selective agonists and antagonists are still under development. PMID:9841859

Treatment and prognosis of breast cancer patients with brain metastases according to intrinsic subtype.

PubMed

Kuba, Sayaka; Ishida, Mayumi; Nakamura, Yoshiaki; Yamanouchi, Kosho; Minami, Shigeki; Taguchi, Kenichi; Eguchi, Susumu; Ohno, Shinji

2014-11-01

How breast cancer subtypes should affect treatment decisions for breast cancer patients with brain metastases is unclear. We analyzed local brain metastases treatments and their outcomes according to subtype in patients with breast cancer and brain metastases. We reviewed records and database information for women treated at the National Kyushu Cancer Center between 2001 and 2010. Patients were divided into three breast cancer subtype groups: Luminal (estrogen receptor positive and/or progesterone receptor positive, but human epidermal growth factor receptor 2 negative); human epidermal growth factor receptor 2 positive and triple negative (estrogen receptor negative, progesterone receptor negative and human epidermal growth factor receptor 2 negative). Of 524 advanced breast cancer patients, we reviewed 65 (12%) with brain metastases and records showing estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 status, as well as outcome data; there were 26 (40%) Luminal, 26 (40%) had human epidermal growth factor receptor 2 and 13 (20%) had triple negative subtypes. There was no statistical difference in the number of brain metastases among subtypes; however, rates of stereotactic radiosurgery or surgery for brain metastases differed significantly by subtype (human epidermal growth factor receptor 2: 81%, Luminal: 42% and triple negative: 47%; P = 0.03). Patients having the human epidermal growth factor receptor 2 subtype, a performance status of ≤1 and ≤4 brain metastases, who underwent systemic therapy after brain metastases and underwent stereotactic radiosurgery or surgery, were predicted to have longer overall survival after brain metastases. Multivariate analysis demonstrated that not having systemic therapy and not having the human epidermal growth factor receptor 2 subtype were independent factors associated with an increased risk of death (hazard ratio 2.4, 95% confidence interval 1.01-5.6; P = 0.05 and hazard ratio 2.9, 95% confidence interval 1.5-5.8; P = 0.003, respectively). Our study showed that local brain treatments and prognosis differed by subtype in breast cancer patients with brain metastases. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genetics and Pathogenesis of Diffuse Large B-Cell Lymphoma.

PubMed

Schmitz, Roland; Wright, George W; Huang, Da Wei; Johnson, Calvin A; Phelan, James D; Wang, James Q; Roulland, Sandrine; Kasbekar, Monica; Young, Ryan M; Shaffer, Arthur L; Hodson, Daniel J; Xiao, Wenming; Yu, Xin; Yang, Yandan; Zhao, Hong; Xu, Weihong; Liu, Xuelu; Zhou, Bin; Du, Wei; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Connors, Joseph M; Campo, Elias; Lopez-Guillermo, Armando; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa M; Tay Kuang Wei, Kevin; Zelenetz, Andrew D; Leonard, John P; Bartlett, Nancy L; Tran, Bao; Shetty, Jyoti; Zhao, Yongmei; Soppet, Dan R; Pittaluga, Stefania; Wilson, Wyndham H; Staudt, Louis M

2018-04-12

Diffuse large B-cell lymphomas (DLBCLs) are phenotypically and genetically heterogeneous. Gene-expression profiling has identified subgroups of DLBCL (activated B-cell-like [ABC], germinal-center B-cell-like [GCB], and unclassified) according to cell of origin that are associated with a differential response to chemotherapy and targeted agents. We sought to extend these findings by identifying genetic subtypes of DLBCL based on shared genomic abnormalities and to uncover therapeutic vulnerabilities based on tumor genetics. We studied 574 DLBCL biopsy samples using exome and transcriptome sequencing, array-based DNA copy-number analysis, and targeted amplicon resequencing of 372 genes to identify genes with recurrent aberrations. We developed and implemented an algorithm to discover genetic subtypes based on the co-occurrence of genetic alterations. We identified four prominent genetic subtypes in DLBCL, termed MCD (based on the co-occurrence of MYD88 L265P and CD79B mutations), BN2 (based on BCL6 fusions and NOTCH2 mutations), N1 (based on NOTCH1 mutations), and EZB (based on EZH2 mutations and BCL2 translocations). Genetic aberrations in multiple genes distinguished each genetic subtype from other DLBCLs. These subtypes differed phenotypically, as judged by differences in gene-expression signatures and responses to immunochemotherapy, with favorable survival in the BN2 and EZB subtypes and inferior outcomes in the MCD and N1 subtypes. Analysis of genetic pathways suggested that MCD and BN2 DLBCLs rely on "chronic active" B-cell receptor signaling that is amenable to therapeutic inhibition. We uncovered genetic subtypes of DLBCL with distinct genotypic, epigenetic, and clinical characteristics, providing a potential nosology for precision-medicine strategies in DLBCL. (Funded by the Intramural Research Program of the National Institutes of Health and others.).

The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland.

PubMed

Grandclément, B; Ronsin, B; Morel, G

1997-03-01

Atrial natriuretic peptide (ANP) actions are mediated by highly selective and specific receptors. Three subtypes have been characterized and cloned: ANP receptor-A (or GC-A), -B (or GC-B) and -C (the so-called clearance receptor). In rat adrenal gland, the mRNA for each subtype was detected using 35S-dUTP or digoxigenin-11-dUTP specific labeled probes, and in situ hybridization at light and electron microscopic levels respectively. The three subtypes were expressed the most abundantly in the zona glomerulosa. The amount of GC-A mRNA expression, quantified using macro-autoradiography and densitometry, was higher than the amounts of GC-B mRNA and ANPR-C mRNA both in zona glomerulosa and medullary of adrenal gland. At electron microscopic level, the three subtypes of ANPR were revealed in glomerulosa cells. A noticeable signal was also present in the medullary area, especially for GC-A mRNA, in adrenaline-containing chromaffin cells. No signal was detected in noradrenaline-containing chromaffin cells. The subcellular localization of the three mRNAs is similar: in the cytoplasmic matrix and in the euchromatin of the nucleus in each cell of glomerulosa, and in the same compartments of the adrenaline-containing chromaffin cells. These data indicate that the adrenal gland is an important target tissue for ANP action both in glomerulosa cells and adrenaline-containing chromaffin cells. The mRNA expression levels were different for each ANPR subtype.

O6-methylguanine-DNA methyltransferase as a prognostic and predictive marker for basal-like breast cancer treated with cyclophosphamide-based chemotherapy.

PubMed

Isono, Sayuri; Fujishima, Makoto; Azumi, Tatsuya; Hashimoto, Yukihiko; Komoike, Yoshifumi; Yukawa, Masao; Watatani, Masahiro

2014-06-01

The O 6 -methylguanine-DNA methyltransferase (MGMT) protein protects cells from alkylating agents by removing alkyl groups from the O 6 -position of guanine. However, its effect on DNA damage induced by cyclophosphamide (CPM) is unclear. The present study investigated whether MGMT expression was correlated with prognosis in patients with breast cancer that was managed according to a common therapeutic protocol or treated with CPM-based chemotherapy. The intrinsic subtypes and MGMT protein expression levels were assessed in 635 consecutive patients with breast cancer using immunohistochemistry. In total, 425 (67%) luminal A, 95 (15%) luminal B, 47 (7%) human epidermal growth factor receptor-2 + /estrogen receptor - (HER2 + /ER - ) and 48 (8%) basal-like subtypes were identified. Of these, MGMT positivity was identified in 398 (63%) of 635 breast cancers; 68% of luminal A, 67% of luminal B, 30% of HER2 + /ER - and 46% of basal-like subtypes were positive. The overall survival (OS) and disease-free survival (DFS) rates did not significantly differ according to the MGMT status among patients with luminal A, luminal B or HER2 + /ER - subtypes, and patients with MGMT-negative basal-like cancers tended to have a longer DFS, but not a significantly longer OS time. CPM-containing chemotherapy was administered to 26%, 40%, 47% and 31% of patients with luminal A, luminal B, HER2 + /ER - and basal-like tumors, respectively. Although the MGMT status and clinical outcomes of patients with the luminal A, luminal B or HER2 + /ER - subtypes treated with CPM were not significantly correlated, the patients with MGMT-negative basal-like tumors who received CPM exhibited significantly improved DFS and OS compared with the CPM-treated patients with MGMT-positive tumors. MGMT may be a useful prognostic and predictive marker for CPM-containing chemotherapy in basal-like breast cancer.

Characterization of [3H]LS-3-134, a Novel Arylamide Phenylpiperazine D3 Dopamine Receptor Selective Radioligand

PubMed Central

Rangel-Barajas, Claudia; Malik, Maninder; Taylor, Michelle; Neve, Kim A.; Mach, Robert H.; Luedtke, Robert R.

2014-01-01

LS-3-134 is a substituted N-phenylpiperazine derivative that has been reported to exhibit a) high-affinity binding (Ki value 0.2 nM) at human D3 dopamine receptors, b) >100-fold D3 vs. D2 dopamine receptor subtype binding selectivity and c) low-affinity binding (Ki values >5,000 nM) at sigma 1 and sigma 2 receptors. Based upon a forskolin-dependent activation of the adenylyl cyclase inhibition assay, LS-3-134 is a weak partial agonist at both D2 and D3 dopamine receptor subtypes (29% and 35% of full agonist activity, respectively). In this study, [3H]-labeled LS-3-134 was prepared and evaluated to further characterize its use as a D3 dopamine receptor selective radioligand. Kinetic and equilibrium radioligand binding studies were performed. This radioligand rapidly reaches equilibrium (10-15 min at 37°C) and binds with high affinity to both human (Kd = 0.06 ± 0.01 nM) and rat (Kd = 0.2 ± 0.02 nM) D3 receptors expressed in HEK-293 cells. Direct and competitive radioligand binding studies using rat caudate and nucleus accumbens tissue indicate that [3H]LS-3-134 selectively binds a homogeneous population of binding sites with a dopamine D3 receptor pharmacological profile. Based upon these studies we propose that [3H]LS-3-134 represents a novel D3 dopamine receptor selective radioligand that can be used for studying the expression and regulation of the D3 dopamine receptor subtype. PMID:25041389

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study.

PubMed

Troester, Melissa A; Sun, Xuezheng; Allott, Emma H; Geradts, Joseph; Cohen, Stephanie M; Tse, Chiu-Kit; Kirk, Erin L; Thorne, Leigh B; Mathews, Michelle; Li, Yan; Hu, Zhiyuan; Robinson, Whitney R; Hoadley, Katherine A; Olopade, Olufunmilayo I; Reeder-Hayes, Katherine E; Earp, H Shelton; Olshan, Andrew F; Carey, Lisa A; Perou, Charles M

2018-02-01

African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood. Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests. Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women. Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

H2O2 attenuates IGF-1R tyrosine phosphorylation and its survival signaling properties in neuronal cells via NR2B containing NMDA receptor.

PubMed

Zeng, Zhiwen; Wang, Dejun; Gaur, Uma; Rifang, Liao; Wang, Haitao; Zheng, Wenhua

2017-09-12

Impairment of insulin-like growth factor I (IGF-I) signaling plays an important role in the development of neurodegeneration. In the present study, we investigated the effect of H 2 O 2 on the survival signaling of IGF-1 and its underlying mechanisms in human neuronal cells SH-SY5Y. Our results showed that IGF-1 promoted cell survival and stimulated phosphorylation of IGF-1R as well as its downstream targets like AKT and ERK1/2 in these cells. Meanwhile, these effects of IGF-1 were abolished by H 2 O 2 at 200μM concentration which did not cause any significant toxicity to cells itself in our experiments. Moreover, studies using various glutamate receptor subtype antagonists displayed that N-methyl-D -aspartate (NMDA) receptor antagonist dizocilpine maleate (MK-801) blocked the effects of H 2 O 2 , whereas other glutamate receptor subtype antagonists, such as non-NMDA receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX), metabolic glutamate receptor antagonists LY341495 and CPCCOEt, had no effect. Further studies revealed that NR2B-containing NMDARs are responsible for these effects as its effects were blocked by pharmacological inhibitor Ro25-698 or specific siRNA for NR2B, but not NR2A. Finally, our data also showed that Ca 2+ influx contributes to the effects of H 2 O 2 . Similar results were obtained in primary cultured cortical neurons. Taken together, the results from the present study suggested that H 2 O 2 attenuated IGF-1R tyrosine phosphorylation and its survival signaling properties via NR2B containing NMDA receptors and Ca 2+ influx in SH-SY5Y cells. Therefore, NMDAR antagonists, especially NR2B-selective ones, combined with IGF-1 may serve as an alternative therapeutic agent for oxidative stress related neurodegenerative disease.

A family of photoswitchable NMDA receptors

PubMed Central

Berlin, Shai; Szobota, Stephanie; Reiner, Andreas; Carroll, Elizabeth C; Kienzler, Michael A; Guyon, Alice; Xiao, Tong; Trauner, Dirk; Isacoff, Ehud Y

2016-01-01

NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity. DOI: http://dx.doi.org/10.7554/eLife.12040.001 PMID:26929991

Molecular Subtypes of Indonesian Breast Carcinomas - Lack of Association with Patient Age and Tumor Size

PubMed Central

Rahmawati, Yeni; Setyawati, Yunita; Widodo, Irianiwati; Ghozali, Ahmad; Purnomosari, Dewajani

2018-01-01

Objective: Breast carcinoma (BC) is a heterogeneous disease that exhibits variation in biological behaviour, prognosis and response to therapy. Molecular classification is generally into Luminal A, Luminal B, HER2+ and triple negative/basal-like, depending on receptor characteristics. Clinical factors that determined the BC prognosis are age and tumor size. Since information on molecular subtypes of Indonesian BCs is limited, the present study was conducted, with attention to subtypes in relation to age and tumor size. Methods: A retrospective cross-sectional study of 247 paraffin-embedded samples of invasive BC from Dr. Sardjito General Hospital Yogyakarta in the year 2012- 2015 was performed. Immunohistochemical staining using anti- ER, PR, HER2, Ki-67 and CK 5/6 antibodies was applied to classify molecular subtypes. Associations with age and tumor size were analyzed using the Chi Square Test. Results: The Luminal A was the most common subtype of Indonesian BC (41.3%), followed by triple negative (25.5%), HER2 (19.4%) and luminal B (13.8%). Among the triple negative lesions, the basal-like subtype was more frequent than the non basal-like (58.8 % vs 41.2%). Luminal B accounted for the highest percentage of younger age cases (< 40 years old) while HER2+ was most common in older age (> 50 years old) patients. Triple negative/basal-like were commonly large in size. Age (p = 0.080) and tumor size (p = 0.462) were not significantly associated with molecular subtypes of BC. Conclusion: The most common molecular subtype of Indonesian BC is luminal A, followed by triple-negative, HER2+ and luminal B. The majority of triple-negative lesions are basal-like. There are no association between age and tumor size with molecular subtypes of Indonesian BCs. PMID:29373908

Molecular Subtypes of Indonesian Breast Carcinomas - Lack of Association with Patient Age and Tumor Size

PubMed

Rahmawati, Yeni; Setyawati, Yunita; Widodo, Irianiwati; Ghozali, Ahmad; Purnomosari, Dewajani

2018-01-27

Objective: Breast carcinoma (BC) is a heterogeneous disease that exhibits variation in biological behaviour, prognosis and response to therapy. Molecular classification is generally into Luminal A, Luminal B, HER2+ and triple negative/basal-like, depending on receptor characteristics. Clinical factors that determined the BC prognosis are age and tumor size. Since information on molecular subtypes of Indonesian BCs is limited, the present study was conducted, with attention to subtypes in relation to age and tumor size. Methods: A retrospective cross-sectional study of 247 paraffin-embedded samples of invasive BC from Dr. Sardjito General Hospital Yogyakarta in the year 2012- 2015 was performed. Immunohistochemical staining using anti- ER, PR, HER2, Ki-67 and CK 5/6 antibodies was applied to classify molecular subtypes. Associations with age and tumor size were analyzed using the Chi Square Test. Results: The Luminal A was the most common subtype of Indonesian BC (41.3%), followed by triple negative (25.5%), HER2 (19.4%) and luminal B (13.8%). Among the triple negative lesions, the basal-like subtype was more frequent than the non basal-like (58.8 % vs 41.2%). Luminal B accounted for the highest percentage of younger age cases (< 40 years old) while HER2+ was most common in older age (> 50 years old) patients. Triple negative/basal-like were commonly large in size. Age (p = 0.080) and tumor size (p = 0.462) were not significantly associated with molecular subtypes of BC. Conclusion: The most common molecular subtype of Indonesian BC is luminal A, followed by triple-negative, HER2+ and luminal B. The majority of triple-negative lesions are basal-like. There are no association between age and tumor size with molecular subtypes of Indonesian BCs. Creative Commons Attribution License

Temporal Profiling of Orexin Receptor-Arrestin-Ubiquitin Complexes Reveals Differences between Receptor Subtypes*

PubMed Central

Dalrymple, Matthew B.; Jaeger, Werner C.; Eidne, Karin A.; Pfleger, Kevin D. G.

2011-01-01

Orexin G protein-coupled receptors (OxRs) and their cognate agonists have been implicated in a number of disorders since their recent discovery, ranging from narcolepsy to formation of addictive behavior. Bioluminescence resonance energy transfer assays of agonist-occupied OxRs provided evidence for a strong dose-dependent interaction with both trafficking proteins β-arrestin 1 and 2 that required unusually high agonist concentrations compared with inositol phosphate signaling. This appears to be reflected in functional differences in potency with respect to orexin A (OxA) and OxR2-dependent ERK1/2 phosphorylation after 90 min compared with 2 min, potentially consistent with β-arrestin-mediated versus G protein-mediated signaling, respectively. Furthermore, extended bioluminescence resonance energy transfer kinetic data monitoring OxA-dependent receptor-β-arrestin and β-arrestin-ubiquitin proximity suggested subtype-specific differences in receptor trafficking, with OxR2 activation resulting in more sustained receptor-β-arrestin-ubiquitin complex formation than elicited by OxR1 activation. Enzyme-linked immunosorbent assay (ELISA) data also revealed that OxR1 underwent significantly more rapid recycling compared with OxR2. Finally, we have observed sustained OxA-dependent ERK1/2 phosphorylation in the presence of OxR2 compared with OxR1. Although both OxR subtypes could be classified as class B receptors for β-arrestin usage based on the initial strength of interaction with both β-arrestins, our temporal profiling revealed tangible differences between OxR subtypes. Consequently, OxR1 appears to fit uneasily into the commonly used β-arrestin classification scheme. More importantly, it is hoped that this improved profiling capability, enabling the subtleties of protein complex formation, stability, and duration to be assessed in live cells, will help unlock the therapeutic potential of targeting these receptors. PMID:21378163

Intrinsic breast tumor subtypes, race, and long-term survival in the Carolina Breast Cancer Study.

PubMed

O'Brien, Katie M; Cole, Stephen R; Tse, Chiu-Kit; Perou, Charles M; Carey, Lisa A; Foulkes, William D; Dressler, Lynn G; Geradts, Joseph; Millikan, Robert C

2010-12-15

Previous research identified differences in breast cancer-specific mortality across 4 intrinsic tumor subtypes: luminal A, luminal B, basal-like, and human epidermal growth factor receptor 2 positive/estrogen receptor negative (HER2(+)/ER(-)). We used immunohistochemical markers to subtype 1,149 invasive breast cancer patients (518 African American, 631 white) in the Carolina Breast Cancer Study, a population-based study of women diagnosed with breast cancer. Vital status was determined through 2006 using the National Death Index, with median follow-up of 9 years. Cancer subtypes luminal A, luminal B, basal-like, and HER2(+)/ER(-) were distributed as 64%, 11%, 11%, and 5% for whites, and 48%, 8%, 22%, and 7% for African Americans, respectively. Breast cancer mortality was higher for participants with HER2(+)/ER(-) and basal-like breast cancer compared with luminal A and B. African Americans had higher breast cancer-specific mortality than whites, but the effect of race was statistically significant only among women with luminal A breast cancer. However, when compared with the luminal A subtype within racial categories, mortality for participants with basal-like breast cancer was higher among whites (HR = 2.0, 95% CI: 1.2-3.4) than African Americans (HR = 1.5, 95% CI: 1.0-2.4), with the strongest effect seen in postmenopausal white women (HR = 3.9, 95% CI: 1.5-10.0). Our results confirm the association of basal-like breast cancer with poor prognosis and suggest that basal-like breast cancer is not an inherently more aggressive disease in African American women compared with whites. Additional analyses are needed in populations with known treatment profiles to understand the role of tumor subtypes and race in breast cancer mortality, and in particular our finding that among women with luminal A breast cancer, African Americans have higher mortality than whites. ©2010 AACR.

A2A Adenosine Receptor Antagonists as Therapeutic Candidates: are they still an interesting challenge?

PubMed

Cacciari, Barbara; Federico, Stephanie; Spalluto, Giampiero

2018-04-22

In the past decades, many efforts were done to develope ligands for the adenosine receptors, with the purpose to individuate agonists and antagonists affine and selective for each subtypes , named A1, A2A, A2B, and A3. These intense studies allowed a deeper and deeper knowledge of the nature and, moreover, of the pathophysiological roles of all the adenosine receptor subtypes. In particular, the involvment of the A2A adenosine receptor subtype in some physiological mechanisms in the brain, that could be related to important diseases such as the Parkinson's disease, encouraged the research in this field. Particular attention was given to the antagonists endowed with high affinity and selectivity since they could have a real employment in the treatment of Parkinson's disease, and some compounds, such as istradefylline, preladenant and tozadenant, are already studied in clinical trials. Actually, the role of A2A antagonists in Parkinson's disease is becoming contradictory due to contrasting results in the last studies, but, at the same time, new possible employments are emerging for this class of antagonists in cancer pathologies as much interesting to legitimate further efforts in the research of A2A ligands. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone-secreting adrenal phaeochromocytoma: review of the literature and report of a case

PubMed Central

Ruggeri, R.M.; Ferraù, F.; Campennì, A.; Simone, A.; Barresi, V.; Giuffrè, G.; Tuccari, G.; Baldari, S.; Trimarchi, F.

2009-01-01

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochromocytoma, causing ectopic Cushing’s syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous, 9-cm mass in the right adrenal gland, and [111In-DTPA0] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region, corresponding to the adrenal mass. The patient underwent laparoscopic surgery and formalin-fixed and paraffin-embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed. Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma, which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR1, SSTR2A, SSTR2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing’s disease was dependent on CRH overproduction by the pheochromocytoma, in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas, and provide further evidence for functional SSTR subtype SSTR2a in a subgroup of pheochromocytomas, suggesting that these tumours may represent potential target for octreotide treatment.

Immunohistochemical localization and functional characterization of somatostatin receptor subtypes in a corticotropin releasing hormone- secreting adrenal phaeochromocytoma: review of the literature and report of a case.

PubMed

Ruggeri, Rosaria M; Ferraù, F; Campennì, A; Simone, A; Barresi, V; Giuffrè, G; Tuccari, G; Baldari, S; Trimarchi, F

2009-01-01

Somastostatin receptors are frequently expressed in phaeochromocytoma but data on somatostatin receptor subtyping are scanty and the functional response to the somatostatin analogue octretide is still debated.We report an unusual case of pheochro-mocytoma,causing ectopic Cushing's syndrome due to CRH production by the tumour cells, in a 50-yr-old woman. Abdominal computed tomography revealed an inhomogeneous,9-cm mass in the right adrenal gland,and [111In-DTPA0] octreotide scintigraphy showed an abnormal uptake of the radiotracer in the right perirenal region,corresponding to the adrenal mass.The patient underwent laparoscopic surgery and formalin-fixed and paraffin embedded samples were studied. The tumour was extensively characterized by immunohistochemistry and somatostatin receptor (SSTRs) subtypes expression was analyzed.Histological and immunohistochemical examination of the surgical specimens displayed a typical pheochromocytoma,which was found to be immunoreative to S-100, chromogranin A and neurofilaments. Immunostaining for SSTR subtypes showed a positive reaction for SSTR1, SSTR2A, SSTR2B, antisera on tumour cells. The intense and diffuse immunostaining for corticotropin releasing hormone (CRH) antiserum indicated that Cushing's disease was dependent on CRH overproduction by the pheochromocytoma,in which no immunostaining for adrenocorticotropic hormone was found. Our report confirms the heterogeneity of the pattern of SSTR expression in pheochromocytomas,and provide further evidence for functional SSTR subtype SSTR2a in a subgroup of pheochromocytomas,suggesting that these tumours may represent potential target for octreotide treatment.

Periaqueductal gray knockdown of V2, not V1a and V1b receptor influences nociception in the rat. yj6676@yahoo.com.

PubMed

Yang, Jun; Yang, Yu; Chen, Jian-Min; Wang, Gen; Xu, Hong-Tao; Liu, Wen-Yan; Lin, Bao-Cheng

2007-01-01

Our pervious study has proved that arginine vasopressin (AVP) in periaqueductal gray (PAG) plays a role in antinociception. After establishing a model of local special gene knockdown, the nociceptive effect of vasopressin receptor subunit in PAG was investigated in the rat. Microinjection of short-interfering RNA (siRNA) into PAG, which targeted vasopressin receptor subtypes (V(1a), V(1b) and V(2)), locally weakened the associated mRNA expression and depressed the related receptor synthesis in a dose-dependent manner, in which the significant inhibitive effect occurred on from 7th day to 14th day following 1microg or 2microg siRNA administration. PAG knockdown of V(2) receptor gene markedly decreased pain threshold in from 6th day to 13th day after siRNA administration, whereas local knockdown of either V(1a) or V(1b) receptor gene could not influence pain threshold. The data suggest that V(2) rather than V(1a) and V(1b) receptor in PAG involves in nociception.

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

Electrophysiological effects of tachykinins and capsaicin on guinea-pig bronchial parasympathetic ganglion neurones.

PubMed Central

Myers, A C; Undem, B J

1993-01-01

1. We evaluated the effects of neurokinins, tachykinin analogues, or capsaicin on passive membrane properties of guinea-pig bronchial parasympathetic neurones using intracellular recording techniques. 2. Substance P (SP) and the tachykinin analogue, acetyl-[Arg6,Sar9,Met(O2)11]-SP(6-11) (ASMSP), at concentrations selective for the neurokinin (NK)-1 receptor subtype, depolarized the resting potential (3 and 5 mV, respectively) with no change in input resistance. Neurokinin A and beta Ala8NKA(4-10), at concentrations selective for the NK-2 receptor subtype (0.1 microM), were without effect. 3. Neurokinin B (NKB) and [Asp5,6,methyl-Phe8]SP(5-11) (senktide analogue), at concentrations selective for NK-3 receptor subtype, elicited maximum depolarizations of 16 +/- 2 mV for both agonists. The peak of the depolarization was associated with an decrease in membrane resistance (35 +/- 4 and 50 +/- 7%, respectively). 4. Capsaicin (1 microM) elicited a 3-24 mV depolarization of the resting potential of thirteen of eighteen bronchial ganglion neurones and decreased the input resistance of seven of thirteen of these neurones. The effects of capsaicin were reduced by desensitization with senktide analogue at a concentration selective for the NK-3 receptor subtype, whereas a non-peptide NK-1 receptor antagonist had no effect. 5. Using voltage clamp analysis, capsaicin and senktide analogue evoked an inward current and an increase in membrane conductance at the resting membrane potential. The reversal potential for senktide analogue was estimated to be + 4 mV. 6. These data support the hypothesis that neurokinin-containing nerve terminals are localized within guinea-pig bronchial parasympathetic ganglia and, when released, the predominant effect of the neurokinins is by activation of NK-3 receptors. PMID:7508508

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor-Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint.

PubMed

Myburgh, Ettienne J; Langenhoven, Lizanne; Grant, Kathleen A; van der Merwe, Lize; Kotze, Maritha J

2017-08-01

Human epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping. Clinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor-positive and/or progesterone receptor-positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study. The median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal ( P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors ( P = .0002; 95% CI, 0.40 to 1.06). Of the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value.

Pathophysiological roles of P2 receptors in glial cells.

PubMed

Abbracchio, Maria P; Verderio, Claudia

2006-01-01

Extracellular nucleotides act through specific receptors on target cells: the seven ionotropic P2X and the eight G protein-coupled P2Y receptors. All these receptors are expressed by brain astroglia and microglia. In astrocytes, P2 receptors have been implicated in short-term calcium-dependent cell-cell communication. Upon mechanical stimulation or activation by other transmitters, astrocytes release ATP and respond to ATP with a propagating wave of intracellular calcium increases, allowing a homotypic astrocyte-astrocyte communication, as well as an heterotypic signalling which also involves neurons, oligodendrocytes and microglia. Astrocytic P2 receptors also mediate reactive astrogliosis, a reaction contributing to neuronal death in neurodegenerative diseases. Signalling leading to inflammatory astrogliosis involves induction of cyclo-oxygenase 2 through stimulation of ERK1,2 and of the transcriptional factors AP-1 and NF-kappaB. Microglia also express several P2 receptors linked to intracellular calcium increases. P2 receptor subtypes are differentially regulated by typical proinflammatory signals for these cells (e.g. lipopolysaccharide), suggesting specific roles in brain immune responses. Globally, these findings highlight the roles of P2 receptors in glial cell pathophysiology suggesting a contribution to neurodegenerative diseases characterized by excessive gliosis and neuro-inflammation. They also open up the possibility of modulating brain damage by ligands selectively targeting the specific P2 receptor subtypes involved in the gliotic response.

Geographic differences in the distribution of molecular subtypes of breast cancer in Brazil

PubMed Central

2014-01-01

Background To compare the distribution of the intrinsic molecular subtypes of breast cancer based on immunohistochemical profile in the five major geographic regions of Brazil, a country of continental dimension, with a wide racial variation of people. Methods The study was retrospective observational. We classified 5,687 invasive breast cancers by molecular subtype based on immunohistochemical expression of estrogen-receptor (ER), progesterone-receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki-67 proliferation index. Cases were classified as luminal A (ER and/or PR positive and HER2 negative, Ki-67 < 14%), luminal B (ER and/or PR positive, HER2 negative, and Ki-67 > 14%), triple-positive (ER and/or PR positive and HER2 positive), HER2-enriched (ER and PR negative, and HER2- positive), and triple-negative (TN) (ER negative, PR negative, and HER2- negative). Comparisons of the ages of patients and molecular subtypes between different geographic regions were performed. Results South and Southeast regions with a higher percentage of European ancestry and higher socioeconomic status presented with the highest proportion of luminal tumors. The North region presented with more aggressive subtypes (HER2-enriched and triple-negative), while the Central-West region predominated triple-positive carcinomas. The Northeast—a region with a high African influence—presented intermediate frequency of the different molecular subtypes. The differences persisted in subgroups of patients under and over 50 years. Conclusions The geographic regions differ according to the distribution of molecular subtypes of breast cancer. However, other differences, beside those related to African ancestry, such as socioeconomic, climatic, nutritional, and geographic, have to be considered to explain our results. The knowledge of the differences in breast cancer characteristics among the geographic regions may help to organize healthcare programs in large countries like Brazil with diverse economic and race composition among different geographic regions. PMID:25174527

Pattern of recurrence of early breast cancer is different according to intrinsic subtype and proliferation index

PubMed Central

2013-01-01

Introduction Recurrence risk in breast cancer varies throughout the follow-up time. We examined if these changes are related to the level of expression of the proliferation pathway and intrinsic subtypes. Methods Expression of estrogen and progesterone receptor, Ki-67, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR) and cytokeratin 5/6 (CK 5/6) was performed on tissue-microarrays constructed from a large and uniformly managed series of early breast cancer patients (N = 1,249). Subtype definitions by four biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14), HER2-enriched (any ER, any PR, HER2+, any Ki-67), triple-negative (ER-, PR-, HER2-, any Ki-67). Subtype definitions by six biomarkers were as follows: luminal A (ER + and/or PR+, HER2-, Ki-67 <14, any CK 5/6, any EGFR), luminal B (ER + and/or PR+, HER2-, Ki-67 ≥14, any CK 5/6, any EGFR), HER2-enriched (ER-, PR-, HER2+, any Ki-67, any CK 5/6, any EGFR), Luminal-HER2 (ER + and/or PR+, HER2+, any Ki-67, any CK 5/6, any EGFR), Basal-like (ER-, PR-, HER2-, any Ki-67, CK5/6+ and/or EGFR+), triple-negative nonbasal (ER-, PR-, HER2-, any Ki-67, CK 5/6-, EGFR-). Each four- or six-marker defined intrinsic subtype was divided in two groups, with Ki-67 <14% or with Ki-67 ≥14%. Recurrence hazard rate function was determined for each intrinsic subtype as a whole and according to Ki-67 value. Results Luminal A displayed a slow risk increase, reaching its maximum after three years and then remained steady. Luminal B presented most of its relapses during the first five years. HER2-enriched tumors show a peak of recurrence nearly twenty months post-surgery, with a greater risk in Ki-67 ≥14%. However a second peak occurred at 72 months but the risk magnitude was greater in Ki-67 <14%. Triple negative tumors with low proliferation rate display a smooth risk curve, but with Ki-67 ≥14% show sharp peak at nearly 18 months. Conclusions Each intrinsic subtype has a particular pattern of relapses over time which change depending on the level of activation of the proliferation pathway assessed by Ki-67. These findings could have clinical implications both on adjuvant treatment trial design and on the recommendations concerning the surveillance of patients. PMID:24148581

High prevalence of luminal B breast cancer intrinsic subtype in Colombian women

PubMed Central

Serrano-Gomez, Silvia Juliana; Sanabria-Salas, Maria Carolina; Hernández-Suarez, Gustavo; García, Oscar; Silva, Camilo; Romero, Alejandro; Mejía, Juan Carlos; Miele, Lucio; Fejerman, Laura; Zabaleta, Jovanny

2016-01-01

Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Little is known about the prevalence of breast cancer intrinsic subtypes and their association with clinicopathological data and genetic ancestry in Latin Americans. Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were used to classify breast cancers in 301 patients from Colombia into intrinsic subtypes. We analyzed the distribution of subtypes by clinicopathological variables. Genetic ancestry was estimated from a panel of 80 ancestry informative markers. Luminal B breast cancer subtype was the most prevalent in our population (37.2%) followed by luminal A (26.3%), non-basal triple negative (NBTN) (11.6%), basal like (9%), human epidermal growth factor receptor 2 (HER2) enriched (8.6%) and unknown (7.3%). We found statistical significant differences in distribution between Colombian region (P = 0.007), age at diagnosis (P = 0.0139), grade (P < 0.001) and recurrence (P < 0.001) according to intrinsic subtype. Patients diagnosed with HER2-enriched, basal-like and NBTN breast cancer had the highest African ancestry. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian patients. PMID:27207651

Presynaptic muscarinic acetylcholine autoreceptors (M1, M2 and M4 subtypes), adenosine receptors (A1 and A2A) and tropomyosin-related kinase B receptor (TrkB) modulate the developmental synapse elimination process at the neuromuscular junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Santafé, Manel; Tomàs, Josep

2016-06-23

The development of the nervous system involves an initially exuberant production of neurons that make an excessive number of synaptic contacts. The initial overproduction of synapses promotes connectivity. Hebbian competition between axons with different activities (the least active are punished) leads to the loss of roughly half of the overproduced elements and this refines connectivity and increases specificity. The neuromuscular junction is innervated by a single axon at the end of the synapse elimination process and, because of its relative simplicity, has long been used as a model for studying the general principles of synapse development. The involvement of the presynaptic muscarinic ACh autoreceptors may allow for the direct competitive interaction between nerve endings through differential activity-dependent acetylcholine release in the synaptic cleft. Then, the most active ending may directly punish the less active ones. Our previous results indicate the existence in the weakest axons on the polyinnervated neonatal NMJ of an ACh release inhibition mechanism based on mAChR coupled to protein kinase C and voltage-dependent calcium channels. We suggest that this mechanism plays a role in the elimination of redundant neonatal synapses. Here we used confocal microscopy and quantitative morphological analysis to count the number of brightly fluorescent axons per endplate in P7, P9 and P15 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. We investigate the involvement of individual mAChR M1-, M2- and M4-subtypes in the control of axonal elimination after the Levator auris longus muscle had been exposed to agonist and antagonist in vivo. We also analysed the role of adenosine receptor subtypes (A1 and A2A) and the tropomyosin-related kinase B receptor. The data show that postnatal axonal elimination is a regulated multireceptor mechanism that guaranteed the monoinnervation of the neuromuscular synapses. The three receptor sets considered (mAChR, AR and TrkB receptors) intervene in modulating the conditions of the competition between nerve endings, possibly helping to determine the winner or the lossers but, thereafter, the final elimination would occur with some autonomy and independently of postsynaptic maturation.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa 1b subtype.

PubMed

Lai, J; Ma, S W; Zhu, R H; Rothman, R B; Lentes, K U; Porreca, F

1994-10-27

Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

PubMed Central

ABBRACCHIO, MARIA P.; BURNSTOCK, GEOFFREY; BOEYNAEMS, JEAN-MARIE; BARNARD, ERIC A.; BOYER, JOSÉ L.; KENNEDY, CHARLES; KNIGHT, GILLIAN E.; FUMAGALLI, MARTA; GACHET, CHRISTIAN; JACOBSON, KENNETH A.; WEISMAN, GARY A.

2012-01-01

There have been many advances in our knowledge about different aspects of P2Y receptor signaling since the last review published by our International Union of Pharmacology subcommittee. More receptor subtypes have been cloned and characterized and most orphan receptors deorphanized, so that it is now possible to provide a basis for a future subdivision of P2Y receptor subtypes. More is known about the functional elements of the P2Y receptor molecules and the signaling pathways involved, including interactions with ion channels. There have been substantial developments in the design of selective agonists and antagonists to some of the P2Y receptor subtypes. There are new findings about the mechanisms underlying nucleotide release and ectoenzymatic nucleotide breakdown. Interactions between P2Y receptors and receptors to other signaling molecules have been explored as well as P2Y-mediated control of gene transcription. The distribution and roles of P2Y receptor subtypes in many different cell types are better understood and P2Y receptor-related compounds are being explored for therapeutic purposes. These and other advances are discussed in the present review. PMID:16968944

Effects of targeted deletion of A1 adenosine receptors on postischemic cardiac function and expression of adenosine receptor subtypes.

PubMed

Morrison, R Ray; Teng, Bunyen; Oldenburg, Peter J; Katwa, Laxmansa C; Schnermann, Jurgen B; Mustafa, S Jamal

2006-10-01

To examine ischemic tolerance in the absence of A(1) adenosine receptors (A(1)ARs), isolated wild-type (WT) and A(1)AR knockout (A(1)KO) murine hearts underwent global ischemia-reperfusion, and injury was measured in terms of functional recovery and efflux of lactate dehydrogenase (LDH). Hearts were analyzed by real-time RT-PCR both at baseline and at intervals during ischemia-reperfusion to determine whether compensatory expression of other adenosine receptor subtypes occurs with either A(1)AR deletion and/or ischemia-reperfusion. A(1)KO hearts had higher baseline coronary flow (CF) and left ventricular developed pressure (LVDP) than WT hearts, whereas heart rate was unchanged by A(1)AR deletion. After 20 min of ischemia, CF was attenuated in A(1)KO compared with WT hearts, and this reduction persisted throughout reperfusion. Final recovery of LVDP was decreased in A(1)KO hearts (54.4 +/- 5.1 vs. WT 81.1 +/- 3.4% preischemic baseline) and correlated with higher diastolic pressure during reperfusion. Postischemic efflux of LDH was greater in A(1)KO compared with WT hearts. Real-time RT-PCR demonstrated the absence of A(1)AR transcript in A(1)KO hearts, and the message for A(2A), A(2B), and A(3) adenosine receptors was similar in uninstrumented A(1)KO and WT hearts. Ischemia-reperfusion increased A(2B) mRNA expression 2.5-fold in both WT and A(1)KO hearts without changing A(1) or A(3) expression. In WT hearts, ischemia transiently doubled A(2A) mRNA, which returned to preischemic level upon reperfusion, a pattern not observed in A(1)KO hearts. Together, these data affirm the cardioprotective role of A(1)ARs and suggest that induced expression of other adenosine receptor subtypes may participate in the response to ischemia-reperfusion in isolated murine hearts.

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells

PubMed Central

Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A.; Yang, Jay; Emala, Charles W.

2013-01-01

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [β-Ala8]-neurokinin A(4–10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase. PMID:18203813

Expression and coupling of neurokinin receptor subtypes to inositol phosphate and calcium signaling pathways in human airway smooth muscle cells.

PubMed

Mizuta, Kentaro; Gallos, George; Zhu, Defen; Mizuta, Fumiko; Goubaeva, Farida; Xu, Dingbang; Panettieri, Reynold A; Yang, Jay; Emala, Charles W

2008-03-01

Neuropeptide tachykinins (substance P, neurokinin A, and neurokinin B) are present in peripheral terminals of sensory nerve fibers within the respiratory tract and cause airway contractile responses and hyperresponsiveness in humans and most mammalian species. Three subtypes of neurokinin receptors (NK1R, NK2R, and NK3R) classically couple to Gq protein-mediated inositol 1,4,5-trisphosphate (IP3) synthesis and liberation of intracellular Ca2+, which initiates contraction, but their expression and calcium signaling mechanisms are incompletely understood in airway smooth muscle. All three subtypes were identified in native and cultured human airway smooth muscle (HASM) and were subsequently overexpressed in HASM cells using a human immunodeficiency virus-1-based lentivirus transduction system. Specific NKR agonists {NK1R, [Sar9,Met(O2)11]-substance P; NK2R, [beta-Ala8]-neurokinin A(4-10); NK3R, senktide} stimulated inositol phosphate synthesis and increased intracellular Ca2+ concentration ([Ca2+]i) in native HASM cells and in HASM cells transfected with each NKR subtype. These effects were blocked by NKR-selective antagonists (NK1R, L-732138; NK2R, GR-159897; NK3R, SB-222200). The initial transient and sustained phases of increased [Ca2+]i were predominantly inhibited by the IP3 receptor antagonist 2-aminoethoxydiphenyl borate (2-APB) or the store-operated Ca2+ channel antagonist SKF-96365, respectively. These results show that all three subtypes of NKRs are expressed in native HASM cells and that IP3 levels are the primary mediators of NKR-stimulated initial [Ca2+]i increases, whereas store-operated Ca2+ channels mediate the sustained phase of the [Ca2+]i increase.

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

Campbell, K.S.

Norepinephrine has previously been demonstrated by this laboratory to potentiate the in vitro T-dependent antibody response through the stimulation of {beta}-adrenergic receptors. The role of {beta}-adrenergic receptor subtypes in norepinephrine-induced potentiation of the antibody responses was examined with selective {beta}-adrenergic antagonists. The antagonists were metoprolol ({beta}{sub 1}-selective), ICI 118-551 ({beta}{sub 2}-selective), and propranolol ({beta}-non-selective). Both propranolol and ICI 118-551 blocked norepinephrine-induced potentiation of the antibody response, but metoprolol was ineffective. Receptor binding competition of antagonists with the radioligant, ({sup 3}H)CGP-12177 was examined and results were analyzed with the computer program, LIGAND. Competition by ICI 118-551 identified 75% {beta}{sub 2}- andmore » 25% {beta}{sub 1}-adrenergic receptors on splenic mononuclear cells. Enriched T lymphocytes exhibited 75% {beta}{sub 2}-adrenergic receptors, while enriched B lymphocytes contained 90% {beta}{sub 2}-adrenergic receptors as identified by ICI 118-551. Greater than twice as many total receptors were identified on B lymphocytes than T lymphocytes. A T cell lymphoma contained about 60% {beta}{sub 2}-receptors, while 100% were {beta}{sub 2} receptors on a B cell lymphoma, as assessed by ICI 118-551. Results support a heterogeneous {beta}-adrenergic receptor population on T lymphocytes and a more homogeneous {beta}{sub 2}-population on B lymphocytes.« less

Prognostic Significance of Progesterone Receptor–Positive Tumor Cells Within Immunohistochemically Defined Luminal A Breast Cancer

PubMed Central

Prat, Aleix; Cheang, Maggie Chon U.; Martín, Miguel; Parker, Joel S.; Carrasco, Eva; Caballero, Rosalía; Tyldesley, Scott; Gelmon, Karen; Bernard, Philip S.; Nielsen, Torsten O.; Perou, Charles M.

2013-01-01

Purpose Current immunohistochemical (IHC)-based definitions of luminal A and B breast cancers are imperfect when compared with multigene expression-based assays. In this study, we sought to improve the IHC subtyping by examining the pathologic and gene expression characteristics of genomically defined luminal A and B subtypes. Patients and Methods Gene expression and pathologic features were collected from primary tumors across five independent cohorts: British Columbia Cancer Agency (BCCA) tamoxifen-treated only, Grupo Español de Investigación en Cáncer de Mama 9906 trial, BCCA no systemic treatment cohort, PAM50 microarray training data set, and a combined publicly available microarray data set. Optimal cutoffs of percentage of progesterone receptor (PR) –positive tumor cells to predict survival were derived and independently tested. Multivariable Cox models were used to test the prognostic significance. Results Clinicopathologic comparisons among luminal A and B subtypes consistently identified higher rates of PR positivity, human epidermal growth factor receptor 2 (HER2) negativity, and histologic grade 1 in luminal A tumors. Quantitative PR gene and protein expression were also found to be significantly higher in luminal A tumors. An empiric cutoff of more than 20% of PR-positive tumor cells was statistically chosen and proved significant for predicting survival differences within IHC-defined luminal A tumors independently of endocrine therapy administration. Finally, no additional prognostic value within hormonal receptor (HR) –positive/HER2-negative disease was observed with the use of the IHC4 score when intrinsic IHC-based subtypes were used that included the more than 20% PR-positive tumor cells and vice versa. Conclusion Semiquantitative IHC expression of PR adds prognostic value within the current IHC-based luminal A definition by improving the identification of good outcome breast cancers. The new proposed IHC-based definition of luminal A tumors is HR positive/HER2 negative/Ki-67 less than 14%, and PR more than 20%. PMID:23233704

Identification of the dopamine autoreceptor in the guinea-pig retina as D2 receptor using novel subtype-selective antagonists

PubMed Central

Weber, Bernd; Schlicker, Eberhard; Sokoloff, Pierre; Stark, Holger

2001-01-01

Dopamine release in the retina is subject to modulation via autoreceptors, which belong to the D2 receptor family (encompassing the D2, D3 and D4 receptors). The aim of the present study was to determine the receptor subtype (D2 vs D3) involved in the inhibition of dopamine release in guinea-pig retinal discs, using established (haloperidol, (S)-nafadotride) and novel dopamine receptor antagonists (ST-148, ST-198). hD2L and hD3 receptors were expressed in CHO cells and the pKi values determined in binding studies with [125I]-iodosulpride were: haloperidol 9.22 vs 8.54; ST-148 7.85 vs 6.60; (S)-nafadotride 8.52 vs 9.51; ST-198 6.14 vs 7.92. The electrically evoked tritium overflow from retinal discs preincubated with [3H]-noradrenaline (which represents quasi-physiological dopamine release) was inhibited by the dopamine receptor agonists B-HT 920 (talipexole) and quinpirole (maximally by 82 and 71%; pEC50 5.80 and 5.83). The concentration-response curves of these agonists were shifted to the right by haloperidol (apparent pA2 8.69 and 8.23) and ST-148 (7.52 and 7.66). (S)-Nafadotride 0.01 μM and ST-198 0.32 μM did not affect the concentration-response curve of B-HT 920. The dopamine autoreceptor in the guinea-pig retina can be classified as a D2 receptor. ST-148 and ST-198 show an improved selectivity for D2 and D3 receptors when compared to haloperidol and (S)-nafadotride, respectively. PMID:11498509

Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype-targeted drugs.

PubMed

Meltzer, Herbert Y; Roth, Bryan L

2013-12-01

Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtype-selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson's disease psychosis, respectively.

Effects of muscarinic receptor antagonists on cocaine discrimination in wild-type mice and in muscarinic receptor M1, M2, and M4 receptor knockout mice.

PubMed

Joseph, Lauren; Thomsen, Morgane

2017-06-30

Muscarinic M 1 /M 4 receptor stimulation can reduce abuse-related effects of cocaine and may represent avenues for treating cocaine addiction. Muscarinic antagonists can mimic and enhance effects of cocaine, including discriminative stimulus (S D ) effects, but the receptor subtypes mediating those effects are not known. A better understanding of the complex cocaine/muscarinic interactions is needed to evaluate and develop potential muscarinic-based medications. Here, knockout mice lacking M 1 , M 2 , or M 4 receptors (M 1 -/- , M 2 -/- , M 4 -/- ), as well as control wild-type mice and outbred Swiss-Webster mice, were trained to discriminate 10mg/kg cocaine from saline. Muscarinic receptor antagonists with no subtype selectivity (scopolamine), or preferential affinity at the M 1 , M 2 , or M 4 subtype (telenzepine, trihexyphenidyl; methoctramine, AQ-RA 741; tropicamide) were tested alone and in combination with cocaine. In intact animals, antagonists with high affinity at M 1 /M 4 receptors partially substituted for cocaine and increased the S D effect of cocaine, while M 2 -preferring antagonists did not substitute, and reduced the S D effect of cocaine. The cocaine-like effects of scopolamine were absent in M 1 -/- mice. The cocaine S D attenuating effects of methoctramine were absent in M 2 -/- mice and almost absent in M 1 -/- mice. The findings indicate that the cocaine-like S D effects of muscarinic antagonists are primarily mediated through M 1 receptors, with a minor contribution of M 4 receptors. The data also support our previous findings that stimulation of M 1 receptors and M 4 receptors can each attenuate the S D effect of cocaine, and show that this can also be achieved by blocking M 2 autoreceptors, likely via increased acetylcholine release. Copyright © 2017 Elsevier B.V. All rights reserved.

Oxytocin receptor mRNA expression in rat brain: implications for behavioral integration and reproductive success.

PubMed

Ostrowski, N L

1998-11-01

The nonapeptide, oxytocin (OT), has been implicated in a wide range of physiological, behavioral and pharmacological effects related to learning and memory, parturition and lactation, maternal and sexual behavior, and the formation of social attachments. Specific G-protein linked membrane bound OT receptors mediate OTs effects. The unavailability of highly selective pharmacological ligands that discriminate the OT receptor from the highly homologous vasopressin receptors (V1a, V1b and V2 subtypes) has made it difficult to confirm specific effects of oxytocin, particularly in brain regions where OT and multiple AVP receptor subtypes may be coexpressed. Here, data on the oxytocin receptor (OTR) messenger ribonucleic acid (mRNA) localization in brain are presented in the context of a model that proposes a reproductive state-dependent role for steroid-hormone restructuring of neural circuits, and a role for oxytocin in the integration of neural transmission in pathways subserving: (1) steroid-sensitive reproductive behaviors; (2) learning; and (3) reinforcement. It is hypothesized that social attachments emerge as a consequence of a conditioned association between OT-related activity in these pathways and the eliciting stimulus.

Identification of two H3-histamine receptor subtypes

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

West, R.E. Jr.; Zweig, A.; Shih, N.Y.

The H3-histamine receptor provides feedback inhibition of histamine synthesis and release as well as inhibition of other neurotransmitter release. We have characterized this receptor by radioligand binding studies with the H3 agonist N alpha-(3H)methylhistamine ((3H)NAMHA). The results of (3H)NAMHA saturation binding and NAMHA inhibition of (3H)NAMHA binding were consistent with an apparently single class of receptors (KD = 0.37 nM, Bmax = 73 fmol/mg of protein) and competition assays with other agonists and the antagonists impromidine and dimaprit disclosed only a single class of sites. In contrast, inhibition of (3H)NAMHA binding by the specific high affinity H3 antagonist thioperamide revealedmore » two classes of sites (KiA = 5 nM, BmaxA = 30 fmol/mg of protein; KiB = 68 nM, BmaxB = 48 fmol/mg of protein). Burimamide, another antagonist that, like thioperamide, contains a thiourea group, likewise discriminated between two classes of sites. In addition to differences between some antagonist potencies for the two receptors, there is a differential guanine nucleotide sensitivity of the two. The affinity of the H3A receptor for (3H) NAMHA was reduced less than 2-fold, whereas (3H)NAMHA binding to the H3B receptor was undetectable in the presence of guanosine 5'-O-(3-thiotriphosphate). The distinction between H3A and H3B receptor subtypes, the former a high affinity and the latter a low affinity thioperamide site, draws support from published in vitro data.« less

Functional assessment of recombinant human alpha(2)-adrenoceptor subtypes with cytosensor microphysiometry.

PubMed

Pihlavisto, M; Scheinin, M

1999-12-03

We applied the Cytosensor Microphysiometry system to study the three human alpha(2)-adrenoceptor subtypes, alpha(2A), alpha(2B) and alpha(2C), expressed in Chinese hamster ovary (CHO) cells, and assessed its potential in the quantitative monitoring of agonist activity. The natural full agonist, (-)-noradrenaline, was used to define agonist efficacy. The imidazole derivative dexmedetomidine was a potent full agonist of all three receptor subtypes. The imidazolines clonidine and UK 14,304 (5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine) appeared to be partial agonists at alpha(2B)-adrenoceptors (E(max) approximately 60% of (-)-noradrenaline) but full agonists at alpha(2A)- and alpha(2C)-adrenoceptors. The responses mediated by all three alpha(2)-adrenoceptor subtypes were partly inhibited by the sodium-hydrogen (Na(+)/H(+)) exchange inhibitor, MIA (5-(N-methyl-N-isobutyl)-amiloride). The agonist responses were totally abolished by pretreatment with pertussis toxin in cells with alpha(2A)- and alpha(2C)-adrenoceptors, and partly abolished in cells with alpha(2B)-adrenoceptors. The residual signal in alpha(2B)-cells was sensitive to the intracellular Ca(2+)chelator, BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester). Cholera toxin (which acts on G(s)-proteins) had no effect on the agonist responses. The results suggest that the extracellular acidification responses mediated by all three human alpha(2)-adrenoceptor subtypes are dependent on Na(+)/H(+)exchange and G(i/o) pathways, and that alpha(2B)-adrenoceptors are capable of coupling to another, G(i/o)-independent and Ca(2+)-dependent signaling pathway.

α(2) noradrenergic receptor suppressed CaMKII signaling in spinal dorsal horn of mice with inflammatory pain.

PubMed

Wang, Xin-Tai; Lian, Xia; Xu, Ying-Ming; Suo, Zhan-Wei; Yang, Xian; Hu, Xiao-Dong

2014-02-05

Intrathecal application of α2 noradrenergic receptor agonists effectively alleviates the pathological pain induced by peripheral tissue injury. However, the spinal antinociceptive mechanisms of α2 noradrenergic receptors remain to be characterized. The present study performed immunohistochemistry and western blot to elucidate the signaling pathway initiated by α2 noradrenergic receptors in spinal dorsal horn of mice, and identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an important target for noradrenergic suppression of inflammatory pain. Our data showed that intraplantar injection of Complete Freund's Adjuvant (CFA) substantially enhanced CaMKII autophosphorylation at Threonine 286, which could be abolished by intrathecal administration of α2 noradrenergic receptor agonist clonidine. Gi protein-coupled α2 noradrenergic receptor might inhibit cAMP-dependent protein kinase (PKA) to disturb CaMKII signaling. We found that pharmacological activation of PKA in intact mice also enhanced spinal CaMKII autophosphorylation level, which was completely antagonized by clonidine. Moreover, direct PKA inhibition in CFA-injected mice mimicked the suppressive effect of α2 noradrenergic receptors on CaMKII. PKA inhibition has been shown to downregulate CaMKII by enhancing protein phosphatase activity. Consistent with this notion, spinal treatment with protein phosphatase inhibitor okadaic acid ruled out clonidine-mediated CaMKII dephosphorylation in CFA-injected mice. Through PKA/protein phosphatase/CaMKII pathway, clonidine noticeably decreased CFA-evoked phosphorylation of N-methyl-d-aspartate subtype glutamate receptor GluN1 and GluN2B subunit as well as α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid subtype glutamate receptor GluA1 subunit. These data suggested that interference with CaMKII signaling might represent an important mechanism underlying noradrenergic suppression of inflammatory pain. Copyright © 2013 Elsevier B.V. All rights reserved.

K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

PubMed

Linden, Anni-Maija; Aller, M Isabel; Leppä, Elli; Rosenberg, Per H; Wisden, William; Korpi, Esa R

2008-10-01

TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

Cerebral Artery Alpha-1 AR Subtypes: High Altitude Long-Term Acclimatization Responses

PubMed Central

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D.

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10−5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function. PMID:25393740

Cerebral artery alpha-1 AR subtypes: high altitude long-term acclimatization responses.

PubMed

Goyal, Ravi; Goyal, Dipali; Chu, Nina; Van Wickle, Jonathan; Longo, Lawrence D

2014-01-01

In response to hypoxia and other stress, the sympathetic (adrenergic) nervous system regulates arterial contractility and blood flow, partly through differential activities of the alpha1 (α1) - adrenergic receptor (AR) subtypes (α1A-, α1B-, and α1D-AR). Thus, we tested the hypothesis that with acclimatization to long-term hypoxia (LTH), contractility of middle cerebral arteries (MCA) is regulated by changes in expression and activation of the specific α1-AR subtypes. We conducted experiments in MCA from adult normoxic sheep maintained near sea level (300 m) and those exposed to LTH (110 days at 3801 m). Following acclimatization to LTH, ovine MCA showed a 20% reduction (n = 5; P<0.05) in the maximum tension achieved by 10-5 M phenylephrine (PHE). LTH-acclimatized cerebral arteries also demonstrated a statistically significant (P<0.05) inhibition of PHE-induced contractility in the presence of specific α1-AR subtype antagonists. Importantly, compared to normoxic vessels, there was significantly greater (P<0.05) α1B-AR subtype mRNA and protein levels in LTH acclimatized MCA. Also, our results demonstrate that extracellular regulated kinase 1 and 2 (ERK1/2)-mediated negative feedback regulation of PHE-induced contractility is modulated by α1B-AR subtype. Overall, in ovine MCA, LTH produces profound effects on α1-AR subtype expression and function.

High prevalence of luminal B breast cancer intrinsic subtype in Colombian women.

PubMed

Serrano-Gomez, Silvia Juliana; Sanabria-Salas, Maria Carolina; Hernández-Suarez, Gustavo; García, Oscar; Silva, Camilo; Romero, Alejandro; Mejía, Juan Carlos; Miele, Lucio; Fejerman, Laura; Zabaleta, Jovanny

2016-07-01

Breast cancer is the most frequent malignancy in women worldwide. Distinct intrinsic subtypes of breast cancer have different prognoses, and their relative prevalence varies significantly among ethnic groups. Little is known about the prevalence of breast cancer intrinsic subtypes and their association with clinicopathological data and genetic ancestry in Latin Americans. Immunohistochemistry surrogates from the 2013 St. Gallen International Expert Consensus were used to classify breast cancers in 301 patients from Colombia into intrinsic subtypes. We analyzed the distribution of subtypes by clinicopathological variables. Genetic ancestry was estimated from a panel of 80 ancestry informative markers. Luminal B breast cancer subtype was the most prevalent in our population (37.2%) followed by luminal A (26.3%), non-basal triple negative (NBTN) (11.6%), basal like (9%), human epidermal growth factor receptor 2 (HER2) enriched (8.6%) and unknown (7.3%). We found statistical significant differences in distribution between Colombian region (P = 0.007), age at diagnosis (P = 0.0139), grade (P < 0.001) and recurrence (P < 0.001) according to intrinsic subtype. Patients diagnosed with HER2-enriched, basal-like and NBTN breast cancer had the highest African ancestry. Future studies analyzing the molecular profiles of breast cancer in Colombian women will help us understand the molecular basis of this subtype distribution and compare the molecular characteristics of the different intrinsic subtypes in Colombian patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain.

PubMed

Dziedzic, Barbara; Prevot, Vincent; Lomniczi, Alejandro; Jung, Heike; Cornea, Anda; Ojeda, Sergio R

2003-02-01

Hypothalamic astroglial erbB tyrosine kinase receptors are required for the timely initiation of mammalian puberty. Ligand-dependent activation of these receptors sets in motion a glia-to-neuron signaling pathway that prompts the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development, from hypothalamic neuroendocrine neurons. The neuronal systems that may regulate this growth factor-mediated back signaling to neuroendocrine neurons have not been identified. Here we demonstrate that hypothalamic astrocytes contain metabotropic receptors of the metabotropic glutamate receptor 5 subtype and the AMPA receptor subunits glutamate receptor 2 (GluR2) and GluR3. As in excitatory synapses, these receptors are in physical association with their respective interacting/clustering proteins Homer and PICK1. In addition, they are associated with erbB-1 and erbB-4 receptors. Concomitant activation of astroglial metabotropic and AMPA receptors results in the recruitment of erbB tyrosine kinase receptors and their respective ligands to the glial cell membrane, transactivation of erbB receptors via a mechanism requiring metalloproteinase activity, and increased erbB receptor gene expression. By facilitating erbB-dependent signaling and promoting erbB receptor gene expression in astrocytes, a neuron-to-glia glutamatergic pathway may represent a basic cell-cell communication mechanism used by the neuroendocrine brain to coordinate the facilitatory transsynaptic and astroglial input to LHRH neurons during sexual development.

G protein γ (Gγ) subtype dependent targeting of GRK2 to M3 receptor by Gβγ.

PubMed

Samaradivakara, Saroopa; Kankanamge, Dinesh; Senarath, Kanishka; Ratnayake, Kasun; Karunarathne, Ajith

2018-06-11

Interactions of cytosolic G protein coupled receptor kinase 2 (GRK2) with activated G protein coupled receptors (GPCRs) induce receptor phosphorylation and desensitization. GRK2 is recruited to active M3-muscarinic receptors (M3R) with the participation of the receptor, Gαq and Gβγ. Since we have shown that signaling efficacy of Gβγ is governed by its Gγ subtype identity, the present study examined whether recruitment of GRK2 to M3R is also Gγ subtype dependent. To capture the dynamics of GRK2-recruitment concurrently with GPCR-G protein activation, we employed live cell confocal imaging and a novel assay based on Gβγ translocation. Data show that M3R activation-induced GRK2 recruitment is Gγ subtype dependent in which Gβγ dimers with low PM-affinity Gγ9 exhibited a two-fold higher GRK2-recruitment compared to high PM affinity Gγ3 expressing cells. Since 12-mammalian Gγ types exhibit a cell and tissue specific expressions and the PM-affinity of a Gγ is linked to its subtype identity, our results indicate a mechanism by which Gγ profile of a cell controls GRK2 signaling and GPCR desensitization. Copyright © 2018 Elsevier Inc. All rights reserved.

Outcomes by Tumor Subtype and Treatment Pattern in Women With Small, Node-Negative Breast Cancer: A Multi-Institutional Study

PubMed Central

Vaz-Luis, Ines; Ottesen, Rebecca A.; Hughes, Melissa E.; Mamet, Rizvan; Burstein, Harold J.; Edge, Stephen B.; Gonzalez-Angulo, Ana M.; Moy, Beverly; Rugo, Hope S.; Theriault, Richard L.; Weeks, Jane C.; Winer, Eric P.; Lin, Nancy U.

2014-01-01

Purpose Treatment decisions for patients with T1a,bN0M0 breast cancer are challenging. We studied the time trends in use of adjuvant chemotherapy and survival outcomes among these patients. Patients and Methods This was a prospective cohort study within the National Comprehensive Cancer Network Database that included 4,113 women with T1a,bN0M0 breast cancer treated between 2000 and 2009. Tumors were grouped by size (T1a, T1b), biologic subtype defined by hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status, and receipt of chemotherapy with or without trastuzumab. Results Median follow-up time was 5.5 years. Eight percent of patients with HR-positive/HER2-negative tumors were treated with chemotherapy. Fifty-two percent of those with HER2-positive or HR-negative/HER2-negative breast cancers received chemotherapy, with an increase over the last decade. Survival outcomes diverged by subtype and size, but the 5-year distant relapse-free survival (DRFS) did not exceed 10% in any subgroup. The 5-year DRFS for patients with T1a tumors untreated with chemotherapy ranged from 93% to 98% (n = 49 to 972), and for patients with T1b tumors, it ranged from 90% to 96% (n = 17 to 2,005). Patients with HR-positive/HER2-negative disease had the best DRFS estimates, and patients with HR-negative/HER2-negative tumors had the lowest. In this observational, nonrandomized cohort study, the 5-year DRFS for treated patients with T1a tumors was 100% for all subgroups (n = 12 to 33), and for patients with T1b tumors, it ranged from 94% to 96% (n = 88 to 241). Conclusion Women with T1a,b tumors have an excellent prognosis without chemotherapy. Size and tumor subtype may identify patients in whom the rate of recurrence justifies consideration of chemotherapy. These patients represent an optimal group for evaluating less toxic adjuvant regimens to maintain efficacy while minimizing short- and long-term risks. PMID:24888816

Selective increases in serotonin 5-HT1B/1D and 5-HT2A/2C binding sites in adult rat basal ganglia following lesions of serotonergic neurons.

PubMed

Compan, V; Segu, L; Buhot, M C; Daszuta, A

1998-05-18

Quantitative autoradiography was used to examine possible adaptive changes in serotonin 5-HT1B/1D and 5-HT2A/2C receptor binding sites in adult rat basal ganglia, after partial or severe lesions of serotonergic neurons produced by intraraphe injections of variable amounts of 5,7-dihydroxytryptamine. In controls, the 5-HT1B/1D sites labeled with S-CM-G[125I]TNH2 were evenly distributed in the core and the shell of the nucleus accumbens. The density of 5-HT1B/1D sites was higher in the ventral than dorsal part of the striatum and no regional differences were detected along the rostrocaudal axis of the structure. The 5-HT2A/2C sites labeled with [125I]DOI were preferentially distributed in the mediodorsal striatum and higher densities were detected in the shell than core of the nucleus accumbens. Following 5,7-dihydroxytryptamine injections, there were no changes in binding of either receptor subtype after partial lesions entailing 80-90% 5-HT depletions. After severe 5-HT depletions (over 95%), large increases in 5-HT1B/1D binding were observed in the substantia nigra (78%), but no changes took place in the globus pallidus. Increases in 5-HT1B/1D binding were also detected in the shell of the nucleus accumbens (27%). Similar sized increases in 5-HT2A/2C binding (22%) were restricted to the medial striatum. The present results suggest a preferential association between 5-HT1B/1D receptors and the striatonigral neurons containing substance P, as indicated by the striatal distribution of these receptors and their selective increases in the substantia nigra after severe 5-HT deprivation. We recently proposed a similar relationship between the 5-HT4 receptors and the striatopallidal neurons containing met-enkephalin. Moreover, the increases in 5-HT1B/1D binding in the substantia nigra and in the shell of the nucleus accumbens reinforce the view of an implication of this receptor subtype in motor functions. In contrast, the prominent increases in 5-HT2A/2C binding after severe 5-HT deprivation as restricted to the medial region of the striatum and suggest up-regulation of most probably 5-HT2C receptors in a region implicated in cognitive functions. Copyright 1998 Elsevier Science B. V.

Variations in endothelin receptor B subtype 2 (EDNRB2) coding sequences and mRNA expression levels in 4 Muscovy duck plumage colour phenotypes.

PubMed

Wu, N; Qin, H; Wang, M; Bian, Y; Dong, B; Sun, G; Zhao, W; Chang, G; Xu, Q; Chen, G

2017-04-01

1. Endothelin receptor B subtype 2 (EDNRB2) is a paralog of EDNRB, which encodes a 7-transmembrane G-protein coupled receptor. Previous studies reported that EDNRB was essential for melanoblast migration in mammals and ducks. 2. Muscovy ducks have different plumage colour phenotypes. Variations in EDNRB2 coding sequences (CDSs) and mRNA expression levels were investigated in 4 different Muscovy duck plumage colour phenotypes, including black, black mutant, silver and white head. 3. The EDNRB2 gene from Muscovy duck was cloned; it had a length of 6435 bp and encoded 437 amino acids. The coding region was screened and potential single nucleotide polymorphisms were identified. Eight mutations were obtained, including one missense variant (c.64C > T) and 7 synonymous substitutions. The substitutions were associated with plumage colour phenotypes. 4. The EDNRB2 mRNA expression levels were compared between feather pulp from black birds and black mutant birds. The results indicated that EDNRB2 transcripts in feather pulp were significantly higher in black feathers than in white feathers. 5. The results determined the variation of EDNRB2 CDS and mRNA expression in Muscovy ducks of various plumage colours.

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.

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces.

PubMed

Zhu, Shujia; Riou, Morgane; Yao, C Andrea; Carvalho, Stéphanie; Rodriguez, Pamela C; Bensaude, Olivier; Paoletti, Pierre; Ye, Shixin

2014-04-22

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.

Discovery of a new class of ionotropic glutamate receptor antagonists by the rational design of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid.

PubMed

Larsen, Ann M; Venskutonytė, Raminta; Valadés, Elena Antón; Nielsen, Birgitte; Pickering, Darryl S; Bunch, Lennart

2011-02-16

The kainic acid (KA) receptors belong to the class of glutamate (Glu) receptors in the brain and constitute a promising target for the treatment of neurological and/or psychiatric diseases such as schizophrenia, major depression, and epilepsy. Five KA subtypes have been identified and named GluK1-5. In this article, we present the discovery of (2S,3R)-3-(3-carboxyphenyl)-pyrrolidine-2-carboxylic acid (1) based on a rational design process. Target compound 1 was synthesized by a stereoselective strategy in 10 steps from commercially available starting materials. Binding affinities of 1 at native ionotropic Glu receptors were determined to be in the micromolar range (AMPA, 51 μM; KA, 22 μM; NMDA 6 μM), with the highest affinity for cloned homomeric KA receptor subtypes GluK1,3 (3.0 and 8.1 μM, respectively). Functional characterization of 1 by two electrode voltage clamp (TEVC) electrophysiology at a nondesensitizing mutant of GluK1 showed full competitive antagonistic behavior with a K(b) of 11.4 μM.

Risk Differences Between Prediabetes And Diabetes According To Breast Cancer Molecular Subtypes.

PubMed

Crispo, A; Augustin, L S A; Grimaldi, M; Nocerino, F; Giudice, A; Cavalcanti, E; Di Bonito, M; Botti, G; De Laurentiis, M; Rinaldo, M; Esposito, E; Riccardi, G; Amore, A; Libra, M; Ciliberto, G; Jenkins, D J A; Montella, M

2017-05-01

Hyperglycemia and hyperinsulinemia may play a role in breast carcinogenesis and prediabetes and diabetes have been associated with increased breast cancer (BC) risk. However, whether BC molecular subtypes may modify these associations is less clear. We therefore investigated these associations in all cases and by BC molecular subtypes among women living in Southern Italy. Cases were 557 patients with non-metastatic incident BC and controls were 592 outpatients enrolled during the same period as cases and in the same hospital for skin-related non-malignant conditions. Adjusted multivariate logistic regression models were built to assess the risks of developing BC in the presence of prediabetes or diabetes. The analyses were repeated by strata of BC molecular subtypes: Luminal A, Luminal B, HER2+, and Triple Negative (TN). Prediabetes and diabetes were significantly associated with higher BC incidence after controlling for known risk factors (OR = 1.94, 95% CI 1.32-2.87 and OR = 2.46, 95% CI 1.38-4.37, respectively). Similar results were seen in Luminal A and B while in the TN subtype only prediabetes was associated with BC (OR = 2.43, 95% CI 1.11-5.32). Among HER2+ patients, only diabetes was significantly associated with BC risk (OR = 3.04, 95% CI 1.24-7.47). Furthermore, when postmenopausal HER2+ was split into hormone receptor positive versus negative, the association with diabetes remained significant only in the former (OR = 5.13, 95% CI 1.53-17.22). These results suggest that prediabetes and diabetes are strongly associated with BC incidence and that these metabolic conditions may be more relevant in the presence of breast cancer molecular subtypes with positive hormone receptors. J. Cell. Physiol. 232: 1144-1150, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Negative Allosteric Modulators Selective for The NR2B Subtype of The NMDA Receptor Impair Cognition in Multiple Domains

PubMed Central

Weed, Michael R; Bookbinder, Mark; Polino, Joseph; Keavy, Deborah; Cardinal, Rudolf N; Simmermacher-Mayer, Jean; Cometa, Fu-ni L; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E; Bristow, Linda J

2016-01-01

Antidepressant activity of N-methyl-D-aspartate (NMDA) receptor antagonists and negative allosteric modulators (NAMs) has led to increased investigation of their behavioral pharmacology. NMDA antagonists, such as ketamine, impair cognition in multiple species and in multiple cognitive domains. However, studies with NR2B subtype-selective NAMs have reported mixed results in rodents including increased impulsivity, no effect on cognition, impairment or even improvement of some cognitive tasks. To date, the effects of NR2B-selective NAMs on cognitive tests have not been reported in nonhuman primates. The current study evaluated two selective NR2B NAMs, CP101,606 and BMT-108908, along with the nonselective NMDA antagonists, ketamine and AZD6765, in the nonhuman primate Cambridge Neuropsychological Test Automated Battery (CANTAB) list-based delayed match to sample (list-DMS) task. Ketamine and the two NMDA NR2B NAMs produced selective impairments in memory in the list-DMS task. AZD6765 impaired performance in a non-specific manner. In a separate cohort, CP101,606 impaired performance of the nonhuman primate CANTAB visuo-spatial Paired Associates Learning (vsPAL) task with a selective impairment at more difficult conditions. The results of these studies clearly show that systemic administration of a selective NR2B NAM can cause transient cognitive impairment in multiple cognitive domains. PMID:26105137

Evidence for Alpha Receptors in the Human Ureter

NASA Astrophysics Data System (ADS)

Madeb, Ralph; Knopf, Joy; Golijanin, Dragan; Bourne, Patricia; Erturk, Erdal

2007-04-01

An immunohistochemical and western blot expression analysis of human ureters was performed in order to characterize the alpha-1-adrenergic receptor distribution along the length of the human ureteral wall. Mapping the distribution will assist in understanding the potential role alpha -1-adrenergic receptors and their subtype density might have in the pathophysiology of ureteral colic and stone passage. Patients diagnosed with renal cancer or bladder cancer undergoing nephrectomy, nephroureterectomy, or cystectomy had ureteral specimens taken from the proximal, mid, distal and tunneled ureter. Tissues were processed for fresh frozen examination and fixed in formalin. None of the ureteral specimens were involved with cancer. Serial histologic sections and immunohistochemical studies were performed using antibodies specific for alpha-1-adrenergic receptor subtypes (alpha 1a, alpha 1b, alpha 1d). The sections were examined under a light microscope and scored as positive or negative. In order to validate and quantify the alpha receptor subtypes along the human ureter. Western blotting techniques were applied. Human ureter stained positively for alpha -1-adrenergic receptors. Immunostaining appeared red, with intense reaction in the smooth muscle of the ureter and endothelium of the neighboring blood vessels. There was differential expression between all the receptors with the highest staining for alpha-1D subtype. The highest protein expression for all three subtypes was in the renal pelvis and decreased with advancement along the ureter to the distal ureter. At the distal ureter, there was marked increase in expression as one progressed towards the ureteral orifice. The same pattern of protein expression was exhibited for all three alpha -1-adrenergic receptor subtypes. We provide preliminary evidence for the ability to detect and quantify the alpha-1-receptor subtypes along the human ureter which to the best of our knowledge has never been done with immunohistochemistry and molecular techniques. These findings may lend support to the preliminary studies of the effectiveness of alpha-receptor blockade on ureteral colic and stone passage.

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.

GABAA receptor: Positive and negative allosteric modulators.

PubMed

Olsen, Richard W

2018-01-31

gamma-Aminobutyric acid (GABA)-mediated inhibitory neurotransmission and the gene products involved were discovered during the mid-twentieth century. Historically, myriad existing nervous system drugs act as positive and negative allosteric modulators of these proteins, making GABA a major component of modern neuropharmacology, and suggesting that many potential drugs will be found that share these targets. Although some of these drugs act on proteins involved in synthesis, degradation, and membrane transport of GABA, the GABA receptors Type A (GABA A R) and Type B (GABA B R) are the targets of the great majority of GABAergic drugs. This discovery is due in no small part to Professor Norman Bowery. Whereas the topic of GABA B R is appropriately emphasized in this special issue, Norman Bowery also made many insights into GABA A R pharmacology, the topic of this article. GABA A R are members of the ligand-gated ion channel receptor superfamily, a chloride channel family of a dozen or more heteropentameric subtypes containing 19 possible different subunits. These subtypes show different brain regional and subcellular localization, age-dependent expression, and potential for plastic changes with experience including drug exposure. Not only are GABA A R the targets of agonist depressants and antagonist convulsants, but most GABA A R drugs act at other (allosteric) binding sites on the GABA A R proteins. Some anxiolytic and sedative drugs, like benzodiazepine and related drugs, act on GABA A R subtype-dependent extracellular domain sites. General anesthetics including alcohols and neurosteroids act at GABA A R subunit-interface trans-membrane sites. Ethanol at high anesthetic doses acts on GABA A R subtype-dependent trans-membrane domain sites. Ethanol at low intoxicating doses acts at GABA A R subtype-dependent extracellular domain sites. Thus GABA A R subtypes possess pharmacologically specific receptor binding sites for a large group of different chemical classes of clinically important neuropharmacological agents. Copyright © 2018 Elsevier Ltd. All rights reserved.

NK-2 is the predominant tachykinin receptor subtype in the swine ureter.

PubMed

Jerde, T J; Saban, R; Bjorling, D E; Nakada, S Y

1999-02-01

To determine which of the known tachykinin receptor subtypes is predominant in the swine ureter. Ureters from adult pigs were harvested, cut into longitudinal strips and placed in 10 mL tissue baths containing Krebs buffer, under 4 g of initial tension. The magnitude and frequency of contractions were recorded. Tissues were incubated with 1 micromol/L solutions of peptidase inhibitors (phosphoramidon and captopril) for 1 h to inhibit degradation of peptides and treated with either CP 96,345 (NK-1 receptor antagonist), SR 48,968 (NK-2 receptor antagonist) or saline (control). Concentration-response curves to the tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB) were determined. Ureteric segments showed a concentration-dependent response to all tachykinins; NKA stimulated increased contractions at a lower concentration than either SP or NKB (P<0.05). This was reflected by the difference in the effective concentration required to obtain half the maximal response (EC50 ) for each of the peptides. The mean (sd) EC50 values were (micromol/L): NKA, 0.2 (0.02); SP, 3.5 (0.7); and NKB, 4.5 (1.7). In addition, the selective NK-2 antagonist (SR 48,968) significantly reduced contractile responses to all peptides, as indicated by a 10-fold rightward shift of the concentration-response curves (P<0. 05), whereas the NK-1 antagonist (CP 96,345) had no significant effect. These results indicate that NK-2 is the predominant tachykinin receptor subtype responsible for contraction of ureteric smooth muscle. The use of mediators which act on NK-2 receptors may have clinical applications for the treatment of ureteric disease.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M 1 -, M 2 - and M 4 -subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo . Our previous results show that M 1 , M 2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M 1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M 2 receptor is largely independent of both M 1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination.

Presynaptic Muscarinic Acetylcholine Receptors and TrkB Receptor Cooperate in the Elimination of Redundant Motor Nerve Terminals during Development

PubMed Central

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria A.; Cilleros, Victor; Tomàs, Josep

2017-01-01

The development of the nervous system involves the overproduction of synapses but connectivity is refined by Hebbian activity-dependent axonal competition. The newborn skeletal muscle fibers are polyinnervated but, at the end of the competition process, some days later, become innervated by a single axon. We used quantitative confocal imaging of the autofluorescent axons from transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice to investigate the possible cooperation of the muscarinic autoreceptors (mAChR, M1-, M2- and M4-subtypes) and the tyrosine kinase B (TrkB) receptor in the control of axonal elimination after the mice Levator auris longus (LAL) muscle had been exposed to several selective antagonist of the corresponding receptor pathways in vivo. Our previous results show that M1, M2 and TrkB signaling individually increase axonal loss rate around P9. Here we show that although the M1 and TrkB receptors cooperate and add their respective individual effects to increase axonal elimination rate even more, the effect of the M2 receptor is largely independent of both M1 and TrkB receptors. Thus both, cooperative and non-cooperative signaling mechanisms contribute to developmental synapse elimination. PMID:28228723

Effects of selective type I and II adrenal steroid agonists on immune cell distribution.

PubMed

Miller, A H; Spencer, R L; hassett, J; Kim, C; Rhee, R; Ciurea, D; Dhabhar, F; McEwen, B; Stein, M

1994-11-01

Adrenal steroids exert their effects through two distinct adrenal steroid receptor subtypes; the high affinity type I, or mineralocorticoid, receptor and the lower affinity type II, or glucocorticoid, receptor. Adrenal steroids have well known effects on immune cell distribution, and although both type I and II receptors are expressed in immune cells and tissues, few data exist on the relative effects mediated through these two receptor subtypes. Accordingly, we administered selective type I and II adrenal steroid receptor agonists to young adult male Sprague-Dawley rats for 7 days and then measured immune cell distribution in the peripheral blood and spleen. Results were compared with those of similar studies using the naturally occurring glucocorticoid of the rat, corticosterone, which binds both type I and II receptors. The majority of the well characterized effects of adrenal steroids on peripheral blood immune cells (increased neutrophils and decreased lymphocytes and monocytes) were reproduced by the type II receptor agonist, RU28362. RU28362 decreased the numbers of all lymphocyte subsets [T-cells, B-cells, and natural killer (NK) cells] to very low absolute levels. The largest relative decrease (i.e. in percentage) was seen in B-cells, whereas NK cells exhibited the least relative decrease and actually showed a 2-fold increase in relative percentage during RU28362 treatment. Similar to RU28362, the type I receptor agonist, aldosterone, significantly reduced the number of lymphocytes and monocytes. In contrast to RU28362, however, aldosterone significantly decreased the number of neutrophils. Moreover, aldosterone decreased the number of T-helper cells and NK cells, while having no effect on the number of B-cells or T-suppressor/cytotoxic cells. Corticosterone at physiologically relevant concentrations had potent effects on immune cell distribution, which were indistinguishable from those of the type II receptor agonist, RU28362. Taken together, these results indicate that effects of adrenal steroids on immune cell distribution are dependent on the receptor subtype involved as well as the specific cell type targeted. These factors allow for varied and complex effects of adrenal steroids on the immune system under physiological conditions.

Flexible synthesis of poison-frog alkaloids of the 5,8-disubstituted indolizidine-class. II: Synthesis of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and an epimer of 193E and pharmacological effects at neuronal nicotinic acetylcholine receptors.

PubMed

Kobayashi, Soushi; Toyooka, Naoki; Zhou, Dejun; Tsuneki, Hiroshi; Wada, Tsutomu; Sasaoka, Toshiyasu; Sakai, Hideki; Nemoto, Hideo; Garraffo, H Martin; Spande, Thomas F; Daly, John W

2007-01-01

The 5,8-disubstituted indolizidines constitute the largest class of poison-frog alkaloids. Some alkaloids have been shown to act as noncompetitive blockers at nicotinic acetylcholine receptors but the proposed structures and the biological activities of most of the 5,8-disubstituted indolizidines have not been determined because of limited supplies of the natural products. We have therefore conducted experiments to confirm proposed structures and determine biological activities using synthetic compounds. Recently, we reported that one of this class of alkaloids, (-)-235B', acts as a noncompetitive antagonist for α4β2 nicotinic receptors, and its sensitivity is comparable to that of the classical competitive antagonist for this receptor, dihydro-β-erythroidine. The enantioselective syntheses of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and what proved to be an epimer of natural 193E, starting from common chiral lactams have been achieved. When we performed electrophysiological recordings to examine the effects of the synthetic alkaloids on two major subtypes of nicotinic receptors (α4β2 and α7) expressed in Xenopus laevis oocytes, (-)-231C effectively blocked α4β2 receptor responses (IC(50 )value, 1.5 μM) with a 7.0-fold higher potency than for blockade of α7 receptor responses. In contrast, synthetic (-)-221I and (-)-epi-193E were more potent in blocking α7 receptor responses (IC(50 )value, 4.4 μM and 9.1 μM, respectively) than α4β2 receptor responses (5.3-fold and 2.0-fold, respectively). We achieved the total synthesis of (-)-209B, (-)-231C, (-)-233D, (-)-235B", (-)-221I, and an epimer of 193E starting from common chiral lactams, and the absolute stereochemistry of natural (-)-233D was determined. Furthermore, the relative stereochemistry of (-)-231C and (-)-221I was also determined. The present asymmetric synthesis of the proposed structure for 193E revealed that the C-8 configuration of natural 193E should be revised. The selectivity for α4β2 and α7 nicotinic receptors differed markedly for the 5,8-disubstituted indolizidines tested, and thus it appears that the nature of the side chains in these indolizidines is crucial with regard to subtype-selectivity.

Development of a human vasopressin V1a-receptor antagonist from an evolutionary-related insect neuropeptide

NASA Astrophysics Data System (ADS)

di Giglio, Maria Giulia; Muttenthaler, Markus; Harpsøe, Kasper; Liutkeviciute, Zita; Keov, Peter; Eder, Thomas; Rattei, Thomas; Arrowsmith, Sarah; Wray, Susan; Marek, Ales; Elbert, Tomas; Alewood, Paul F.; Gloriam, David E.; Gruber, Christian W.

2017-02-01

Characterisation of G protein-coupled receptors (GPCR) relies on the availability of a toolbox of ligands that selectively modulate different functional states of the receptors. To uncover such molecules, we explored a unique strategy for ligand discovery that takes advantage of the evolutionary conservation of the 600-million-year-old oxytocin/vasopressin signalling system. We isolated the insect oxytocin/vasopressin orthologue inotocin from the black garden ant (Lasius niger), identified and cloned its cognate receptor and determined its pharmacological properties on the insect and human oxytocin/vasopressin receptors. Subsequently, we identified a functional dichotomy: inotocin activated the insect inotocin and the human vasopressin V1b receptors, but inhibited the human V1aR. Replacement of Arg8 of inotocin by D-Arg8 led to a potent, stable and competitive V1aR-antagonist ([D-Arg8]-inotocin) with a 3,000-fold binding selectivity for the human V1aR over the other three subtypes, OTR, V1bR and V2R. The Arg8/D-Arg8 ligand-pair was further investigated to gain novel insights into the oxytocin/vasopressin peptide-receptor interaction, which led to the identification of key residues of the receptors that are important for ligand functionality and selectivity. These observations could play an important role for development of oxytocin/vasopressin receptor modulators that would enable clear distinction of the physiological and pathological responses of the individual receptor subtypes.

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.

Molecular basis for subtype-specificity and high-affinity zinc inhibition in the GluN1-GluN2A NMDA receptor amino terminal domain

PubMed Central

Romero-Hernandez, Annabel; Simorowski, Noriko; Karakas, Erkan

2016-01-01

Summary Zinc is vastly present in the mammalian brain and controls functions of various cell surface receptors to regulate neurotransmission. A distinctive characteristic of N-methyl-D-aspartate (NMDA) receptors containing a GluN2A subunit is that their ion channel activity is allosterically inhibited by a nano-molar concentration of zinc that binds to an extracellular domain called an amino terminal domain (ATD). Despite physiological importance, the molecular mechanism underlying the high-affinity zinc inhibition has been incomplete due to lack of a GluN2A ATD structure. Here we show the first crystal structures of the heterodimeric GluN1-GluN2A ATD, which provide the complete map of the high-affinity zinc binding site and reveals distinctive features from the ATD of the GluN1-GluN2B subtype. Perturbation of hydrogen bond networks at the hinge of the GluN2A bi-lobe structure affects both zinc inhibition and open probability supporting the general model where the bi-lobe motion in ATD regulates the channel activity in NMDA receptors. PMID:27916457

Bidirectional modulation of visual plasticity by cholinergic receptor subtypes in the frog optic tectum

PubMed Central

Yu, Chuan-Jiang; Butt, Christopher M.; Debski, Elizabeth A.

2008-01-01

Cholinergic input to the optic tectum is necessary for visual map maintenance. To understand why, we examined the effects of activation of the different cholinergic receptor subtypes in tectal brain slices and determined whether the retinotectal map was affected by manipulations of their activity in vivo. Both α-bungarotoxin sensitive and insensitive nicotinic receptor agonists increased spontaneous postsynaptic currents (sPSCs) in a subpopulation of patch-clamped tectal cells; application of subtype selective receptor antagonists reduced nicotine-induced increases in sPSCs. Activation of α-bungarotoxin insensitive nicotinic receptors also induced substantial inward current in some cells. Muscarinic receptor mediated outward current responses were blocked by the M2-like muscarinic receptor antagonists himbacine or AF-DX 384 and mimicked by application of the M2-like agonist oxotremorine. A less frequently observed muscarinic response involving a change in sPSC frequency appeared to be mediated by M1-like muscarinic receptors. In separate experiments, pharmacological manipulation of cholinergic receptor subtype activation led to changes in the activity-dependent visual map created in the tectum by retinal ganglion cell terminals. Chronic exposure of the tectum to either α-bungarotoxin insensitive, α-bungarotoxin sensitive or M1-like receptor antagonists resulted in map disruption. However, treatment with the M2-like receptor antagonist, AF-DX 384, compressed the map. We conclude that nicotinic or M1-like muscarinic receptors control input to tectal cells while α-bungarotoxin insensitive nicotinic receptors and M2-like muscarinic receptors change tectal cell responses to that input. Blockade of the different cholinergic receptor subtypes can have opposing effects on map topography that are consistent with expected effects on tectal cell activity levels. PMID:12670313

Mapping the central effects of (±)-ketamine and traxoprodil using pharmacological magnetic resonance imaging in awake rats.

PubMed

Tang, Haiying; Kukral, Daniel; Li, Yu-Wen; Fronheiser, Matthew; Malone, Harold; Pena, Adrienne; Pieschl, Rick; Sidik, Kurex; Tobon, Gabriel; Chow, Patrick L; Bristow, Linda J; Hayes, Wendy; Luo, Feng

2018-02-01

Major depressive disorder is a leading cause of disability globally. Improvements in the efficacy of antidepressant therapy are needed as a high proportion (>40%) of individuals with major depressive disorder fail to respond adequately to current treatments. The non-selective N-methyl-D-aspartate receptor channel blocker, (±)-ketamine, has been reported to produce a rapid and long-lasting antidepressant response in treatment-resistant major depressive disorder patients, which provides a unique opportunity for investigation of mechanisms that mediate its therapeutic effect. Efforts have also focused on the development of selective N-methyl-D-aspartate receptor subtype 2B antagonists which may retain antidepressant activity but have lower potential for dissociative/psychotomimetic effects. In the present study, we examined the central nervous system effects of acute, intravenous administration of (±)-ketamine or the N-methyl-D-aspartate receptor subtype 2B antagonist, traxoprodil, in awake rats using pharmacological magnetic resonance imaging. The study contained five treatment groups: vehicle, 3 mg/kg (±)-ketamine, and three doses of traxoprodil (0.3 mg/kg, 5 mg/kg, and 15 mg/kg). Non-linear model fitting was performed on the temporal hemodynamic pharmacological magnetic resonance imaging data to generate brain activation maps as well as regional responses based on blood oxygen level dependent signal changes for group analysis. Traxoprodil at 5 mg/kg and 15 mg/kg produced a dose-dependent pharmacological magnetic resonance imaging signal in rat forebrain regions with both doses achieving >80% N-methyl-D-aspartate receptor subtype 2B occupancy determined by ex vivo [ 3 H]Ro 25-6981 binding. The middle dose of traxoprodil (5 mg/kg) generated region-specific activations in medial prefrontal cortex, ventral orbital cortex, and anterior cingulate cortex whereas the high dose (15 mg/kg) produced a widespread pharmacological magnetic resonance imaging response in both cortical and subcortical brain regions which was similar to that produced by (±)-ketamine (3 mg/kg, intravenous).

Important roles of P2Y receptors in the inflammation and cancer of digestive system.

PubMed

Wan, Han-Xing; Hu, Jian-Hong; Xie, Rei; Yang, Shi-Ming; Dong, Hui

2016-05-10

Purinergic signaling is important for many biological processes in humans. Purinoceptors P2Y are widely distributed in human digestive system and different subtypes of P2Y receptors mediate different physiological functions from metabolism, proliferation, differentiation to apoptosis etc. The P2Y receptors are essential in many gastrointestinal functions and also involve in the occurrence of some digestive diseases. Since different subtypes of P2Y receptors are present on the same cell of digestive organs, varying subtypes of P2Y receptors may have opposite or synergetic functions on the same cell. Recently, growing lines of evidence strongly suggest the involvement of P2Y receptors in the pathogenesis of several digestive diseases. In this review, we will focus on their important roles in the development of digestive inflammation and cancer. We anticipate that as the special subtypes of P2Y receptors are studied in depth, specific modulators for them will have good potentials to become promising new drugs to treat human digestive diseases in the near future.

Intrinsic subtypes from PAM50 gene expression assay in a population-based breast cancer cohort: differences by age, race, and tumor characteristics.

PubMed

Sweeney, Carol; Bernard, Philip S; Factor, Rachel E; Kwan, Marilyn L; Habel, Laurel A; Quesenberry, Charles P; Shakespear, Kaylynn; Weltzien, Erin K; Stijleman, Inge J; Davis, Carole A; Ebbert, Mark T W; Castillo, Adrienne; Kushi, Lawrence H; Caan, Bette J

2014-05-01

Data are lacking to describe gene expression-based breast cancer intrinsic subtype patterns for population-based patient groups. We studied a diverse cohort of women with breast cancer from the Life After Cancer Epidemiology and Pathways studies. RNA was extracted from 1 mm punches from fixed tumor tissue. Quantitative reverse-transcriptase PCR was conducted for the 50 genes that comprise the PAM50 intrinsic subtype classifier. In a subcohort of 1,319 women, the overall subtype distribution based on PAM50 was 53.1% luminal A, 20.5% luminal B, 13.0% HER2-enriched, 9.8% basal-like, and 3.6% normal-like. Among low-risk endocrine-positive tumors (i.e., estrogen and progesterone receptor positive by immunohistochemistry, HER2 negative, and low histologic grade), only 76.5% were categorized as luminal A by PAM50. Continuous-scale luminal A, luminal B, HER2-enriched, and normal-like scores from PAM50 were mutually positively correlated. Basal-like score was inversely correlated with other subtypes. The proportion with non-luminal A subtype decreased with older age at diagnosis, P Trend < 0.0001. Compared with non-Hispanic Whites, African American women were more likely to have basal-like tumors, age-adjusted OR = 4.4 [95% confidence intervals (CI), 2.3-8.4], whereas Asian and Pacific Islander women had reduced odds of basal-like subtype, OR = 0.5 (95% CI, 0.3-0.9). Our data indicate that over 50% of breast cancers treated in the community have luminal A subtype. Gene expression-based classification shifted some tumors categorized as low risk by surrogate clinicopathologic criteria to higher-risk subtypes. Subtyping in a population-based cohort revealed distinct profiles by age and race. ©2014 AACR.

Actions of subtype-specific purinergic ligands on rat spiral ganglion neurons.

PubMed

Ito, Ken; Iwasaki, Shinichi; Kondo, Kenji; Dulon, Didier; Kaga, Kimitaka

2004-08-01

In a previous study we showed that, in rat spiral ganglion neurons (SGNs), the adenosine 5'-triphosphate (ATP)-evoked currents were a combination of the activation of ionotropic receptors (the first fast current) and the activation of metabotropic receptors which secondarily opened non-selective cation channels. These two conductances imply the involvement of different receptor subtypes. In the present study, we tested three subtype-specific purinergic ligands: alpha,beta-methylene ATP (a;pha,beta-meATP) for P2X receptors, uridine 5'-triphosphate (UTP) for P2Y receptors and 2'-3'-O-(4-benzoylbenzoyl) ATP (Bz-ATP) for P2Z (P2X(7)) receptors. Application of 100 microM alpha,beta-meATP did not trigger any significant change in membrane conductance, while the SGNs were responsive to ATP. Pressure application of UTP (100 microM, 1 s) evoked an inward current averaging 344+/-169 pA at a holding potential of -50 mV. The conductance developed after a latency averaging 1.5+/-0.6 s, took 4-6 s to peak and reversed slowly within 15-30 s. The current-voltage curve reversed near 0 mV, suggesting a non-selective cation conductance, like the second component of the ATP conductance. Bz-ATP evoked an inward current which developed without latency, was sustained during ligand application and was rapidly inactivated at the end of application: the same characteristics as the first component of the ATP-evoked current. The Bz-ATP conductance reversed around -10 mV, indicating also a non-selective cation conductance. These results suggest that, in SGNs, ATP acts via two different receptor subtypes, ionotropic P2Z receptors and metabotropic P2Y receptors, and that these two receptor subtypes can assume different physiological roles.

BCR-ABL1-like acute lymphoblastic leukaemia: From bench to bedside.

PubMed

Boer, Judith M; den Boer, Monique L

2017-09-01

Acute lymphoblastic leukaemia (ALL) occurs in approximately 1:1500 children and is less frequently found in adults. The most common immunophenotype of ALL is the B cell lineage and within B cell precursor ALL, specific genetic aberrations define subtypes with distinct biological and clinical characteristics. With more advanced genetic analysis methods such as whole genome and transcriptome sequencing, novel genetic subtypes have recently been discovered. One novel class of genetic aberrations comprises tyrosine kinase-activating lesions, including translocations and rearrangements of tyrosine kinase and cytokine receptor genes. These newly discovered genetic aberrations are harder to detect by standard diagnostic methods such as karyotyping, fluorescent in situ hybridisation (FISH) or polymerase chain reaction (PCR) because they are diverse and often cryptic. These lesions involve one of several tyrosine kinase genes (among others, v-abl Abelson murine leukaemia viral oncogene homologue 1 (ABL1), v-abl Abelson murine leukaemia viral oncogene homologue 2 (ABL2), platelet-derived growth factor receptor beta polypeptide (PDGFRB)), each of which can be fused to up to 15 partner genes. Together, they compose 2-3% of B cell precursor ALL (BCP-ALL), which is similar in size to the well-known fusion gene BCR-ABL1 subtype. These so-called BCR-ABL1-like fusions are mutually exclusive with the sentinel translocations in BCP-ALL (BCR-ABL1, ETV6-RUNX1, TCF3-PBX1, and KMT2A (MLL) rearrangements) and have the promising prospect to be sensitive to tyrosine kinase inhibitors similar to BCR-ABL1. In this review, we discuss the types of tyrosine kinase-activating lesions discovered, and the preclinical and clinical evidence for the use of tyrosine kinase inhibitors in the treatment of this novel subtype of ALL. Copyright © 2017 Elsevier Ltd. All rights reserved.

Tonic B-cell receptor signaling in diffuse large B-cell lymphoma.

PubMed

Havranek, Ondrej; Xu, Jingda; Köhrer, Stefan; Wang, Zhiqiang; Becker, Lisa; Comer, Justin M; Henderson, Jared; Ma, Wencai; Man Chun Ma, John; Westin, Jason R; Ghosh, Dipanjan; Shinners, Nicholas; Sun, Luhong; Yi, Allen F; Karri, Anusha R; Burger, Jan A; Zal, Tomasz; Davis, R Eric

2017-08-24

We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL. © 2017 by The American Society of Hematology.

Characterization of kappa 1 and kappa 2 opioid binding sites in frog (Rana esculenta) brain membrane preparation

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

Benyhe, S.; Varga, E.; Hepp, J.

1990-09-01

The distribution and properties of frog brain kappa-opioid receptor subtypes differ not only from those of the guinea pig brain, but also from that of the rat brain. In guinea pig cerebellum the kappa 1 is the dominant receptor subtype, frog brain contains mainly the kappa 2 subtype, and the distribution of the rat brain subtypes is intermediate between the two others. In competition experiments it has been established that ethylketocyclazocine and N-cyclopropylmethyl-norazidomorphine, which are nonselective kappa-ligands, have relatively high affinities to frog brain membranes. The kappa 2 ligands (Met5)enkephalin-Arg6-Phe7 and etorphine also show high affinities to the frog brain.more » Kappa 1 binding sites measured in the presence of 5 microM/D-Ala2-Leu5/enkephalin represent 25-30% of (3H)ethylketocyclazocine binding in frog brain membranes. The kappa 2 subtype in frog brain resembles more to the mu subtype than the delta subtype of opioid receptors, but it differs from the mu subtype in displaying low affinity toward beta-endorphin and /D-Ala2-(Me)Phe4-Gly5-ol/enkephalin (DAGO). From our data it is evident that the opioid receptor subtypes are already present in the amphibian brain but the differences among them are less pronounced than in mammalian brain.« less

Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

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

Yang, Hua; Chen, Li-Mei; Carney, Paul J.

2012-02-21

Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avianmore » receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.« less

Chemosensitivity and Endocrine Sensitivity in Clinical Luminal Breast Cancer Patients in the Prospective Neoadjuvant Breast Registry Symphony Trial (NBRST) Predicted by Molecular Subtyping.

PubMed

Whitworth, Pat; Beitsch, Peter; Mislowsky, Angela; Pellicane, James V; Nash, Charles; Murray, Mary; Lee, Laura A; Dul, Carrie L; Rotkis, Michael; Baron, Paul; Stork-Sloots, Lisette; de Snoo, Femke A; Beatty, Jennifer

2017-03-01

Hormone receptor-positive (HR+) tumors have heterogeneous biology and present a challenge for determining optimal treatment. In the Neoadjuvant Breast Registry Symphony Trial (NBRST) patients were classified according to MammaPrint/BluePrint subtyping to provide insight into the response to neoadjuvant endocrine therapy (NET) or neoadjuvant chemotherapy (NCT). The purpose of this predefined substudy was to compare MammaPrint/BluePrint with conventional 'clinical' immunohistochemistry/fluorescence in situ hybridization (IHC/FISH) subtyping in 'clinical luminal' [HR+/human epidermal growth factor receptor 2-negative (HER2-)] breast cancer patients to predict treatment sensitivity. NBRST IHC/FISH HR+/HER2- breast cancer patients (n = 474) were classified into four molecular subgroups by MammaPrint/BluePrint subtyping: Luminal A, Luminal B, HER2, and Basal type. Pathological complete response (pCR) rates were compared with conventional IHC/FISH subtype. The overall pCR rate for 'clinical luminal' patients to NCT was 11 %; however, 87 of these 474 patients were reclassified as Basal type by BluePrint, with a high pCR rate of 32 %. The MammaPrint index was highly associated with the likelihood of pCR (p < 0.001). Fifty-three patients with BluePrint Luminal tumors received NET with an aromatase inhibitor and 36 (68 %) had a clinical response. With BluePrint subtyping, 18 % of clinical 'luminal' patients are classified in a different subgroup, compared with conventional assessment, and these patients have a significantly higher response rate to NCT compared with BluePrint Luminal patients. MammaPrint/BluePrint subtyping can help allocate effective treatment to appropriate patients. In addition, accurate identification of subtype biology is important in the interpretation of neoadjuvant treatment response since lack of pCR in luminal patients does not portend the worse prognosis associated with residual disease in Basal and HER2 subtypes.

Allosteric modulation of alpha4beta2 nicotinic acetylcholine receptors by HEPES.

PubMed

Weltzin, Maegan M; Huang, Yanzhou; Schulte, Marvin K

2014-06-05

A number of new positive allosteric modulators (PAMs) have been reported that enhance responses of neuronal alpha7 and alpha4beta2 nicotinic acetylcholine receptor subtypes to orthosteric ligands. PAMs represent promising new leads for the development of therapeutic agents for disorders involving alterations in nicotinic neurotransmission including Autism, Alzheimer's and Parkinson's disease. During our recent studies of alpha4beta2 PAMs, we identified a novel effect of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The effects of HEPES were evaluated in a phosphate buffered recording solution using two-electrode voltage clamp techniques and alpha4beta2 and alpha7 nicotinic acetylcholine receptor subtypes expressed in Xenopus laevis oocytes. Acetylcholine induced responses of high-sensitivity alpha4beta2 receptors were potentiated 190% by co-exposure to HEPES. Responses were inhibited at higher concentrations (bell-shaped concentration/response curve). Coincidentally, at concentrations of HEPES typically used in oocyte recording (5-10mM), the potentiating effects of HEPES are matched by its inhibitory effects, thus producing no net effect. Mutagenesis results suggest HEPES potentiates the high-sensitivity stoichiometry of the alpha4beta2 receptors through action at the beta2+/beta2- interface and is dependent on residue beta2D218. HEPES did not potentiate low-sensitivity alpha4beta2 receptors and did not produce any observable effect on acetylcholine induced responses on alpha7 nicotinic acetylcholine receptors. Copyright © 2012 Elsevier B.V. All rights reserved.

Purinergic Signalling: Therapeutic Developments

PubMed Central

Burnstock, Geoffrey

2017-01-01

Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990’s when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine) receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A2A receptor antagonists are promising for the treatment of Parkinson’s disease. Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer. PMID:28993732

Renal atrial natriuretic factor receptors in hamster cardiomyopathy.

PubMed

Mukaddam-Daher, S; Jankowski, M; Dam, T V; Quillen, E W; Gutkowska, J

1995-12-01

Hamsters with cardiomyopathy (CMO), an experimental model of congestive heart failure, display stimulated renin-angiotensin-aldosterone and enhanced sympathetic nervous activity, all factors that lead to sodium retention, volume expansion and subsequent elevation of plasma atrial natriuretic factor (ANF) by the cardiac atria. However, sodium and water retention persist in CMO, indicating hyporesponsiveness to endogenous ANF. These studies were undertaken to fully characterize renal ANF receptor subtypes in normal hamsters and to evaluate whether alterations in renal ANF receptors may contribute to renal resistance to ANF in cardiomyopathy. Transcripts of the guanylyl cyclase-A (GC-A) and guanylyl cyclase B (GC-B) receptors were detected by quantitative polymerase chain reaction (PCR) in renal cortex, and outer and inner medullas. Compared to normal controls, the cardiomyopathic hamster's GC-A mRNA was similar in cortex but significantly increased in outer and inner medulla. Levels of GC-B mRNA were not altered by the disease. On the other hand, competitive binding studies, autoradiography, and affinity cross-linking demonstrated the absence of functional GC-B receptors in the kidney glomeruli and inner medulla. Also, C-type natriuretic peptide (CNP), the natural ligand for the GC-B receptors, failed to stimulate glomerular production of its second messenger cGMP. In CMO, sodium and water excretion were significantly reduced despite elevated plasma ANF (50.5 +/- 11.1 vs. 309.4 +/- 32.6 pg/ml, P < 0.001). Competitive binding studies of renal glomerular ANF receptors revealed no change in total receptor density, Bmax (369.6 +/- 27.4 vs. 282.8 +/- 26.2 fmol/mg protein), nor in dissociation constant, Kd (647.4 +/- 79.4 vs. 648.5 +/- 22.9 pM). Also, ANF-C receptor density (254.3 +/- 24.8 vs. 233.8 +/- 23.5 fmol/mg protein), nor affinity were affected by heart failure. Inner medullary receptors were exclusively of the GC-A subtype with Bmax (153.2 +/- 26.4 vs. 134.5 +/- 21.2 fmol/mg protein) and Kd (395.7 +/- 148.0 vs. 285.8 +/- 45.0 pM) not altered by cardiomyopathy. The increase in ANF-stimulated glomerular cGMP production was similar in normal and CMO hamsters (94- vs. 75-fold). These results demonstrate that renal ANF receptors do not contribute to the attenuated renal responses to ANF in hamster cardiomyopathy.

The serologic decoy receptor 3 (DcR3) levels are associated with slower disease progression in HIV-1/AIDS patients.

PubMed

Lin, Yu-Ting; Yen, Chia-Hung; Chen, Heng-Li; Liao, Yi-Jen; Lin, I-Feng; Chen, Marcelo; Lan, Yu-Ching; Chuang, Shao-Yuan; Hsieh, Shie-Liang; Chen, Yi-Ming Arthur

2015-06-01

The decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor (TNFR) super-family. It counteracts the biological effects of Fas ligands and inhibits apoptosis. The goals of this study were to understand the associations between serologic DcR3 (sDcR3) levels and different human immunodeficiency virus type 1 (HIV-1) subtypes, as well as the AIDS disease progression. Serum samples from 61 HIV/AIDS patients, who had been followed up every 6 months for 3 years, were collected. sDcR3 levels were quantified using an enzyme immunoassay (EIA). The sDcR3 levels in patients with HIV-1 subtype B were significantly higher than those in patients infected with subtype CRF01_AE (p < 0.001). In addition, multivariable linear mixed model analysis demonstrated that HIV-1 subtype B and slow disease progression were associated with higher levels of sDcR3, adjusting for potential predictors (p = 0.0008 and 0.0455, respectively). HIV-1-infected cells may gain a survival advantage by activating DcR3, which prevents infected cell detection by the host immune system. These data indicate that the sDcR3 level is a biomarker for AIDS disease progression. Copyright © 2013. Published by Elsevier B.V.

Histamine receptors in human detrusor smooth muscle cells: physiological properties and immunohistochemical representation of subtypes.

PubMed

Neuhaus, Jochen; Weimann, Annett; Stolzenburg, Jens-Uwe; Dawood, Waled; Schwalenberg, Thilo; Dorschner, Wolfgang

2006-06-01

The potent inflammatory mediator histamine is released from activated mast cells in interstitial cystitis (IC). Here, we report on the histamine receptor subtypes involved in the intracellular calcium response of cultured smooth muscle cells (cSMC). Fura-2 was used to monitor the calcium response in cSMC, cultured from human detrusor biopsies. The distribution of histamine receptor subtypes was addressed by immunocytochemistry in situ and in vitro. Histamine stimulated a maximum of 92% of the cells (n=335), being more effective than carbachol (70%, n=920). HTMT (H1R-agonist), dimaprit (H2R) and MTH (H3R) lead to significant lower numbers of reacting cells (60, 48 and 54%). Histamine receptor immunoreactivity (H1R, H2R, H3R, H4R) was found in situ and in vitro. Histamine-induced calcium increase is mediated by distinct histamine receptors. Thus, pre-therapeutic evaluation of histamine receptor expression in IC patients may help to optimize therapy by using a patient-specific cocktail of subtype-specific histamine receptor antagonists.

Differential distribution of adenosine receptors in rat cochlea.

PubMed

Vlajkovic, Srdjan M; Abi, Shukri; Wang, Carol J H; Housley, Gary D; Thorne, Peter R

2007-06-01

Adenosine is a constitutive cell metabolite that can be released from cells via specific bi-directional transporters and is an end-point for nucleotide hydrolysis. In the extracellular space, adenosine becomes a signalling molecule for P1 (adenosine) receptors that modulate physiological responses in a wide range of mammalian tissues. Whereas adenosine signalling has been implicated in the regulation of cochlear blood flow and in cochlear protection from oxidative damage, the potential roles for adenosine signalling in the modulation of sound transduction and auditory neurotransmission have not been established. We have characterised the expression and distribution of adenosine receptors in the rat cochlea. mRNA transcripts for all four subtypes of adenosine receptors (A(1), A(2A), A(2B) and A(3)) were detected in dissected cochlear tissue by using reverse transcription/polymerase chain reaction analysis. The protein distribution for the A(1), A(2A) and A(3) receptor subtypes was identified by immunoperoxidase histochemistry and confocal immunofluorescence labelling. These receptors were differentially expressed in the organ of Corti, spiral ganglion neurones, lateral wall tissues and cochlear blood vessels. The distribution of adenosine receptors in sensory and neural tissues and in the vasculature coincided with other elements of purinergic signalling (P2X and P2Y receptors, ectonucleotidases), consistent with the integrative regulation of many physiological processes in the cochlea by extracellular nucleotides and nucleosides. Our study provides a framework for further investigation of adenosine signalling in the inner ear, including putative roles in oxidative stress responses.

Studies on the role of serotonin receptor subtypes in the effect of sibutramine in various feeding paradigms in rats

PubMed Central

Grignaschi, G; Fanelli, E; Scagnol, I; Samanin, R

1999-01-01

The effect of the 5-hydroxytryptamine (5-HT) and noradrenaline (NA) reuptake inhibitor sibutramine was studied in food deprived, neuropeptide Y (NPY)- or muscimol-injected rats. Sibutramine dose-dependently reduced feeding caused by food-deprivation (ED50=5.1±0.8 mg kg−1) or by NPY injection into the paraventricular nucleus of the hypothalamus (ED50=6.0±0.5 mg kg−1). The increase in food intake caused by muscimol injected into the dorsal raphe was not modified by sibutramine (1–10 mg kg−1). The hypophagic effect of 5.1 mg kg−1 sibutramine in food-deprived rats was studied in rats pretreated with different serotonin receptor antagonists. Metergoline (non-selective, 0.3 and 1.0 mg kg−1), ritanserin (5-HT2A/2C, 0.5 and 1.0 mg kg−1) and GR127935 (5-HT1B/1D, 0.5 and 1.0 mg kg−1) did not modify the hypophagic effect of sibutramine, while SB206553 (5-HT2B/2C, 5 and 10 mg kg−1) slightly but significantly reduced it (Fint(2.53)=3.4; P<0.05). The reduction in food intake caused by 6.0 mg kg−1 sibutramine in NPY-injected rats was not modified by GR127935 (1.0 mg kg−1). The results suggest that, with the possible exception of a partial involvement of 5-HT2B/2C receptors in sibutramine's hypophagia in food-deprived rats, 5-HT1 and 5-HT2 receptor subtypes do not play an important role in the hypophagic effect of sibutramine, at least in the first 2 h after injection. PMID:10455265

Thieno[3,2-b]- and thieno[2,3-b]pyrrole bioisosteric analogues of the hallucinogen and serotonin agonist N,N-dimethyltryptamine.

PubMed

Blair, J B; Marona-Lewicka, D; Kanthasamy, A; Lucaites, V L; Nelson, D L; Nichols, D E

1999-03-25

The synthesis and biological activity of 6-[2-(N, N-dimethylamino)ethyl]-4H-thieno[3,2-b]pyrrole (3a) and 4-[2-(N, N-dimethylamino)ethyl]-6H-thieno[2,3-b]pyrrole (3b), thienopyrroles as potential bioisosteres of N,N-dimethyltryptamine (1a), are reported. Hallucinogen-like activity was evaluated in the two-lever drug discrimination paradigm using LSD- and DOI-trained rats. Neither 3a nor 3b substituted for LSD or DOI up to doses of 50 micromol/kg. By comparison, 1a fully substituted in LSD-trained rats. However, 3a and 3b fully substituted for the 5-HT1A agonist LY293284 ((-)-(4R)-6-acetyl-4-(di-n-propylamino)-1,3,4, 5-tetrahydrobenz[c,d]indole). Both 3a and 3b induced a brief "serotonin syndrome" and salivation, an indication of 5-HT1A receptor activation. At the cloned human 5-HT2A receptor 3b had about twice the affinity of 3a. At the cloned human 5-HT2B and 5-HT2C receptors, however, 3a had about twice the affinity of 3b. Therefore, thiophene lacks equivalence as a replacement for the phenyl ring in the indole nucleus of tryptamines that bind to 5-HT2 receptor subtypes and possess LSD-like behavioral effects. Whereas both of the thienopyrroles had lower affinity than the corresponding 1a at 5-HT2 receptors, 3a and 3b had significantly greater affinity than 1a at the 5-HT1A receptor. Thus, thienopyrrole does appear to serve as a potent bioisostere for the indole nucleus in compounds that bind to the serotonin 5-HT1A receptor. These differences in biological activity suggest that serotonin receptor isoforms are very sensitive to subtle changes in the electronic character of the aromatic systems of indole compounds.

Molecular recognition at adenine nucleotide (P2) receptors in platelets.

PubMed

Jacobson, Kenneth A; Mamedova, Liaman; Joshi, Bhalchandra V; Besada, Pedro; Costanzi, Stefano

2005-04-01

Transmembrane signaling through P2Y receptors for extracellular nucleotides controls a diverse array of cellular processes, including thrombosis. Selective agonists and antagonists of the two P2Y receptors present on the platelet surface-the G (q)-coupled P2Y (1) subtype and the G (i)-coupled P2Y (12) subtype-are now known. High-affinity antagonists of each have been developed from nucleotide structures. The (N)-methanocarba bisphosphate derivatives MRS2279 and MRS2500 are potent and selective P2Y (1) receptor antagonists. The carbocyclic nucleoside AZD6140 is an uncharged, orally active P2Y (12) receptor antagonist of nM affinity. Another nucleotide receptor on the platelet surface, the P2X (1) receptor, the activation of which may also be proaggregatory, especially under conditions of high shear stress, has high-affinity ligands, although high selectivity has not yet been achieved. Although alpha,beta-methylene-adenosine triphosphate (ATP) is the classic agonist for the P2X (1) receptor, where it causes rapid desensitization, the agonist BzATP is among the most potent in activating this subtype. The aromatic sulfonates NF279 and NF449 are potent antagonists of the P2X (1) receptor. The structures of the two platelet P2Y receptors have been modeled, based on a rhodopsin template, to explain the basis for nucleotide recognition within the putative transmembrane binding sites. The P2Y (1) receptor model, especially, has been exploited in the design and optimization of antagonists targeted to interact selectively with that subtype.

Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications.

PubMed

Lambrecht, G

2000-11-01

P2X receptors are nucleotide-gated cation channels composed of homomeric or heteromeric assemblies of three subunits. In the past 7 years, an extended series (P2X1-7) of P2X subunits has been cloned from vertebrate tissues. In this rapidly expanding field, one of the main current challenges is to relate the cloned P2X receptor subtypes to the diverse physiological responses mediated by the native P2X receptors. However, the paucity of useful ligands, especially subtype-selective agonists and antagonists as well as radioligands, acts as a considerable impediment to progress. Most of the ligands available are highly limited in terms of their kinetics of action, receptor-affinity, subtype-selectivity and P2X receptor-specificity. Their suspected ability to be a substrate for ecto-nucleotidases or to inhibit these enzymes also complicates their use. A number of new antagonists at P2X receptors have recently been described which to some degree are more potent and more selective than earlier antagonists like suramin or pyridoxal-5'-phosphate-6-azophenyl 2',4'-disulfonate (PPADS). This work moves us closer to the ideal goal of classifying the recombinant and native P2X receptor subtypes on the basis of antagonist profiles. This review begins with a brief account of the current status of P2X receptors. It then focuses on the pharmacological properties of a series of key P2 receptor agonists and antagonists and will finish with the discussion of some related therapeutic possibilities.

Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B

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

Ploux, O.; Lavielle, S.; Chassaing, G.

1987-11-01

The activities of two groups of cyclic agonists of substance P (SP) have been studied. The disulfide bridge constraints have been designed on the basis of conformational studies on SP and physalaemin indicating an ..cap alpha..-helical structure for the core of these two tachykinins (group I) and a folding of the C-terminal carboxamide towards the side chains of the glutamines 5 and 6 (group II). Only peptides simulating the ..cap alpha..-helix present substantial potencies. (Cys/sup 3,6/)SP is as active as SP in inhibiting /sup 125/I-labeled Bolton and Hunter SP-specific binding on rat brain synaptosomes and on dog carotid bioassay, twomore » assays specific for the neurokinin 1 receptor. Moreover, (Cys/sup 3,6/)SP is a potent as neurokinin B in inhibiting /sup 125/I-labeled Bolton and Hunter eledoisin-specific binding on rat cortical synaptosomes as well as in stimulating rat portal vein, two tests specific for the neurokinin 3 receptor. Interestingly, in contrast to neurokinin B, (Cys/sup 3,6/)SP is a weak agonist of the neurokinin 2 receptor subtype, as evidenced by its binding potency in inhibiting /sup 3/H-labeled neurokinin A-specific binding on rat duodenum and in inducing the contractions of the rabbit pulmonary artery, a neurokinin 2-type bioassay. To increase the specificity of the cyclic analogue (Cys/sup 3,6/)SP positions 8 and 9 were modified. Collectively, these results suggest that the neurokinin 1 and neurokinin 3 tachykinin receptors may recognize a similar three-dimensional structure of the core of the tachykinins. Different orientations of the common C-terminal tripeptide may be related to the selectivity for the different receptor subtypes.« less

Oxytocin induces penile erection and yawning when injected into the bed nucleus of the stria terminalis: Involvement of glutamic acid, dopamine, and nitric oxide.

PubMed

Sanna, Fabrizio; Bratzu, Jessica; Argiolas, Antonio; Melis, Maria Rosaria

2017-11-01

Oxytocin (5-100ng), but not Arg 8 -vasopressin (100ng), injected unilaterally into the bed nucleus of the stria terminalis (BNST) induces penile erection and yawning in a dose-dependent manner in male rats. The minimal effective dose was 20ng for penile erection and 5ng for yawning. Oxytocin responses were abolished not only by the oxytocin receptor antagonist d(CH 2 ) 5 Tyr(Me) 2 -Orn 8 -vasotocin (1μg), but also by (+) MK-801 (1μg), an excitatory amino acid receptor antagonist of the N-methyl-d-aspartic acid (NMDA) subtype, SCH 23390 (1μg), a D1 receptor antagonist, but not haloperidol (1μg), a D2 receptor antagonist, and SMTC (40μg), an inhibitor of neuronal nitric oxide synthase, injected into the BNST 15min before oxytocin. Oxytocin-induced penile erection, but not yawning, was also abolished by CNQX (1μg), an excitatory amino acid receptor antagonist of the AMPA subtype. In contrast, oxytocin responses were not reduced by bicuculline (20ng), a GABA A receptor antagonist, phaclofen (5μg), a GABA B receptor antagonist, CP 376395, a CRF receptor-1 antagonist (5μg), or astressin 2B, a CRF receptor-2 antagonist (150ng). Considering the ability of NMDA (100ng) to induce penile erection and yawning when injected into the BNST and the available evidence showing possible interaction among oxytocin, glutamic acid, and dopamine in the BNST, oxytocin possibly activates glutamatergic neurotransmission in the BNST. This in turn leads to the activation of neural pathways projecting back to the paraventricular nucleus, medial preoptic area, ventral tegmental area, and/or ventral subiculum/amygdala, thereby inducing penile erection and yawning. Copyright © 2017 Elsevier Inc. All rights reserved.

8-(2-Furyl)adenine derivatives as A₂A adenosine receptor ligands.

PubMed

Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Thomas, Ajiroghene; Klotz, Karl-Norbert; Federico, Stephanie; Cacciari, Barbara; Spalluto, Giampiero; Volpini, Rosaria

2013-01-01

Selective adenosine receptor modulators are potential tools for numerous therapeutic applications, including cardiovascular, inflammatory, and neurodegenerative diseases. In this work, the synthesis and biological evaluation at the four human adenosine receptor subtypes of a series of 9-substituted 8-(2-furyl)adenine derivatives are reported. Results show that 8-(2-furyl)-9-methyladenine is endowed with high affinity at the A₂A subtype. Further modification of this compound with introduction of arylacetyl or arylcarbamoyl groups in N(6)-position takes to different effects on the A₂A affinity and in particular on the selectivity versus the other three adenosine receptor subtypes. A molecular modelling analysis at three different A₂A receptor crystal structures provides an interpretation of the obtained biological results. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Association of Hormone-Related Characteristics and Breast Cancer Risk by Estrogen Receptor/Progesterone Receptor Status in the Shanghai Breast Cancer Study

PubMed Central

Bao, Ping-Ping; Shu, Xiao Ou; Gao, Yu-Tang; Zheng, Ying; Cai, Hui; Deming, Sandra L.; Ruan, Zhi-Xian; Gu, Kai; Lu, Wei; Zheng, Wei

2011-01-01

Etiologic differences between subtypes of breast cancer defined by estrogen receptor (ER) and progesterone receptor (PR) status are not well understood. The authors evaluated associations of hormone-related factors with breast cancer subtypes in a population-based case-control study involving 1,409 ER-positive (ER+)/PR-positive (PR+) cases, 712 ER-negative (ER−)/PR-negative (PR−) cases, 301 ER+/PR− cases, 254 ER−/PR+ cases, and 3,474 controls aged 20–70 years in Shanghai, China (phase I, 1996–1998; phase II, 2002–2005). Polytomous logistic regression and Wald tests for heterogeneity across subtypes were conducted. Breast cancer risks associated with age at menarche, age at menopause, breastfeeding, age at first livebirth, waist-to-hip ratio, and oral contraceptive use did not differ by hormone receptor status. Among postmenopausal women, higher parity (≥2 children vs. 1) was associated with reduced risk (odds ratio (OR) = 0.69, 95% confidence interval (CI): 0.52, 0.91) and higher body mass index (BMI; weight (kg)/height (m)2) with increased risk (highest quartile: OR = 2.40, 95% CI: 1.65, 3.47) of the ER+/PR+ subtype but was unrelated to the ER−/PR− subtype (for parity, Pheterogeneity = 0.02; for BMI, Pheterogeneity < 0.01). Hormone replacement therapy (OR = 2.25, 95% CI: 1.40, 3.62) and alcohol consumption (OR = 1.59, 95% CI: 1.01, 2.51) appeared to be preferentially associated with the ER+/PR− subtype. These findings indicate that BMI, parity, hormone replacement therapy, and alcohol consumption may play different roles in subtypes of breast cancer. More research is needed to better understand the etiology of 2 relatively rare subtypes, ER+/PR− tumors and ER−/PR+ tumors. PMID:21768404

Functional characterization of the beta-adrenergic receptor subtypes expressed by CA1 pyramidal cells in the rat hippocampus.

PubMed

Hillman, Kristin L; Doze, Van A; Porter, James E

2005-08-01

Recent studies have demonstrated that activation of the beta-adrenergic receptor (AR) using the selective beta-AR agonist isoproterenol (ISO) facilitates pyramidal cell long-term potentiation in the cornu ammonis 1 (CA1) region of the rat hippocampus. We have previously analyzed beta-AR genomic expression patterns of 17 CA1 pyramidal cells using single cell reverse transcription-polymerase chain reaction, demonstrating that all samples expressed the beta2-AR transcript, with four of the 17 cells additionally expressing mRNA for the beta1-AR subtype. However, it has not been determined which beta-AR subtypes are functionally expressed in CA1 for these same pyramidal neurons. Using cell-attached recordings, we tested the ability of ISO to increase pyramidal cell action potential (AP) frequency in the presence of subtype-selective beta-AR antagonists. ICI-118,551 [(+/-)-1-[2,3-(dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol] and butoxamine [alpha-[1-(t-butylamino)ethyl]-2,5-dimethoxybenzyl alcohol) hydrochloride], agents that selectively block the beta2-AR, produced significant parallel rightward shifts in the concentration-response curves for ISO. From these curves, apparent equilibrium dissociation constant (K(b)) values of 0.3 nM for ICI-118,551 and 355 nM for butoxamine were calculated using Schild regression analysis. Conversely, effective concentrations of the selective beta1-AR antagonists CGP 20712A [(+/-)-2-hydroxy-5-[2-([2-hydroxy-3-(4-[1-methyl-4-(trifluoromethyl)-1H-imidazol-2-yl]phenoxy)propyl]amino)ethoxy]-benzamide methanesulfonate] and atenolol [4-[2'-hydroxy-3'-(isopropyl-amino)propoxy]phenylacetamide] did not significantly affect the pyramidal cell response to ISO. However, at higher concentrations, atenolol significantly decreased the potency for ISO-mediated AP frequencies. From these curves, an apparent atenolol K(b) value of 3162 nM was calculated. This pharmacological profile for subtype-selective beta-AR antagonists indicates that beta2-AR activation is mediating the increased AP frequency. Knowledge of functional AR expression in CA1 pyramidal neurons will aid future long-term potentiation studies by allowing selective manipulation of specific beta-AR subtypes.

Overall survival and the response to radiotherapy among molecular subtypes of breast cancer brain metastases treated with targeted therapies.

PubMed

Miller, Jacob A; Kotecha, Rupesh; Ahluwalia, Manmeet S; Mohammadi, Alireza M; Chao, Samuel T; Barnett, Gene H; Murphy, Erin S; Vogelbaum, Michael A; Angelov, Lilyana; Peereboom, David M; Suh, John H

2017-06-15

The current study was conducted to investigate survival and the response to radiotherapy among patients with molecular subtypes of breast cancer brain metastases treated with or without targeted therapies. Patients diagnosed with breast cancer brain metastases at a single tertiary care institution were included. The primary outcome was overall survival, whereas secondary outcomes included the cumulative incidences of distant intracranial failure, local failure, and radiation necrosis. Competing risks regression was used to model secondary outcomes. Within the study period, 547 patients presented with 3224 brain metastases and met inclusion criteria. Among patients with human epidermal growth factor receptor 2 (HER2)-amplified disease, 80% received HER2 antibodies and 38% received HER2/epidermal growth factor receptor tyrosine kinase inhibitors (TKIs). The median survival was significantly shorter in the basal cohort (8.4 months), and progressively increased in the luminal A (12.3 months), HER2-positive (15.4 months), and luminal B (18.8 months) cohorts (P<.001). Among patients with HER2-amplified disease, the median survival was extended with the use of both HER2 antibodies (17.9 months vs 15.1 months; P = .04) and TKIs (21.1 months vs 15.4 months; P = .03). The 12-month cumulative incidences of local failure among molecular subtypes were 6.0% in the luminal A cohort, 10.3% in the luminal B cohort, 15.4% in the HER2-positive cohort, and 9.9% in the basal cohort (P = .01). Concurrent HER2/epidermal growth factor receptor TKIs with stereotactic radiosurgery significantly decreased the 12-month cumulative incidence of local failure from 15.1% to 5.7% (P<.001). Molecular subtypes appear to be prognostic for survival and predictive of the response to radiotherapy. TKIs were found to improve survival and local control, and may decrease the rate of distant failure. To preserve neurocognition, these results support a paradigm of upfront radiosurgery and HER2-directed therapy in the HER2-amplified population, reserving whole-brain radiotherapy for salvage. Cancer 2017;123:2283-2293. © 2017 American Cancer Society. © 2017 American Cancer Society.

Dopamine D(3) receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: role of hyperthermia.

PubMed

Baladi, Michelle G; Newman, Amy H; Nielsen, Shannon M; Hanson, Glen R; Fleckenstein, Annette E

2014-06-05

Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. Copyright © 2014 Elsevier B.V. All rights reserved.

Differential Expression of Glutamate Receptors in Avian Neural Pathways for Learned Vocalization

PubMed Central

WADA, KAZUHIRO; SAKAGUCHI, HIRONOBU; JARVIS, ERICH D.; HAGIWARA, MASATOSHI

2008-01-01

Learned vocalization, the substrate for human language, is a rare trait. It is found in three distantly related groups of birds—parrots, hummingbirds, and songbirds. These three groups contain cerebral vocal nuclei for learned vocalization not found in their more closely related vocal nonlearning relatives. Here, we cloned 21 receptor subunits/subtypes of all four glutamate receptor families (AMPA, kainate, NMDA, and metabotropic) and examined their expression in vocal nuclei of songbirds. We also examined expression of a subset of these receptors in vocal nuclei of hummingbirds and parrots, as well as in the brains of dove species as examples of close vocal nonlearning relatives. Among the 21 subunits/subtypes, 19 showed higher and/or lower prominent differential expression in songbird vocal nuclei relative to the surrounding brain subdivisions in which the vocal nuclei are located. This included relatively lower levels of all four AMPA subunits in lMAN, strikingly higher levels of the kainite subunit GluR5 in the robust nucleus of the arcopallium (RA), higher and lower levels respectively of the NMDA subunits NR2A and NR2B in most vocal nuclei and lower levels of the metabotropic group I subtypes (mGluR1 and -5) in most vocal nuclei and the group II subtype (mGluR2), showing a unique expression pattern of very low levels in RA and very high levels in HVC. The splice variants of AMPA subunits showed further differential expression in vocal nuclei. Some of the receptor subunits/subtypes also showed differential expression in hummingbird and parrot vocal nuclei. The magnitude of differential expression in vocal nuclei of all three vocal learners was unique compared with the smaller magnitude of differences found for nonvocal areas of vocal learners and vocal nonlearners. Our results suggest that evolution of vocal learning was accompanied by differential expression of a conserved gene family for synaptic transmission and plasticity in vocal nuclei. They also suggest that neural activity and signal transduction in vocal nuclei of vocal learners will be different relative to the surrounding brain areas. PMID:15236466

Cloning and functional analysis of P2X1b, a new variant in rat optic nerve that regulates the P2X1 receptor in a use-dependent manner.

PubMed

Rangel-Yescas, Gisela E; Vazquez-Cuevas, Francisco G; Garay, Edith; Arellano, Rogelio O

2012-01-01

P2X receptors are trimeric, ATP-gated cation channels. In mammals seven P2X subtypes have been reported (P2X1-P2X7), as well as several variants generated by alternative splicing. Variants confer to the homomeric or heteromeric channels distinct functional and/or pharmacological properties. Molecular biology, biochemical, and functional analysis by electrophysiological methods were used to identify and study a new variant of the P2X1 receptor named P2X1b. This new variant, identified in rat optic nerve, was also expressed in other tissues. P2X1b receptors lack amino acids 182 to 208 of native P2X1, a region that includes residues that are highly conserved among distinct P2X receptors. When expressed in Xenopus oocytes, P2X1b was not functional as a homomer; however, when co-expressed with P2X1, it downregulated the electrical response generated by ATP compared with that of oocytes expressing P2X1 alone, and it seemed to form heteromeric channels with a modestly enhanced ATP potency. A decrease in responses to ATP in oocytes co-expressing different ratios of P2X1b to P2X1 was completely eliminated by overnight pretreatment with apyrase. Thus, it is suggested that P2X1b regulates, through a use-dependent mechanism, the availability, in the plasma membrane, of receptor channels that can be operated by ATP.

Serotonergic modulation of the rat pup ultrasonic isolation call: studies with 5HT1 and 5HT2 subtype-selective agonists and antagonists.

PubMed

Winslow, J T; Insel, T R

1991-01-01

A modulatory role for serotonin has been described for the development and expression of the ultrasonic call of infant rat pups during brief maternal separations. In previous studies, serotonin reuptake inhibitors selectively reduced the rate of calling following acute administration to 9-11-day-old pups and a serotonin neurotoxin (MDMA) systematically disrupted the development of ultrasonic vocalizations but not other measures of motor development. In the current studies, we extended our investigations to include drugs with purported receptor subtype selectivities. Consistent with previous reports, acute administration of 5HT1A agonists buspirone and 8-OH-DPAT [+/-)-8-hydroxy-2-(di-N-propylamino)tetralin) reduced the rate of calling at doses which did not affect motor activity or core body temperature. The rate reducing effects of buspirone persisted up to 1 but not 2 h after injection. Administration of purported 5HT1B receptor agonists, CGS12066B (7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo[1,2-a] quinoxaline) and TFMPP (1-[3-fluoromethyl)phenyl]-piperazine) increased the rate of calling depending on the specificity of the drug for the 5HT1B receptor. d,l-Propranolol, a 5HT1 receptor antagonist, blocked the effects of both 8-OH-DPAT and TFMPP. m-CPP (1-(3-chlorophenyl)piperazine) and DOI [+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane), drugs with putative actions at 5HT1C and 5HT2 receptor sites both decreased calling but differed according to their effects on motor activity. Ritanserin, a 5HT2 and 5HT1C antagonist, produced a dose-related increase in call rate. A dose of ritanserin with no apparent intrinsic effects effectively antagonized DOI rate reducing effects but potentiated the rate reducing effects of m-CPP.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased cyclic guanosine monophosphate production and overexpression of atrial natriuretic peptide A-receptor mRNA in spontaneously hypertensive rats.

PubMed

Tremblay, J; Huot, C; Willenbrock, R C; Bayard, F; Gossard, F; Fujio, N; Koch, C; Kuchel, O; Debinski, W; Hamet, P

1993-11-01

Atrial natriuretic peptide (ANP) specifically stimulates particulate guanylate cyclase, and cyclic guanosine monophosphate (cGMP) has been recognized as its second messenger. Spontaneously hypertensive rats (SHR) have elevated plasma ANP levels, but manifest an exaggerated natriuretic and diuretic response to exogenous ANP when compared to normotensive strains. In isolated glomeruli, the maximal cGMP response to ANP corresponds to a 12- to 14-fold increase over basal levels in normotensive strains (Wistar 13 +/- 2; Wistar-Kyoto 12 +/- 2; Sprague-Dawley 14 +/- 2) while a maximal 33 +/- 3-fold elevation occurs in SHR (P < 0.001). This hyperresponsiveness of cGMP is reproducible in intact glomeruli from SHR from various commercial sources. Furthermore, this abnormality develops early in life, even before hypertension is clearly established, and persists despite pharmacological modulation of blood pressure, indicating that it is a primary event in hypertension. In vitro studies have revealed a higher particulate guanylate cyclase activity in membranes from glomeruli and other tissues from SHR. This increase is not accounted for by different patterns of ANP binding to its receptor subtypes between normotensive and hypertensive strains, as assessed by competitive displacement with C-ANP102-121, an analog which selectively binds to one ANP receptor subtype. The hyperactivity of particulate guanylate cyclase in SHR and its behavior under basal, ligand (ANP), and detergent-enhanced conditions could be attributed either to increased expression or augmented sensitivity of the enzyme. Radiation-inactivation analysis does not evoke a disturbance in the size of regulatory elements normally repressing enzymatic activity, while the expression of particulate guanylate cyclase gene using mutated standard of A- and B-receptors partial cDNAs, quantified by polymerase chain reaction (PCR) transcript titration assay, manifests a selective increase of one guanylate cyclase subtype. Our data suggest that in hypertension, genetic overexpression of the ANP A-receptor subtype is related to the exaggerated biological response to ANP in this disease.

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor.

PubMed

Romagnoli, Romeo; Baraldi, Pier Giovanni; Pavani, Maria Giovanna; Tabrizi, Mojgan Aghazadeh; Moorman, Allan R; Di Virgilio, Francesco; Cattabriga, Elena; Pancaldi, Cecilia; Gessi, Stefania; Borea, Pier Andrea

2004-11-15

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.

Dynamic regulation of glycinergic input to spinal dorsal horn neurones by muscarinic receptor subtypes in rats.

PubMed

Wang, Xiu-Li; Zhang, Hong-Mei; Li, De-Pei; Chen, Shao-Rui; Pan, Hui-Lin

2006-03-01

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

Argyreia nervosa (Burm. f.): receptor profiling of lysergic acid amide and other potential psychedelic LSD-like compounds by computational and binding assay approaches.

PubMed

Paulke, Alexander; Kremer, Christian; Wunder, Cora; Achenbach, Janosch; Djahanschiri, Bardya; Elias, Anderson; Schwed, J Stefan; Hübner, Harald; Gmeiner, Peter; Proschak, Ewgenij; Toennes, Stefan W; Stark, Holger

2013-07-09

The convolvulacea Argyreia nervosa (Burm. f.) is well known as an important medical plant in the traditional Ayurvedic system of medicine and it is used in numerous diseases (e.g. nervousness, bronchitis, tuberculosis, arthritis, and diabetes). Additionally, in the Indian state of Assam and in other regions Argyreia nervosa is part of the traditional tribal medicine (e.g. the Santali people, the Lodhas, and others). In the western hemisphere, Argyreia nervosa has been brought in attention as so called "legal high". In this context, the seeds are used as source of the psychoactive ergotalkaloid lysergic acid amide (LSA), which is considered as the main active ingredient. As the chemical structure of LSA is very similar to that of lysergic acid diethylamide (LSD), the seeds of Argyreia nervosa (Burm. f.) are often considered as natural substitute of LSD. In the present study, LSA and LSD have been compared concerning their potential pharmacological profiles based on the receptor binding affinities since our recent human study with four volunteers on p.o. application of Argyreia nervosa seeds has led to some ambiguous effects. In an initial step computer-aided in silico prediction models on receptor binding were employed to screen for serotonin, norepinephrine, dopamine, muscarine, and histamine receptor subtypes as potential targets for LSA. In addition, this screening was extended to accompany ergotalkaloids of Argyreia nervosa (Burm. f.). In a verification step, selected LSA screening results were confirmed by in vitro binding assays with some extensions to LSD. In the in silico model LSA exhibited the highest affinity with a pKi of about 8.0 at α1A, and α1B. Clear affinity with pKi>7 was predicted for 5-HT1A, 5-HT1B, 5-HT1D, 5-HT6, 5-HT7, and D2. From these receptors the 5-HT1D subtype exhibited the highest pKi with 7.98 in the prediction model. From the other ergotalkaloids, agroclavine and festuclavine also seemed to be highly affine to the 5-HT1D-receptor with pKi>8. In general, the ergotalkaloids of Argyreia nervosa seem to prefer serotonin and dopamine receptors (pKi>7). However, with exception of ergometrine/ergometrinine only for 5-HT3A, and histamine H2 and H4 no affinities were predicted. Compared to LSD, LSA exhibited lower binding affinities in the in vitro binding assays for all tested receptor subtypes. However, with a pKi of 7.99, 7.56, and 7.21 a clear affinity for 5-HT1A, 5-HT2, and α2 could be demonstrated. For DA receptor subtypes and the α1-receptor the pKi ranged from 6.05 to 6.85. Since the psychedelic activity of LSA in the recent human study was weak and although LSA from Argyreia nervosa is often considered as natural exchange for LSD, LSA should not be regarded as LSD-like psychedelic drug. However, vegetative side effects and psychotropic effects may be triggered by serotonin or dopamine receptor subtypes. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Distinct breast cancer subtypes in women with early-onset disease across races

PubMed Central

Singh, Mandeep; Ding, Yi; Zhang, Li-Ying; Song, Dong; Gong, Yun; Adams, Sylvia; Ross, Dara S; Wang, Jin-Hua; Grover, Shruti; Doval, Dinesh Chandra; Shao, Charles; He, Zi-Li; Chang, Victor; Chin, Warren W; Deng, Fang-Ming; Singh, Baljit; Zhang, David; Xu, Ru-Liang; Lee, Peng

2014-01-01

Background: Racial disparities among breast cancer (BCa) patients are known but not well studied in early-onset BCa. We analyzed molecular subtypes in early-onset BCa across five major races. Methods: A total of 2120 cases were included from non-Hispanic White (NHW), African American (AA) and Hispanic, Chinese and Indian. Based on ER, PR and HER-2 status, BCa was classified into 4 intrinsic subtypes as Luminal A, Luminal B, HER2/neu overexpression and Triple negative BCa (TNBC) subtypes. Data was stratified according to race and age as younger/early-onset group (40-years and younger) and older group (50-years and older). Results: In early-onset BCa, incidence of TNBC was significantly higher (p = 0.0369) in Indian women followed by AA, Hispanic, NHW and Chinese women. Incidence of Her2 over-expression subtype also was highest in Indian women, followed by Hispanic, Chinese, AA and NHW women. In contrast, Luminal B subtype was most significantly higher in AA women (p = 0.0000) followed by NHW (p = 0.0002), Chinese (p = 0.0003), Hispanic (0.0128) and Indian (p = 0.0468) women. Luminal A subtype was most significantly reduced in Indian women (p = 0.0113) followed by Hispanic, AA, NHW and Chinese women. These results were based on statistical analysis with the mean of older group populations. Conclusions: These results show significant disparities in receptor subtypes across races. This study will contribute in developing optimal clinical trial protocols and personalized management strategies for early-onset BCa patients. PMID:25057437

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies.

PubMed

Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

2014-08-01

Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. © 2014 The British Pharmacological Society.

Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex.

PubMed

Grandclément, B; Morel, G

1998-06-01

Atrial natriuretic peptide (ANP) and two complementary peptides named brain natriuretic peptide and C-type natriuretic peptide are involved in diuresis, natriuresis, hypotension and vasorelaxation. Their actions are mediated by highly selective and specific ANP receptors. Three subtypes have been characterized and cloned: ANP receptor A, -B and -C. In the present study, the mRNA for each subtype was detected by ultrastructural in situ hybridization on ultrathin sections of Lowicryl-embedded tissue and frozen tissue. The distribution of mRNA (visualized by gold particles) for each subtype was found to differ in different cells of the nephron. The three subtypes of this receptor family were expressed in all the parts of the nephron, but their expression levels were different. The ANPR-A mRNA was the most abundant in cells of glomerulus, proximal and distal tubules. The subtype C was the least expressed mRNA in glomerulus. In contrast, the subcellular localization of the three mRNAs was similar; they were found in the cytoplasmic matrix and the euchromatin of the nucleus. In conclusion, the differential expression of these mRNAs in kidney cortex indicates that these three peptides act directly in differing parts of nephron regions which are the glomerulus, the proximal and distal tubules.

A new class of pyrazolo[5,1-c][1,2,4]triazines as γ-aminobutyric type A (GABAA) receptor subtype ligand: synthesis and pharmacological evaluation.

PubMed

Guerrini, Gabriella; Ciciani, Giovanna; Daniele, Simona; Martini, Claudia; Costagli, Camilla; Guarino, Chiara; Selleri, Silvia

2018-05-15

A comparison between compounds with pyrazolo[1,5-a]pyrimidine structure (series 4-6) and pyrazolo[5,1-c][1,2,4]triazine core (series 9) as ligands at GABA A -receptor subtype, was evaluated. Moreover, for pyrazolotriazine derivatives having binding recognition, the interaction on recombinant rat α(1-3,5) GABA A receptor subtypes, was performed. Among these latter, emerge compounds 9c, 9k, 9l, 9m and 9n as α1-selective and 9h as α2-selective ligands. Copyright © 2018 Elsevier Ltd. All rights reserved.

Muscarinic acetylcholine receptor M1 and M3 subtypes mediate acetylcholine-induced endothelium-independent vasodilatation in rat mesenteric arteries.

PubMed

Tangsucharit, Panot; Takatori, Shingo; Zamami, Yoshito; Goda, Mitsuhiro; Pakdeechote, Poungrat; Kawasaki, Hiromu; Takayama, Fusako

2016-01-01

The present study investigated pharmacological characterizations of muscarinic acetylcholine receptor (AChR) subtypes involving ACh-induced endothelium-independent vasodilatation in rat mesenteric arteries. Changes in perfusion pressure to periarterial nerve stimulation and ACh were measured before and after the perfusion of Krebs solution containing muscarinic receptor antagonists. Distributions of muscarinic AChR subtypes in mesenteric arteries with an intact endothelium were studied using Western blotting. The expression level of M1 and M3 was significantly greater than that of M2. Endothelium removal significantly decreased expression levels of M2 and M3, but not M1. In perfused mesenteric vascular beds with intact endothelium and active tone, exogenous ACh (1, 10, and 100 nmol) produced concentration-dependent and long-lasting vasodilatations. In endothelium-denuded preparations, relaxation to ACh (1 nmol) disappeared, but ACh at 10 and 100 nmol caused long-lasting vasodilatations, which were markedly blocked by the treatment of pirenzepine (M1 antagonist) or 4-DAMP (M1 and M3 antagonist) plus hexamethonium (nicotinic AChR antagonist), but not methoctramine (M2 and M4 antagonist). These results suggest that muscarinic AChR subtypes, mainly M1, distribute throughout the rat mesenteric arteries, and that activation of M1 and/or M3 which may be located on CGRPergic nerves releases CGRP, causing an endothelium-independent vasodilatation. Copyright © 2015 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

Body weight and risk of molecular breast cancer subtypes among postmenopausal Mediterranean women.

PubMed

Crispo, A; Montella, M; Buono, G; Grimaldi, M; D'Aiuto, M; Capasso, I; Esposito, E; Amore, A; Nocerino, F; Augustin, L S A; Giudice, A; Di Bonito, M; Giuliano, M; Forestieri, V; De Laurentiis, M; Rinaldo, M; Ciliberto, G; De Placido, S; Arpino, G

2016-01-01

Breast cancer (BC) is the most common malignant tumor in women, obesity is associated with increased BC incidence and mortality and high levels of circulating insulin may negatively impact on cancer incidence. In the present study, we investigated whether the strength of several anthropometric and metabolic parameters varies between BC molecular subtypes. Eligible cases were 991 non-metastatic BC patients recruited between January 2009 and December 2013. Anthropometric, clinical and immunohistochemical features were measured. Multivariate logistic regression models were built to assess HER2 positive BC risk, comparing (a) triple positive (TP) with luminal A, luminal B and triple negative (TN) and (b) HER2-enriched group with luminal A, luminal B and TN. We stratified patients in pre- and post-menopause: significant differences emerged for luminal A in relation to age: they were more likely to be older compared to other groups. Among postmenopausal patients, the adjusted multivariate analysis showed that high BMI and high waist circumference were inversely correlated to TP subtype when compared to luminal B (OR=0.48 and OR=0.49, respectively). Conversely, HOMA-IR was a risk factor for TP when compared to luminal A and TN (OR=2.47 and OR=3.15, respectively). Our findings suggest a potential role of higher abdominal fat in the development of specific BC molecular subtypes in postmenopausal women. Moreover, they support a potential role of insulin resistance in the development of HER2 positive BC, although this role appears to be stronger when hormone receptors are co-expressed, suggesting a difference in the etiology of these two BC subtypes. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Co-agonists differentially tune GluN2B-NMDA receptor trafficking at hippocampal synapses

PubMed Central

Ferreira, Joana S; Papouin, Thomas; Ladépêche, Laurent; Yao, Andrea; Langlais, Valentin C; Bouchet, Delphine; Dulong, Jérôme; Mothet, Jean-Pierre; Sacchi, Silvia; Pollegioni, Loredano; Paoletti, Pierre; Oliet, Stéphane Henri Richard; Groc, Laurent

2017-01-01

The subunit composition of synaptic NMDA receptors (NMDAR), such as the relative content of GluN2A- and GluN2B-containing receptors, greatly influences the glutamate synaptic transmission. Receptor co-agonists, glycine and D-serine, have intriguingly emerged as potential regulators of the receptor trafficking in addition to their requirement for its activation. Using a combination of single-molecule imaging, biochemistry and electrophysiology, we show that glycine and D-serine relative availability at rat hippocampal glutamatergic synapses regulate the trafficking and synaptic content of NMDAR subtypes. Acute manipulations of co-agonist levels, both ex vivo and in vitro, unveil that D-serine alter the membrane dynamics and content of GluN2B-NMDAR, but not GluN2A-NMDAR, at synapses through a process requiring PDZ binding scaffold partners. In addition, using FRET-based FLIM approach, we demonstrate that D-serine rapidly induces a conformational change of the GluN1 subunit intracellular C-terminus domain. Together our data fuels the view that the extracellular microenvironment regulates synaptic NMDAR signaling. DOI: http://dx.doi.org/10.7554/eLife.25492.001 PMID:28598327

Adenosine receptor desensitization and trafficking.

PubMed

Mundell, Stuart; Kelly, Eamonn

2011-05-01

As with the majority of G-protein-coupled receptors, all four of the adenosine receptor subtypes are known to undergo agonist-induced regulation in the form of desensitization and trafficking. These processes can limit the ability of adenosine receptors to couple to intracellular signalling pathways and thus reduce the ability of adenosine receptor agonists as well as endogenous adenosine to produce cellular responses. In addition, since adenosine receptors couple to multiple signalling pathways, these pathways may desensitize differentially, while the desensitization of one pathway could even trigger signalling via another. Thus, the overall picture of adenosine receptor regulation can be complex. For all adenosine receptor subtypes, there is evidence to implicate arrestins in agonist-induced desensitization and trafficking, but there is also evidence for other possible forms of regulation, including second messenger-dependent kinase regulation, heterologous effects involving G proteins, and the involvement of non-clathrin trafficking pathways such as caveolae. In this review, the evidence implicating these mechanisms is summarized for each adenosine receptor subtype, and we also discuss those issues of adenosine receptor regulation that remain to be resolved as well as likely directions for future research in this field. Copyright © 2010 Elsevier B.V. All rights reserved.

Breast Cancer Subtype is Associated With Axillary Lymph Node Metastasis

PubMed Central

He, Zhen-Yu; Wu, San-Gang; Yang, Qi; Sun, Jia-Yuan; Li, Feng-Yan; Lin, Qin; Lin, Huan-Xin

2015-01-01

Abstract The purpose of this study was to assess whether breast cancer subtype (BCS) as determined by estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 can predict the axillary lymph node metastasis in breast cancer. Patients who received breast conserving surgery or mastectomy and axillary lymph node dissection were identified from 2 cancer centers. The associations between clinicopathological variables and axillary lymph node involvement were evaluated in univariate and multivariate regression analyses. A total of 3471 patients met the inclusion criteria, and 53.0% had axillary lymph node metastases at diagnosis. Patients with hormone receptor (HR)−/human epidermal growth factor receptor 2 (HER2)− subtype had a higher grade disease and the lowest rate of lymphovascular invasion. Univariate and multivariable logistic regression analyses showed that BCS was significantly associated with lymph node involvement. Patients with the HR−/HER2− subtype had the lowest odds of having nodal positivity than those with other BCSs. HR+/HER2− (odds ratio [OR] 1.651, 95% confidence interval [CI]: 1.349–2.021, P < 0.001), HR+/HER2+ (OR 1.958, 95%CI 1.542–2.486, P < 0.001), and HR−/HER2+ (OR 1.525, 95%CI 1.181–1.970, P < 0.001) tumors had higher risk of nodal positivity than the HR−/HER2− subtype. The other independent predictors of nodal metastases included tumor size, tumor grade, and lymphovascular invasion. Breast cancer subtype can predict the presence of axillary lymph node metastasis in breast cancer. HR−/HER2− is associated with a reduced risk of axillary lymph node metastasis compared to other BCSs. Our findings may play an important role in guiding axillary treatment considerations if further confirmed in larger sample size studies. PMID:26632910

Species differences in the localization and number of CNS beta adrenergic receptors: Rat versus guinea pig

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

Booze, R.M.; Crisostomo, E.A.; Davis, J.N.

1989-06-01

The localization and number of beta adrenergic receptors were directly compared in the brains of rats and guinea pigs. The time course of association and saturability of (125I)cyanopindolol (CYP) binding to slide-mounted tissue sections was similar in rats (Kd = 17 pM) and guinea pigs (Kd = 20 pM). The beta-1 and beta-2 receptor subtypes were examined through the use of highly selective unlabeled receptor antagonists, ICI 118,551 (50 nM) and ICI 89,406 (70 nM). Dramatic species differences between rats and guinea pigs were observed in the neuroanatomical regional localization of the beta adrenergic receptor subtypes. For example, in themore » thalamus prominent beta-1 and beta-2 receptor populations were identified in the rat; however, the entire thalamus of the guinea pig had few, if any, beta adrenergic receptors of either subtype. Hippocampal area CA1 had high levels of beta-2 adrenergic receptors in both rats and guinea pigs but was accompanied by a widespread distribution of beta-2 adrenergic receptors only in rats. Quantitative autoradiographic analyses of 25 selected neuroanatomical regions (1) confirmed the qualitative differences in CNS beta adrenergic receptor localization, (2) determined that guinea pigs had significantly lower levels of beta adrenergic receptors than rats and (3) indicated a differential pattern of receptor subtypes between the two species. Knowledge of species differences in receptor patterns may be useful in designing effective experiments as well as in exploring the relationships between receptor and innervation patterns. Collectively, these data suggest caution be used in extrapolation of the relationships of neurotransmitters and receptors from studies of a single species.« less

Muscarinic receptor subtype selectivity of novel heterocyclic QNB analogues

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

Baumgold, J.; Cohen, V.I.; Paek, R.

1991-01-01

In an effort at synthesizing centrally-active subtype-selective antimuscarinic agents, the authors derivatized QNB (quinuclidinyl benzilate), a potent muscarinic antagonist, by replacing one of the phenyl groups with less lipophilic heterocyclic moieties. The displacement of ({sup 3}H)-N-methyl scopolamine binding by these novel compounds to membranes from cells expressing ml - m4 receptor subtypes was determined. Most of the novel 4-bromo-QNB analogues were potent and slightly selective for ml receptors. The 2-thienyl derivative was the most potent, exhibiting a 2-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potency than BrQNB at ml receptors, and a 4-fold greater potencymore » at m2 receptors. This compound was also considerably less lipophilic than BrQNB as determined from its retention time on C18 reverse phase HPLC. This compound may therefore be useful both for pharmacological studies and as a candidate for a radioiodinated SPECT imaging agent for ml muscarinic receptors in human brain.« less

Adenosine receptor subtypes in the airways responses to 5'-adenosine monophosphate inhalation of sensitized guinea-pigs.

PubMed

Smith, N; Broadley, K J

2008-09-01

Endogenous adenosine levels are raised in the lungs during asthma attacks. 5'-adenosine monophosphate (5'-AMP) inhalation in asthmatics causes bronchoconstriction and in sensitized guinea-pigs induces early (EAR) and late asthmatic responses (LAR), airway hyper-reactivity (AHR) and inflammatory cell recruitment to the lungs. The aim of this study was to investigate the roles of A(1), A(2A), A(2B) and A(3) adenosine receptors in these responses to inhaled 5'-AMP in sensitized guinea-pigs. Comparisons were made with the effect of dexamethasone treatment on 5'-AMP-induced responses. Functional airways responses to inhaled 5'-AMP (3 and 300 mM) of actively sensitized, conscious guinea-pigs were determined by whole-body plethysmography following administration of selective adenosine receptor antagonists or their vehicles. AHR to inhaled histamine (1 mM) and inflammatory cell influx in bronchoalveolar lavage fluid were determined. 5'-AMP at 3 mM caused an immediate bronchoconstriction (EAR), whereas 300 mM caused bronchodilatation. Both responses were followed at 6 h by a LAR, together with inflammatory cell influx and AHR to histamine. The A(2A) receptor antagonist, ZM241385, further enhanced cell influx after 5'-AMP inhalation (3 and 300 mM), and blocked the immediate bronchodilator response to 300 mM 5'-AMP, exposing an EAR. The A(2B) receptor antagonist, MRS1706 (in the presence of ZM241385), inhibited the LAR, AHR and cell influx, following inhalation of 5'-AMP (300 mM). The A(3) receptor antagonist, MRS1220, inhibited 5'-AMP-induced inflammatory cell influx. The A(1) receptor antagonist, DPCPX (in the presence of ZM241385), inhibited the EAR following 5'-AMP inhalation (300 mM). Dexamethasone inhibited the LAR, AHR and cell influx following inhalation of 5'-AMP (300 mM). All four adenosine receptor subtypes play various roles in the airways responses to inhaled 5'-AMP in sensitized guinea-pigs.

Targetable genetic features of primary testicular and primary central nervous system lymphomas

PubMed Central

Chapuy, Bjoern; Roemer, Margaretha G. M.; Stewart, Chip; Tan, Yuxiang; Abo, Ryan P.; Zhang, Liye; Dunford, Andrew J.; Meredith, David M.; Thorner, Aaron R.; Jordanova, Ekaterina S.; Liu, Gang; Feuerhake, Friedrich; Ducar, Matthew D.; Illerhaus, Gerald; Gusenleitner, Daniel; Linden, Erica A.; Sun, Heather H.; Homer, Heather; Aono, Miyuki; Pinkus, Geraldine S.; Ligon, Azra H.; Ligon, Keith L.; Ferry, Judith A.; Freeman, Gordon J.; van Hummelen, Paul; Golub, Todd R.; Getz, Gad; Rodig, Scott J.; de Jong, Daphne; Monti, Stefano

2016-01-01

Primary central nervous system lymphomas (PCNSLs) and primary testicular lymphomas (PTLs) are extranodal large B-cell lymphomas (LBCLs) with inferior responses to current empiric treatment regimens. To identify targetable genetic features of PCNSL and PTL, we characterized their recurrent somatic mutations, chromosomal rearrangements, copy number alterations (CNAs), and associated driver genes, and compared these comprehensive genetic signatures to those of diffuse LBCL and primary mediastinal large B-cell lymphoma (PMBL). These studies identify unique combinations of genetic alterations in discrete LBCL subtypes and subtype-selective bases for targeted therapy. PCNSLs and PTLs frequently exhibit genomic instability, and near-uniform, often biallelic, CDKN2A loss with rare TP53 mutations. PCNSLs and PTLs also use multiple genetic mechanisms to target key genes and pathways and exhibit near-uniform oncogenic Toll-like receptor signaling as a result of MYD88 mutation and/or NFKBIZ amplification, frequent concurrent B-cell receptor pathway activation, and deregulation of BCL6. Of great interest, PCNSLs and PTLs also have frequent 9p24.1/PD-L1/PD-L2 CNAs and additional translocations of these loci, structural bases of immune evasion that are shared with PMBL. PMID:26702065

Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster.

PubMed

Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

2017-01-01

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster , a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT 1A , Dm5-HT 1B , and Dm5-HT 7 couple to cAMP signaling cascades, the Dm5-HT 2A receptor leads to Ca 2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT 2B receptor. Knowledge about this receptor's pharmacological properties is very limited. This is quite surprising because Dm5-HT 2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT 2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT 2B 's pharmacology, we evaluated the receptor's response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT 2B signaling in vitro and in vivo .

The effect of distant metastases sites on survival in de novo stage-IV breast cancer: A SEER database analysis.

PubMed

Wu, San-Gang; Li, Hui; Tang, Li-Ying; Sun, Jia-Yuan; Zhang, Wen-Wen; Li, Feng-Yan; Chen, Yong-Xiong; He, Zhen-Yu

2017-06-01

To investigate the effect of distant metastases sites on survival in patients with de novo stage-IV breast cancer. From 2010 to 2013, patients with a diagnosis of de novo stage-IV breast cancer were identified using the Surveillance, Epidemiology, and End Results database. Univariate and multivariate Cox regression analyses were performed to analyze the effect of distant metastases sites on breast cancer-specific survival and overall survival. A total of 7575 patients were identified. The most common metastatic sites were bone, followed by lung, liver, and brain. Patients with hormone receptor+/human epidermal growth factor receptor 2- and hormone receptor+/human epidermal growth factor receptor 2+ status were more prone to bone metastases. Lung and brain metastases were common in hormone receptor-/human epidermal growth factor receptor 2+ and hormone receptor-/human epidermal growth factor receptor 2- subtypes, and patients with hormone receptor+/ human epidermal growth factor receptor 2+ and hormone receptor-/human epidermal growth factor receptor 2+ subtypes were more prone to liver metastases. Patients with liver and brain metastases had unfavorable prognosis for breast cancer-specific survival and overall survival, whereas bone and lung metastases had no effect on patient survival in multivariate analyses. The hormone receptor-/human epidermal growth factor receptor 2- subtype conferred a significantly poorer outcome in terms of breast cancer-specific survival and overall survival. hormone receptor+/human epidermal growth factor receptor 2+ disease was associated with the best prognosis in terms of breast cancer-specific survival and overall survival. Patients with liver and brain metastases were more likely to experience poor prognosis for breast cancer-specific survival and overall survival by various breast cancer subtypes. Distant metastases sites have differential impact on clinical outcomes in stage-IV breast cancer. Follow-up screening for brain and liver metastases might be effective in improving breast cancer-specific survival and overall survival.

Pharmacological rescue of Ras signaling, GluA1-dependent synaptic plasticity, and learning deficits in a fragile X model.

PubMed

Lim, Chae-Seok; Hoang, Elizabeth T; Viar, Kenneth E; Stornetta, Ruth L; Scott, Michael M; Zhu, J Julius

2014-02-01

Fragile X syndrome, caused by the loss of Fmr1 gene function, is the most common form of inherited mental retardation, with no effective treatment. Using a tractable animal model, we investigated mechanisms of action of a few FDA-approved psychoactive drugs that modestly benefit the cognitive performance in fragile X patients. Here we report that compounds activating serotonin (5HT) subtype 2B receptors (5HT2B-Rs) or dopamine (DA) subtype 1-like receptors (D1-Rs) and/or those inhibiting 5HT2A-Rs or D2-Rs moderately enhance Ras-PI3K/PKB signaling input, GluA1-dependent synaptic plasticity, and learning in Fmr1 knockout mice. Unexpectedly, combinations of these 5HT and DA compounds at low doses synergistically stimulate Ras-PI3K/PKB signal transduction and GluA1-dependent synaptic plasticity and remarkably restore normal learning in Fmr1 knockout mice without causing anxiety-related side effects. These findings suggest that properly dosed and combined FDA-approved psychoactive drugs may effectively treat the cognitive impairment associated with fragile X syndrome.

O6-methylguanine-DNA methyltransferase as a prognostic and predictive marker for basal-like breast cancer treated with cyclophosphamide-based chemotherapy

PubMed Central

ISONO, SAYURI; FUJISHIMA, MAKOTO; AZUMI, TATSUYA; HASHIMOTO, YUKIHIKO; KOMOIKE, YOSHIFUMI; YUKAWA, MASAO; WATATANI, MASAHIRO

2014-01-01

The O6-methylguanine-DNA methyltransferase (MGMT) protein protects cells from alkylating agents by removing alkyl groups from the O6-position of guanine. However, its effect on DNA damage induced by cyclophosphamide (CPM) is unclear. The present study investigated whether MGMT expression was correlated with prognosis in patients with breast cancer that was managed according to a common therapeutic protocol or treated with CPM-based chemotherapy. The intrinsic subtypes and MGMT protein expression levels were assessed in 635 consecutive patients with breast cancer using immunohistochemistry. In total, 425 (67%) luminal A, 95 (15%) luminal B, 47 (7%) human epidermal growth factor receptor-2+/estrogen receptor− (HER2+/ER−) and 48 (8%) basal-like subtypes were identified. Of these, MGMT positivity was identified in 398 (63%) of 635 breast cancers; 68% of luminal A, 67% of luminal B, 30% of HER2+/ER− and 46% of basal-like subtypes were positive. The overall survival (OS) and disease-free survival (DFS) rates did not significantly differ according to the MGMT status among patients with luminal A, luminal B or HER2+/ER− subtypes, and patients with MGMT-negative basal-like cancers tended to have a longer DFS, but not a significantly longer OS time. CPM-containing chemotherapy was administered to 26%, 40%, 47% and 31% of patients with luminal A, luminal B, HER2+/ER− and basal-like tumors, respectively. Although the MGMT status and clinical outcomes of patients with the luminal A, luminal B or HER2+/ER− subtypes treated with CPM were not significantly correlated, the patients with MGMT-negative basal-like tumors who received CPM exhibited significantly improved DFS and OS compared with the CPM-treated patients with MGMT-positive tumors. MGMT may be a useful prognostic and predictive marker for CPM-containing chemotherapy in basal-like breast cancer. PMID:24932232

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide-gated channels.

PubMed

Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre-Olivier; Hell, Stefan W; Ngezahayo, Anaclet

2017-04-15

Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell-to-cell diffusion of ions, metabolites and second messengers. Stimulation of the adenosine receptor subtype A 2B increases the gap junction coupling in the human blood-brain barrier endothelial cell line hCMEC/D3. Although the increased gap junction coupling is cAMP-dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase. We found that cAMP activates cyclic nucleotide-gated (CNG) channels and thereby induces a Ca 2+ influx, which leads to the increase in gap junction coupling. The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood-brain barrier. The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood-brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT-PCR. Scrape loading/dye transfer was used to evaluate the impact of the A 2A and A 2B adenosine receptor subtype agonist 2-phenylaminoadenosine (2-PAA) on the gap junction coupling. We found that 2-PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration-dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2-PAA-related enhancement of gap junction coupling. In contrast, the cyclic nucleotide-gated (CNG) channel inhibitor l-cis-diltiazem, as well as the chelation of intracellular Ca 2+ with BAPTA, or the absence of external Ca 2+ , suppressed the 2-PAA-related enhancement of gap junction coupling. Moreover, we observed a 2-PAA-dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca 2+ influx by opening CNG channels in a cAMP-dependent manner. Ca 2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor-dependent signalling of endothelial cells of the blood-brain barrier. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Intrinsic subtypes from PAM50 gene expression assay in a population-based breast cancer cohort: Differences by age, race, and tumor characteristics

PubMed Central

Sweeney, Carol; Bernard, Philip S.; Factor, Rachel E.; Kwan, Marilyn L.; Habel, Laurel A.; Quesenberry, Charles P.; Shakespear, Kaylynn; Weltzien, Erin K.; Stijleman, Inge J.; Davis, Carole A.; Ebbert, Mark T.W.; Castillo, Adrienne; Kushi, Lawrence H.; Caan, Bette J.

2014-01-01

Background Data are lacking to describe gene expression-based breast cancer intrinsic subtype patterns for population-based patient groups. Methods We studied a diverse cohort of women with breast cancer from the Life After Cancer Epidemiology (LACE) and Pathways studies. RNA was extracted from 1 mm punches from fixed tumor tissue. Quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR) was conducted for the 50 genes that comprise the PAM50 intrinsic subtype classifier. Results In a subcohort of 1,319 women, the overall subtype distribution based on PAM50 was 53.1% Luminal A, 20.5% Luminal B, 13.0% HER2-enriched, 9.8% Basal-like, and 3.6% Normal-like. Among low-risk endocrine positive tumors (i.e. estrogen and progesterone receptor positive by immunohistochemistry, Her2 negative, and low histologic grade), only 76.5% were categorized as Luminal A by PAM50. Continuous-scale Luminal A, Luminal B, HER2-enriched, and Normal-like scores from PAM50 were mutually positively correlated; Basal-like score was inversely correlated with other subtypes. The proportion with non-Luminal A subtype decreased with older age at diagnosis, p trend < 0.0001. Compared with non-Hispanic whites, African-American women were more likely to have Basal-like tumors, age-adjusted odds ratio (OR) 4.4 (95% CI 2.3,8.4), whereas Asian and Pacific Islander women had reduced odds of Basal-like subtype, OR 0.5 (95% CI 0.3,0.9). Conclusions Our data indicate that over 50% of breast cancers treated in the community have Luminal A subtype. Gene expression-based classification shifted some tumors categorized as low risk by surrogate clinicopathological criteria to higher-risk subtypes. Impact Subtyping in a population-based cohort revealed distinct profiles by age and race. PMID:24521995

Purinergic P2X receptors: structural models and analysis of ligand-target interaction.

PubMed

Dal Ben, Diego; Buccioni, Michela; Lambertucci, Catia; Marucci, Gabriella; Thomas, Ajiroghene; Volpini, Rosaria

2015-01-07

The purinergic P2X receptors are ligand-gated cation channels activated by the endogenous ligand ATP. They assemble as homo- or heterotrimers from seven cloned subtypes (P2X1-7) and all trimer subunits present a common topology consisting in intracellular N- and C- termini, two transmembrane domains and a large extracellular domain. These membrane proteins are present in virtually all mammalian tissues and regulate a large variety of responses in physio- and pathological conditions. The development of ligands that selectively activate or block specific P2X receptor subtypes hence represents a promising strategy to obtain novel pharmacological tools for the treatment of pain, cancer, inflammation, and neurological, cardiovascular, and endocrine diseases. The publication of the crystal structures of zebrafish P2X4 receptor in inactive and ATP-bound active forms provided structural data for the analysis of the receptor structure, the interpretation of mutagenesis data, and the depiction of ligand binding and receptor activation mechanism. In addition, the availability of ATP-competitive ligands presenting selectivity for P2X receptor subtypes supports the design of new potent and selective ligands with possibly improved pharmacokinetic profiles, with the final aim to obtain new drugs. This study describes molecular modelling studies performed to develop structural models of the human and rat P2X receptors in inactive and active states. These models allowed to analyse the role of some non-conserved residues at ATP binding site and to study the receptor interaction with some non-specific or subtype selective agonists and antagonists. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

The role of striatal NMDA receptors in drug addiction.

PubMed

Ma, Yao-Ying; Cepeda, Carlos; Cui, Cai-Lian

2009-01-01

The past decade has witnessed an impressive accumulation of evidence indicating that the excitatory amino acid glutamate and its receptors, in particular the N-methyl-D-aspartate (NMDA) receptor subtype, play an important role in drug addiction. Various lines of research using animal models of drug addiction have demonstrated that drug-induced craving is accompanied by significant upregulation of NR2B subunit expression. Furthermore, selective blockade of NR2B-containing NMDA receptors in the striatum, especially in the nucleus accumbens (NAc) can inhibit drug craving and reinstatement. The purpose of this review is to examine the role of striatal NMDA receptors in drug addiction. After a brief description of glutamatergic innervation and NMDA receptor subunit distribution in the striatum, we discuss potential mechanisms to explain the role of striatal NMDA receptors in drug addiction by elucidating signaling cascades involved in the regulation of subunit expression and redistribution, phosphorylation of receptor subunits, as well as activation of intracellular signals triggered by drug experience. Understanding the mechanisms regulating striatal NMDA receptor changes in drug addiction will provide more specific and rational targets to counteract the deleterious effects of drug addiction.

The 5-HT{sub 2A} serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

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

Sonier, Brigitte; Arseneault, Madeleine; Institut National de la Recherche Scientifique-Institut Armand-Frappier, Montreal, Que.

2006-05-19

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT{sub 2A} serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTTmore » proliferation assay. We have demonstrated that the 5-HT{sub 2A} receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT{sub 2A} receptor present in this cell line is identical to the 5-HT{sub 2A} receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT{sub 2A} receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT{sub 2A} receptor subtype, which is fully expressed in this cell line.« less

Activation of Adenosine A2A Receptors Inhibits Neutrophil Transuroepithelial Migration ▿

PubMed Central

Säve, Susanne; Mohlin, Camilla; Vumma, Ravi; Persson, Katarina

2011-01-01

Adenosine has been identified as a significant inhibitor of inflammation by acting on adenosine A2A receptors. In this study, we examined the role of adenosine and A2A receptors in the transmigration of human neutrophils across an in vitro model of the transitional bladder urothelium. Human uroepithelial cells (UROtsa) were grown on transwell inserts; uropathogenic Escherichia coli (UPEC) and neutrophils were added to the transwell system; and the number of migrating neutrophils was evaluated. Reverse transcription-PCR (RT-PCR), immunohistochemistry, and flow cytometry were used to investigate the expression of adenosine receptors, the epithelial adhesion molecule ICAM-1, and the neutrophil integrin CD11b. Levels of proinflammatory interleukin-8 (IL-8) and phosphorylated IκBα were measured by enzyme-linked immunosorbent assays (ELISA) and Luminex assays, respectively. The neutrophils expressed all four adenosine receptor subtypes (A1, A2A, A2B, and A3 receptors), but A3 receptors were not expressed by UROtsa cells. UPEC stimulated neutrophil transuroepithelial migration, which was significantly decreased in response to the specific A2A receptor agonist CGS 21680. The inhibitory effect of CGS 21680 on neutrophil migration was reversed by the A2A receptor antagonist SCH 58261. The production of chemotactic IL-8 and the expression of the adhesion molecule ICAM-1 or CD11b were not significantly affected by CGS 21680. However, a significant decrease in the level of phosporylated IκBα was revealed in response to CGS 21680. In conclusion, UPEC infection in vitro evoked neutrophil migration through a multilayered human uroepithelium. The UPEC-evoked neutrophil transmigration decreased in response to A2A receptor activation, possibly through inhibition of NF-κB signaling pathways. PMID:21646447

α1b-Adrenergic Receptor Localization and Relationship to the D1-Dopamine Receptor in the Rat Nucleus Accumbens.

PubMed

Mitrano, Darlene A; Jackson, Kelsey; Finley, Samantha; Seeley, Allison

2018-02-10

The α1-adrenergic receptors (α1ARs) have been implicated in numerous actions of the brain, including attention and wakefulness. Additionally, they have been identified as contributing to disorders of the brain, such as drug addiction, and recent work has shown a role of these receptors in relapse to psychostimulants. While some functionality is known, the actual subcellular localization of the subtypes of the α1ARs remains to be elucidated. Further, their anatomical relationship to receptors for other neurotransmitters, such as dopamine (DA), remains unclear. Therefore, using immunohistochemistry and electron microscopy techniques, this study describes the subcellular localization of the α1b-adrenergic receptor (α1bAR), the subtype most tied to relapse behaviors, as well as its relationship to the D1-dopamine receptor (D1R) in both the shell and core of the rat nucleus accumbens (NAc). Overall, α1bARs were found in unmyelinated axons and axon terminals with some labeling in dendrites. In accordance with other studies of the striatum, the D1R was found mainly in dendrites and spines; therefore, colocalization of the D1R with the α1bAR was rare postsynaptically. However, in the NAc shell, when the receptors were co-expressed in the same neuronal elements there was a trend for both receptors to be found on the plasma membrane, as opposed to the intracellular compartment. This study provides valuable anatomical information about the α1bAR and its relationship to the D1R and the regulation of DA and norepinephrine (NE) neurotransmission in the brain which have been examined previously. Published by Elsevier Ltd.

Learning and Memory Impairments in a Congenic C57BL/6 Strain of Mice That Lacks the M2 Muscarinic Acetylcholine Receptor Subtype

PubMed Central

Bainbridge, Natalie K.; Koselke, Lisa R.; Jeon, Jongrye; Bailey, Kathleen R.; Wess, Jürgen; Crawley, Jacqueline N.; Wrenn, Craige C.

2009-01-01

The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M1-M5). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M2 muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M2 muscarinic receptor gene (M2−/− mice). Comprehensive assessment of general health and neurological function found no major differences between M2−/− and wild-type (M2+/+) mice. In tests of learning and memory, M2−/− mice were impaired in the acquisition (trials to criterion), but not the retention (72 hr) of a passive avoidance task. In a novel open field, M2−/− mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M2−/− mice committed more errors in working memory than M2+/+ mice. Reference memory did not differ between the genotypes. M2−/− mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M2 receptor plays a critical role in specific components of cognitive abilities. PMID:18346798

Regulation of IP 3 Receptors by IP 3 and Ca 2+

NASA Astrophysics Data System (ADS)

Taylor, Colin W.; Swatton, Jane E.

Inositol 1,4,5-trisphosphate ( IP 3) receptors are intracellular Ca 2+ channels that mediate release of Ca 2+ from intracellular stores. The channels are oligomeric assemblies of four subunits, each of which has an N-terminal IP 3-binding domain and each of which contributes to formation of the Ca 2+ channel. In mammals, three different genes encode IP 3 receptors subunits and the type 1 receptor (and perhaps the type 2 receptor) is also expressed as splice variants. Further diversity arises from assembly of the receptor in hetero- and homo-tetrameric channels. The subtypes differ in their expression and regulation, but they share the key property of being regulated by both IP3 and cytosolic Ca 2+. All three mammalian IP 3 subtypes, and probably also the IP 3 receptors expressed in invertebrates, are biphasically regulated by cytosolic Ca2+, although the underlying mechanisms appear to differ between subtypes. The interactions between IP 3 and Ca 2+ in controlling IP 3 receptor gating, and the physiological significance of such regulation will be reviewed.

Tachykinin receptors and the airways.

PubMed

Frossard, N; Advenier, C

1991-01-01

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.

Blocking α4β2 and α7 nicotinic acetylcholine receptors inhibits the reinstatement of morphine-induced CPP by drug priming in mice.

PubMed

Feng, Bin; Xing, Jiang-hao; Jia, Dong; Liu, Shui-bing; Guo, Hong-ju; Li, Xiao-qiang; He, Xiao-sheng; Zhao, Ming-gao

2011-06-20

Investigating the interaction between nicotinic and opioid receptors is of great interest for both basic mechanistic and clinical reasons. Morphine and nicotine, two common drugs of abuse, share several behavioral and rewarding properties. However, little is known about the subtypes of nicotinic acetylcholine receptors (nAChR) in the reinstatement of morphine-induced conditioned place preference (CPP). In this study, we found that a non-specific nAChR agonist, nicotine (0.5mg/kg), had no effects on the reinstatement of morphine-induced CPP. However, we found that pretreatment with specific α(4)β(2) and α(7) nAChR subtype antagonists, dihydroxy-β-erithroidine (DHβE, 5mg/kg) and methyllycaconitine (MLA, 4 mg/kg), 20 min prior to administration of morphine, inhibited the reinstatement of morphine-induced CPP by drug priming in mice. Furthermore, depression of the reinstatement of morphine-induced CPP by a single DHβE or MLA treatment lasted at least three days later when the reinstatement was induced by morphine priming. The data suggest that specific nAChR subtypes, i.e., α(4)β(2) and α(7), may contribute to the reinstatement of morphine-induced CPP by drug priming in mice. Copyright © 2011 Elsevier B.V. All rights reserved.

Tamoxifen therapy improves overall survival in luminal A subtype of ductal carcinoma in situ: a study based on nationwide Korean Breast Cancer Registry database.

PubMed

Hwang, Ki-Tae; Kim, Eun-Kyu; Jung, Sung Hoo; Lee, Eun Sook; Kim, Seung Il; Lee, Seokwon; Park, Heung Kyu; Kim, Jongjin; Oh, Sohee; Kim, Young A

2018-06-01

To determine the prognostic role of tamoxifen therapy for patients with ductal carcinoma in situ (DCIS) according to molecular subtypes. Data of 14,944 patients with DCIS were analyzed. Molecular subtypes were classified into four categories based on expression of estrogen receptor (ER)/progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). Kaplan-Meier estimator was used for overall survival analysis while Cox proportional hazards model was used for univariate and multivariate analyses. Luminal A subtype (ER/PR+, HER2-) showed higher (P = .009) survival rate than triple-negative (TN) subtype. Tamoxifen therapy group showed superior (P < .001) survival than no-tamoxifen therapy group. It had survival benefit only for luminal A subtype (P = .001). Tamoxifen therapy resulted in higher survival rate in subgroups with positive ER (P = .006), positive PR (P = .009), and negative HER2 (P < .001). In luminal A subtype, tamoxifen therapy showed lower hazard ratio (HR) compared to no-tamoxifen therapy (HR, 0.420; 95% CI 0.250-0.705; P = .001). Tamoxifen therapy was a significant independent factor by multivariate analysis (HR, 0.538; 95% CI 0.306-0.946; P = .031) as well as univariate analysis. Tamoxifen therapy group showed superior prognosis than the no-tamoxifen therapy group. Its prognostic influence was only effective for luminal A subtype. Patients with luminal A subtype showed higher survival rate than those with TN subtype. Active tamoxifen therapy is recommended for DCIS patients with luminal A subtype, and routine tests for ER, PR, and HER2 should be considered for DCIS.

Predicting Subtype Selectivity for Adenosine Receptor Ligands with Three-Dimensional Biologically Relevant Spectrum (BRS-3D)

NASA Astrophysics Data System (ADS)

He, Song-Bing; Ben Hu; Kuang, Zheng-Kun; Wang, Dong; Kong, De-Xin

2016-11-01

Adenosine receptors (ARs) are potential therapeutic targets for Parkinson’s disease, diabetes, pain, stroke and cancers. Prediction of subtype selectivity is therefore important from both therapeutic and mechanistic perspectives. In this paper, we introduced a shape similarity profile as molecular descriptor, namely three-dimensional biologically relevant spectrum (BRS-3D), for AR selectivity prediction. Pairwise regression and discrimination models were built with the support vector machine methods. The average determination coefficient (r2) of the regression models was 0.664 (for test sets). The 2B-3 (A2B vs A3) model performed best with q2 = 0.769 for training sets (10-fold cross-validation), and r2 = 0.766, RMSE = 0.828 for test sets. The models’ robustness and stability were validated with 100 times resampling and 500 times Y-randomization. We compared the performance of BRS-3D with 3D descriptors calculated by MOE. BRS-3D performed as good as, or better than, MOE 3D descriptors. The performances of the discrimination models were also encouraging, with average accuracy (ACC) 0.912 and MCC 0.792 (test set). The 2A-3 (A2A vs A3) selectivity discrimination model (ACC = 0.882 and MCC = 0.715 for test set) outperformed an earlier reported one (ACC = 0.784). These results demonstrated that, through multiple conformation encoding, BRS-3D can be used as an effective molecular descriptor for AR subtype selectivity prediction.

Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness.

PubMed

Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

2015-01-01

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel 1a 1.4, mel 1a 1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0-15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

Ultradian oscillation in expression of four melatonin receptor subtype genes in the pineal gland of the grass puffer, a semilunar-synchronized spawner, under constant darkness

PubMed Central

Ikegami, Taro; Maruyama, Yusuke; Doi, Hiroyuki; Hattori, Atsuhiko; Ando, Hironori

2015-01-01

Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel1a1.4, mel1a1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0–15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer. PMID:25688184

Differential Regulation of Endosomal GPCR/β-Arrestin Complexes and Trafficking by MAPK*

PubMed Central

Khoury, Etienne; Nikolajev, Ljiljana; Simaan, May; Namkung, Yoon; Laporte, Stéphane A.

2014-01-01

β-Arrestins are signaling adaptors that bind to agonist-occupied G protein-coupled receptors (GPCRs) and target them for endocytosis; however, the mechanisms regulating receptor/β-arrestin complexes and trafficking in endosomes, remain ill defined. Here we show, in live cells, differential dynamic regulation of endosomal bradykinin B2 receptor (B2R) complexes with either β-arrestin-1 or -2. We find a novel role for MAPK in the B2R/β-arrestin-2 complex formation, receptor trafficking and signaling mediated by an ERK1/2 regulatory motif in the hinge domain of the rat β-arrestin-2 (PET178P), but not rat β-arrestin-1 (PER177P). While the ERK1/2 regulatory motif is conserved between rat and mouse β-arrestin-2, it is surprisingly not conserved in human β-arrestin-2 (PEK178P). However, mutation of lysine 178 to threonine is sufficient to confer MAPK sensitivity to the human β-arrestin-2. Furthermore, substitution for a phosphomimetic residue in both the rat and the human β-arrestin-2 (T/K178D) significantly stabilizes B2R/β-arrestin complexes in endosomes, delays receptor recycling to the plasma membrane and maintains intracellular MAPK signaling. Similarly, the endosomal trafficking of β2-adrenergic, angiotensin II type 1 and vasopressin V2 receptors was altered by the β-arrestin-2 T178D mutant. Our findings unveil a novel subtype specific mode of MAPK-dependent regulation of β-arrestins in intracellular trafficking and signaling of GPCRs, and suggest differential endosomal receptor/β-arrestin-2 signaling roles among species. PMID:25016018

Modulation of the adaptive response to stress by brain activation of selective somatostatin receptor subtypes.

PubMed

Stengel, Andreas; Rivier, Jean; Taché, Yvette

2013-04-01

Somatostatin-14 was discovered in 1973 in the hypothalamus as a peptide inhibiting growth hormone release. Somatostatin interacts with five receptor subtypes (sst(1-5)) which are widely distributed in the brain with a distinct, but overlapping, expression pattern. During the last few years, the development of highly selective peptide agonists and antagonists provided new insight to characterize the role of somatostatin receptor subtypes in the pleiotropic actions of somatostatin. Recent evidence in rodents indicates that the activation of selective somatostatin receptor subtypes in the brain blunts stress-corticotropin-releasing factor (CRF) related ACTH release (sst2/5), sympathetic-adrenal activaton (sst5), stimulation of colonic motility (sst1), delayed gastric emptying (sst5), suppression of food intake (sst2) and the anxiogenic-like (sst2) response. These findings suggest that brain somatostatin signaling pathways may play an important role in dampening CRF-mediated endocrine, sympathetic, behavioral and visceral responses to stress. Published by Elsevier Inc.

Differential expression of muscarinic acetylcholine receptor subtypes in Jurkat cells and their signaling.

PubMed

Alea, Mileidys Perez; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Fuxe, Kjell; Garriga, Pere

2011-08-15

Muscarinic acetylcholine receptors expression and signaling in the human Jurkat T cell line were investigated. Semiquantitative real-time PCR and radioligand binding studies, using a wide set of antagonist compounds, showed the co-existence of M(3), M(4), and M(5) subtypes. Stimulation of these subpopulations caused a concentration and time- dependent activation of second messengers and ERK signaling pathways, with a major contribution of the M(3) subtype in a G(q/11)-mediated response. In addition, we found that T-cell stimulation leads to increased expression of M(3) and M(5) both at transcriptional and protein levels in a PLC/PKCθ dependent manner. Our data clarifies the functional role of AChR subtypes in Jurkat cells and pave the way to future studies on the potential cross-talk among these subpopulations and their regulation of T lymphocytes immune function. Copyright © 2011 Elsevier B.V. All rights reserved.

Antinociceptive synergism of MD-354 and clonidine. Part II. The alpha-adrenoceptor component.

PubMed

Young, Shawquia; Vainio, Minna; Scheinin, Mika; Dukat, Małgorzata

2010-08-01

Previously, we reported that antinociceptive synergism of a 5-HT(3)/alpha(2)-adrenoceptor ligand MD-354 (m-chlorophenylguanidine) and clonidine combination occurs, in part, through a 5-HT(3) receptor antagonist mechanism. In the present investigation, a possible role for alpha(2)-adrenoceptors was examined. Mechanistic studies using yohimbine (a subtype non-selective alpha(2)-adrenoceptor antagonist), BRL 44408 (a preferential alpha(2A)-adrenoceptor antagonist) and imiloxan (a preferential alpha(2B/C)-adrenoceptor antagonist) on the antinociceptive actions of a MD-354/clonidine combination were conducted. Subcutaneous pre-treatment with all three antagonists inhibited the antinociceptive synergism of MD-354 and clonidine in the mouse tail-flick assay in a dose-dependent manner (AD(50) = 0.33, 2.1, and 0.17 mg/kg, respectively). Enhancement of clonidine antinociception by MD-354 did not potentiate clonidine's locomotor suppressant activity in a mouse locomotor assay. When [ethyl-3H]RS-79948-197 was used as radioligand, MD-354 displayed almost equal affinity to alpha(2A)- and alpha(2B)-adrenoceptors (K(i) = 110 and 220 nM) and showed lower affinity at alpha(2C)-adrenoceptors (K(i) = 4,700 nM). MD-354 had no subtype-selectivity for the alpha(2)-adrenoceptor subtypes as an antagonist in functional [35S]GTPgammaS binding assays. MD-354 was a weak partial agonist at alpha(2A)-adrenoceptors. Overall, in addition to the 5-HT(3) receptor component, the present investigation found MD-354 to be a weak partial alpha(2A)-adrenoceptor agonist that enhances clonidine's thermal antinociceptive actions through an alpha(2)-adrenoceptor-mediated mechanism without augmenting sedation.

A Review of the Updated Pharmacophore for the Alpha 5 GABA(A) Benzodiazepine Receptor Model

PubMed Central

Clayton, Terry; Poe, Michael M.; Rallapalli, Sundari; Biawat, Poonam; Savić, Miroslav M.; Rowlett, James K.; Gallos, George; Emala, Charles W.; Kaczorowski, Catherine C.; Stafford, Douglas C.; Arnold, Leggy A.; Cook, James M.

2015-01-01

An updated model of the GABA(A) benzodiazepine receptor pharmacophore of the α5-BzR/GABA(A) subtype has been constructed prompted by the synthesis of subtype selective ligands in light of the recent developments in both ligand synthesis, behavioral studies, and molecular modeling studies of the binding site itself. A number of BzR/GABA(A) α5 subtype selective compounds were synthesized, notably α5-subtype selective inverse agonist PWZ-029 (1) which is active in enhancing cognition in both rodents and primates. In addition, a chiral positive allosteric modulator (PAM), SH-053-2′F-R-CH3 (2), has been shown to reverse the deleterious effects in the MAM-model of schizophrenia as well as alleviate constriction in airway smooth muscle. Presented here is an updated model of the pharmacophore for α5β2γ2 Bz/GABA(A) receptors, including a rendering of PWZ-029 docked within the α5-binding pocket showing specific interactions of the molecule with the receptor. Differences in the included volume as compared to α1β2γ2, α2β2γ2, and α3β2γ2 will be illustrated for clarity. These new models enhance the ability to understand structural characteristics of ligands which act as agonists, antagonists, or inverse agonists at the Bz BS of GABA(A) receptors. PMID:26682068

Pirenzepine binding to membrane-bound, solubilized and purified muscarinic receptor subtypes

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

Baumgold, J.

1986-05-01

Muscarinic receptors were purified to near-homogeneity from bovine cortex, an area rich in the putative M1 subtype, and from bovine pons/medulla, an area rich in the putative M2 subtype. In both cases, the receptors were solubilized in digitonin and purified over an affinity column. Both the cortical and pons/medulla preparations yielded receptor proteins of 70,000 daltons. Pirenzepine binding was deduced from its competition with /sup 3/H-N-methyl scopolamine. The binding of pirenzepine to membrane-bound receptors from cortex was best described by a two site model, with approximately half the sites having a Ki of 6.4 x 10/sup -9/ M and themore » remaining sites having a Ki of 3.5 x 10/sup -7/ M. Membrane-bound receptors from pons/medulla bound pirenzepine according to a one-site model with a Ki of 1.1 x 10/sup -7/ M. After solubilization the two-site binding of cortical receptors became a one-site binding, Ki = 1.1 x 10/sup -7/M. This value was still five-fold lower than that of soluble receptors from pons/medulla. After purification however the affinity of pirenzepine for the pons/medulla receptor increased so that the two putative subtypes bound pirenzepine with approximately the same affinity. These findings suggest that the different pirenzepine binding characteristics used to define muscarinic receptor subtypes are not inherent in the receptor protein itself but may be due to coupling factors associated with the receptor.« less

A Dynamic View of Molecular Switch Behavior at Serotonin Receptors: Implications for Functional Selectivity

PubMed Central

Martí-Solano, Maria; Sanz, Ferran; Pastor, Manuel; Selent, Jana

2014-01-01

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity. PMID:25313636

A dynamic view of molecular switch behavior at serotonin receptors: implications for functional selectivity.

PubMed

Martí-Solano, Maria; Sanz, Ferran; Pastor, Manuel; Selent, Jana

2014-01-01

Functional selectivity is a property of G protein-coupled receptors that allows them to preferentially couple to particular signaling partners upon binding of biased agonists. Publication of the X-ray crystal structure of serotonergic 5-HT1B and 5-HT2B receptors in complex with ergotamine, a drug capable of activating G protein coupling and β-arrestin signaling at the 5-HT1B receptor but clearly favoring β-arrestin over G protein coupling at the 5-HT2B subtype, has recently provided structural insight into this phenomenon. In particular, these structures highlight the importance of specific residues, also called micro-switches, for differential receptor activation. In our work, we apply classical molecular dynamics simulations and enhanced sampling approaches to analyze the behavior of these micro-switches and their impact on the stabilization of particular receptor conformational states. Our analysis shows that differences in the conformational freedom of helix 6 between both receptors could explain their different G protein-coupling capacity. In particular, as compared to the 5-HT1B receptor, helix 6 movement in the 5-HT2B receptor can be constrained by two different mechanisms. On the one hand, an anchoring effect of ergotamine, which shows an increased capacity to interact with the extracellular part of helices 5 and 6 and stabilize them, hinders activation of a hydrophobic connector region at the center of the receptor. On the other hand, this connector region in an inactive conformation is further stabilized by unconserved contacts extending to the intracellular part of the 5-HT2B receptor, which hamper opening of the G protein binding site. This work highlights the importance of considering receptor capacity to adopt different conformational states from a dynamic perspective in order to underpin the structural basis of functional selectivity.

The adrenergic receptor subtypes present in frog (Rana esculenta) skin.

PubMed

Bellantuono, Vito; Cassano, Giuseppe; Lippe, Claudio

2008-08-01

Frog skin transports ions and water under hormonal control. In spite of the fundamental role played by adrenergic stimulation in maintaining the water balance of the organism, the receptor subtype(s) present in the skin have not been identified yet. We measured the increase in short-circuit current (ISC, an estimate of ion transport) induced by cirazoline, clonidine, xamoterol, formoterol, or BRL 37344, in order to verify the presence of alpha1, alpha2, beta1, beta2, or beta3 receptor subtypes, respectively. Only after treatment with formoterol, BRL 37344 and, to a lesser extent, cirazoline was measured a significant increase in ISC (57%, 33.2%, and 4.7%, respectively). The formoterol and BRL 37344 concentrations producing half-maximal effect (EC50) were 1.12 and 70.1 nM, respectively. Moreover, the formoterol effect was inhibited by treatment with ICI 118551 (antagonist of beta2 receptors) while SR 59230A (antagonist of beta3 receptors) had no effect; opposite findings were obtained when the BRL 37344 stimulation was investigated. Finally, by measuring the transepithelial fluxes of 22Na+ and 36Cl-, we demonstrated that Na+ absorption is increased by activation of beta2 and beta3 and is cAMP-sensitive, whereas the Cl- secretion is only increased by activation of beta2 receptors and is cAMP- and calmodulin-sensitive.

Classics in Neuroimaging: The Serotonergic 2A Receptor System-from Discovery to Modern Molecular Imaging.

PubMed

T L'Estrade, Elina; Hansen, Hanne D; Erlandsson, Maria; Ohlsson, Tomas G; Knudsen, Gitte M; Herth, Matthias M

2018-06-20

Already in 1953, Woolley and Shaw speculated that serotonin could be involved in a range of central nervous system (CNS) disorders. Lysergic acid diethylamide (LSD) displayed an important role in this respect. It was used not only to antagonize biological effects of serotonin and to study the system itself, but also to identify serotonergic subtype receptors. The 5-HT 2A receptor was discovered in the 1970s and identified as the responsible receptor mediating psychedelic effects of LSD. The development of positron emission tomography (PET) allowed to study this receptor system in vivo. Parameters such as abundance of 5-HT 2A neuroreceptors or receptor occupancy can be determined using PET. As such, the development of 5-HT 2A receptor tracers started immediately after the introduction of PET in the mid-1970s. In this Viewpoint, we provide a historical overview from the discovery of serotonin to the identification of the 5-HT 2A receptor subtype and the subsequent development of 5-HT 2A receptor subtype specific PET tracers over the last four decades. We emphasize the interplay between pharmacology, medicinal chemistry, radiochemistry, and nuclear medicine that is important while developing a PET tracer. Moreover, we highlight selected examples applying 5-HT 2A receptor PET tracers within neurological diseases and drug occupancy studies.

Partial agonist clonidine mediates alpha(2)-AR subtypes specific regulation of cAMP accumulation in adenylyl cyclase II transfected DDT1-MF2 cells.

PubMed

Limon-Boulez, I; Bouet-Alard, R; Gettys, T W; Lanier, S M; Maltier, J P; Legrand, C

2001-02-01

alpha2-Adrenergic receptor (alpha(2)-AR) activation in the pregnant rat myometrium at midterm potentiates beta(2)-AR stimulation of adenylyl cyclase (AC) via Gbetagamma regulation of the type II isoform of adenylyl cyclase. However, at term, alpha(2)-AR activation inhibits beta(2)-AR stimulation of AC. This phenomenon is associated with changes in alpha(2)-AR subtype expression (midterm alpha(2A/D)-AR > alpha(2B)-AR; term alpha(2B) >or =alpha(2A/D)-AR), without any change in ACII mRNA, suggesting that alpha(2A/D)- and alpha(2B)-AR differentially regulate beta(2)-cAMP production. To address this issue, we have stably expressed the same density of alpha(2A/D)- or alpha(2B)-AR with AC II in DDT1-MF2 cells. Clonidine (partial agonist) increased beta(2)-AR-stimulated cAMP production in alpha(2A/D)-AR-ACII transfectants but inhibited it in alpha(2B)-AR-ACII transfectants. In contrast, epinephrine (full agonist) enhanced beta(2)-stimulated ACII in both alpha(2A)- and alpha(2B)-ACII clonal cell lines. 4-Azidoanilido-[alpha-(32)P]GTP-labeling of activated G proteins indicated that, in alpha(2B)-AR transfectants, clonidine activated only Gi(2), whereas epinephrine, the full agonist, effectively coupled to Gi(2) and Gi(3). Thus, partial and full agonists selectively activate G proteins that lead to drug specific effects on effectors. Moreover, these data indicate that Gi(3) activation is required for potentiation of beta(2)-AR stimulation of AC by alpha(2A/D) and alpha(2B)-AR in DDT1-MF2 cells. This may reflect an issue of the amount of Gbetagamma released upon receptor activation and/or betagamma composition of Gi(3) versus Gi(2).

The 5-HT1-like receptor mediating the increase in canine external carotid blood flow: close resemblance to the 5-HT1D subtype.

PubMed Central

Villalón, C M; Terrón, J A

1994-01-01

1. It has recently been shown that the increase in external carotid blood flow induced by 5-hydroxy-tryptamine (5-HT) in the anaesthetized dog, being mimicked by 5-carboxamidotryptamine (5-CT), inhibited by methiothepin, vagosympathectomy and sympatho-inhibitory drugs, and resistant to blockade by ritanserin and MDL 72222, is mediated by stimulation of prejunctional 5-HT1-like receptors leading to an inhibitory action on carotid sympathetic nerves; these 5-HT1-like receptors are unrelated to either the 5-HT1A, 5-HT1B or 5-HT1C (now 5-HT2C) receptor subtypes. Inasmuch as 5-CT, 5-methoxytryptamine, sumatriptan and metergoline display high affinity, amongst other 5-HT binding sites, for the 5-HT1D subtype, in the present study we have used these drugs in an attempt to determine whether the above inhibitory prejunctional 5-HT1-like receptors correlate with the 5-HT1D subtype. 2. One-minute intracarotid (i.c.) infusions of 5-HT (0.3, 1, 3 and 10 micrograms), 5-CT (0.01, 0.03, 0.1 and 0.3 micrograms), 5-methoxytryptamine (1, 3, 10 and 30 micrograms) and sumatriptan (1, 3, 10, 30 and 100 micrograms) resulted in dose-dependent increases in external carotid blood flow (without changes in mean arterial blood pressure or heart rate) with the following rank order of agonist potency: 5-CT >> 5-HT > 5-methoxytryptamine > or = sumatriptan. Interestingly, sumatriptan-induced vasodilatation was followed by a more pronounced vasoconstriction. 3. The external carotid vasodilator effects of 5-HT, 5-CT, 5-methoxytryptamine and sumatriptan were dose-dependently and specifically antagonized by metergoline (10, 30 and/or 100 micrograms kg-1, i.v.).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7812603

Adenosine receptors regulate gap junction coupling of the human cerebral microvascular endothelial cells hCMEC/D3 by Ca2+ influx through cyclic nucleotide‐gated channels

PubMed Central

Bader, Almke; Bintig, Willem; Begandt, Daniela; Klett, Anne; Siller, Ina G.; Gregor, Carola; Schaarschmidt, Frank; Weksler, Babette; Romero, Ignacio; Couraud, Pierre‐Olivier; Hell, Stefan W.

2017-01-01

Key points Gap junction channels are essential for the formation and regulation of physiological units in tissues by allowing the lateral cell‐to‐cell diffusion of ions, metabolites and second messengers.Stimulation of the adenosine receptor subtype A2B increases the gap junction coupling in the human blood–brain barrier endothelial cell line hCMEC/D3.Although the increased gap junction coupling is cAMP‐dependent, neither the protein kinase A nor the exchange protein directly activated by cAMP were involved in this increase.We found that cAMP activates cyclic nucleotide‐gated (CNG) channels and thereby induces a Ca2+ influx, which leads to the increase in gap junction coupling.The report identifies CNG channels as a possible physiological link between adenosine receptors and the regulation of gap junction channels in endothelial cells of the blood–brain barrier. Abstract The human cerebral microvascular endothelial cell line hCMEC/D3 was used to characterize the physiological link between adenosine receptors and the gap junction coupling in endothelial cells of the blood–brain barrier. Expressed adenosine receptor subtypes and connexin (Cx) isoforms were identified by RT‐PCR. Scrape loading/dye transfer was used to evaluate the impact of the A2A and A2B adenosine receptor subtype agonist 2‐phenylaminoadenosine (2‐PAA) on the gap junction coupling. We found that 2‐PAA stimulated cAMP synthesis and enhanced gap junction coupling in a concentration‐dependent manner. This enhancement was accompanied by an increase in gap junction plaques formed by Cx43. Inhibition of protein kinase A did not affect the 2‐PAA‐related enhancement of gap junction coupling. In contrast, the cyclic nucleotide‐gated (CNG) channel inhibitor l‐cis‐diltiazem, as well as the chelation of intracellular Ca2+ with BAPTA, or the absence of external Ca2+, suppressed the 2‐PAA‐related enhancement of gap junction coupling. Moreover, we observed a 2‐PAA‐dependent activation of CNG channels by a combination of electrophysiology and pharmacology. In conclusion, the stimulation of adenosine receptors in hCMEC/D3 cells induces a Ca2+ influx by opening CNG channels in a cAMP‐dependent manner. Ca2+ in turn induces the formation of new gap junction plaques and a consecutive sustained enhancement of gap junction coupling. The report identifies CNG channels as a physiological link that integrates gap junction coupling into the adenosine receptor‐dependent signalling of endothelial cells of the blood–brain barrier. PMID:28075020

Prognostic Value of Tumor-Infiltrating Lymphocyte Density Assessed Using a Standardized Method Based on Molecular Subtypes and Adjuvant Chemotherapy in Invasive Breast Cancer.

PubMed

Jang, Nuri; Kwon, Hee Jung; Park, Min Hui; Kang, Su Hwan; Bae, Young Kyung

2018-04-01

This study investigated the prognostic value of tumor-infiltrating lymphocyte (TIL) density as determined by molecular subtype and receipt of adjuvant chemotherapy in invasive breast cancer (IBC). Stromal TIL densities were evaluated in 1489 IBC samples using recommendations proposed by the International TILs Working Group. Cases were allocated to high- and low-TIL density groups using a cutoff of 10%. Of the 1489 IBC patients, 427 (28.7%) were assigned to the high-TIL group and 1062 (71.3%) to the low-TIL group. High TIL density was found to be significantly associated with large tumor size (p = 0.001), high histologic grade (p < 0.001), and high Ki-67 labeling index (p < 0.001). Triple-negative and human epidermal growth factor receptor 2 (HER2)-positive subtypes had significantly higher TIL densities than luminal A or B (HER2-negative) subtypes (p < 0.001). High TIL density was significantly associated with prolonged disease-free survival (DFS) by univariate (p < 0.001) and multivariate (p < 0.001) analyses. In the low-TIL-density group, the patients who did not receive adjuvant chemotherapy showed better DFS (p < 0.001), but no such survival difference was observed in the high-TIL group (p = 0.222). For the patients who received adjuvant anthracycline, high-TIL density was found to be an independent prognostic factor of favorable DFS in the luminal B (HER2-negative; p = 0.003), HER2-positive (p = 0.019), and triple-negative (p = 0.017) subtypes. Measurements of TIL density in routine clinical practice could give useful prognostic information for the triple-negative, HER2-positive, and luminal B (HER2-negative) IBC subtypes, especially for patients administered adjuvant anthracycline.

Synthesis, characterization, and first successful monkey imaging studies of metabotropic glutamate receptor subtype 5 (mGluR5) PET radiotracers.

PubMed

Hamill, Terence G; Krause, Stephen; Ryan, Christine; Bonnefous, Celine; Govek, Steve; Seiders, T Jon; Cosford, Nicholas D P; Roppe, Jeffrey; Kamenecka, Ted; Patel, Shil; Gibson, Raymond E; Sanabria, Sandra; Riffel, Kerry; Eng, Waisi; King, Christopher; Yang, Xiaoqing; Green, Mitchell D; O'Malley, Stacey S; Hargreaves, Richard; Burns, H Donald

2005-06-15

Three metabotropic glutamate receptor subtype 5 (mGluR5) PET tracers have been labeled with either carbon-11 or fluorine-18 and their in vitro and in vivo behavior in rhesus monkey has been characterized. Each of these tracers share the common features of high affinity for mGluR5 (0.08-0.23 nM vs. rat mGluR5) and moderate lipophilicity (log P 2.8-3.4). Compound 1b was synthesized using a Suzuki or Stille coupling reaction with [11C]MeI. Compounds 2b and 3b were synthesized by a SNAr reaction using a 3-chlorobenzonitrile precursor. Autoradiographic studies in rhesus monkey brain slices using 2b and 3b showed specific binding in cortex, caudate, putamen, amygdala, hippocampus, most thalamic nuclei, and lower binding in the cerebellum. PET imaging studies in monkey showed that all three tracers readily enter the brain and provide an mGluR5-specific signal in all gray matter regions, including the cerebellum. The specific signal observed in the cerebellum was confirmed by the autoradiographic studies and saturation binding experiments that showed tracer binding in the cerebellum of rhesus monkeys. In vitro metabolism studies using the unlabeled compounds showed that 1a, 2a, and 3a are metabolized slower by human liver microsomes than by monkey liver microsomes. In vivo metabolism studies showed 3b to be long-lived in rhesus plasma with only one other more polar metabolite observed. (c) 2005 Wiley-Liss, Inc.

Discovery of a novel nicotinic receptor antagonist for the treatment of nicotine addiction: 1-(3-Picolinium)-12-triethylammonium-dodecane dibromide (TMPD).

PubMed

Dwoskin, Linda P; Joyce, B Matthew; Zheng, Guangrong; Neugebauer, Nichole M; Manda, Vamshi K; Lockman, Paul; Papke, Roger L; Bardo, Michael T; Crooks, Peter A

2007-10-15

Limitations in efficacy and high relapse rates of currently available smoking cessation agents reveal the need for more efficacious pharmacotherapies. One strategy is to develop subtype-selective nicotinic receptor (nAChR) antagonists that inhibit nicotine-evoked dopamine (DA) release, the primary neurotransmitter involved in nicotine reward. Simple alkylation of the pyridino N-atom converts nicotine from a potent agonist into a potent antagonist. The classical antagonists, hexamethonium and decamethonium, differentiate between peripheral nAChR subtypes. Using a similar approach, we interconnected varying quaternary ammonium moieties with a lipophilic linker to provide N,N'-bis-nicotinium analogs, affording a lead compound, N,N'-dodecyl-1,12-diyl-bis-3-picolinium dibromide (bPiDDB), which inhibited nicotine-evoked DA release and decreased nicotine self-administration. The current work describes a novel compound, 1-(3-picolinium)-12-triethylammonium-dodecane dibromide (TMPD), a hybrid of bPiDDB and decamethonium. TMPD completely inhibited (IC(50)=500 nM) nicotine-evoked DA release from superfused rat striatal slices, suggesting that TMPD acts as a nAChR antagonist at more than one subtype. TMPD (1 microM) inhibited the response to acetylcholine at alpha3beta4, alpha4beta4, alpha4beta2, and alpha1beta1varepsilondelta receptors expressed in Xenopus oocytes. TMPD had a 2-fold higher affinity than choline for the blood-brain barrier choline transporter, suggesting brain bioavailability. TMPD did not inhibit hyperactivity in nicotine sensitized rats, but significantly and specifically decreased nicotine self-administration. Together, the results suggest that TMPD may have the ability to reduce the rewarding effect of nicotine with minimal side effects, a pharmacological profile indicative of potential clinical utility for the treatment of tobacco dependence.

Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFkappaB signal pathways.

PubMed

Chen, Jinghai; Chen, Yuefeng; Zhu, Weiquan; Han, Yu; Han, Bianmei; Xu, Ruixia; Deng, Linzi; Cai, Yan; Cong, Xiangfeng; Yang, Yuejing; Hu, Shengshou; Chen, Xi

2008-04-15

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.

Occurrence of breast cancer subtypes in adolescent and young adult women

PubMed Central

2012-01-01

Introduction Breast cancers are increasingly recognized as heterogeneous based on expression of receptors for estrogen (ER), progesterone (PR), and human epidermal growth factor receptor 2 (HER2). Triple-negative tumors (ER-/PR-/HER2-) have been reported to be more common among younger women, but occurrence of the spectrum of breast cancer subtypes in adolescent and young adult (AYA) women aged between 15 and 39 years is otherwise poorly understood. Methods Data regarding all 5,605 AYA breast cancers diagnosed in California during the period 2005 to 2009, including ER and PR status (referred to jointly as hormone receptor (HR) status) and HER2 status, was obtained from the population-based California Cancer Registry. Incidence rates were calculated by subtype (triple-negative; HR+/HER2-; HR+/HER2+; HR-/HER2+), and logistic regression was used to evaluate differences in subtype characteristics by age group. Results AYAs had higher proportions of HR+/HER2+, triple-negative and HR-/HER2+ breast cancer subtypes and higher proportions of patients of non-White race/ethnicity than did older women. AYAs also were more likely to be diagnosed with stage III/IV disease and high-grade tumors than were older women. Rates of HR+/HER2- and triple-negative subtypes in AYAs varied substantially by race/ethnicity. Conclusions The distribution of breast cancer subtypes among AYAs varies from that observed in older women, and varies further by race/ethnicity. Observed subtype distributions may explain the poorer breast cancer survival previously observed among AYAs. PMID:22452927

Identification and pathogenomic analysis of an Escherichia coli strain producing a novel Shiga toxin 2 subtype

USDA-ARS?s Scientific Manuscript database

Shiga toxin (Stx) is the key virulent factor in Shiga toxin-producing Escherichia coli (STEC). To date, three Stx1 subtypes and Seven Stx2 subtypes have been described in E. coli, which were found to differ in receptor preference and toxin potency. Here, we identified a novel Stx2 subtype designated...

An Inotropic Action Caused by Muscarinic Receptor Subtype 3 in Canine Cardiac Purkinje Fibers

PubMed Central

Urushidani, Tetsuro; Tachibana, Shigehiro

2013-01-01

Objective. The objective of this study was to investigate the inotropic mechanisms and the related muscarinic receptor subtype of acetylcholine (ACh) in canine cardiac Purkinje fibers. Materials and Methods. Isolated Purkinje fiber bundles were used for the measurement of contraction. The receptor subtype was determined using PCR and real-time PCR methods. Results. ACh evoked a biphasic response with a transient negative inotropic effect followed by a positive inotropic effect in a concentration-dependent manner. The biphasic inotropic actions of ACh were inhibited by the pretreatment with atropine. Caffeine inhibited the positive inotropic effect of ACh. ACh increased inositol-1,4,5-trisphosphate content in the Purkinje fibers, which was abolished by atropine. Muscarinic subtypes 2 (M2) and 3 (M3) mRNAs were detected in the canine Purkinje fibers albeit the amount of M3 mRNA was smaller than M2 mRNA. M1 mRNA was not detected. Conclusion. These results suggest that the positive inotropic action of ACh may be mediated by the activation of IP3 receptors through the stimulation of M3 receptors in the canine cardiac Purkinje fibers. PMID:24260719

Asian ethnicity and breast cancer subtypes: a study from the California Cancer Registry.

PubMed

Telli, Melinda L; Chang, Ellen T; Kurian, Allison W; Keegan, Theresa H M; McClure, Laura A; Lichtensztajn, Daphne; Ford, James M; Gomez, Scarlett L

2011-06-01

The distribution of breast cancer molecular subtypes has been shown to vary by race/ethnicity, highlighting the importance of host factors in breast tumor biology. We undertook the current analysis to determine population-based distributions of breast cancer subtypes among six ethnic Asian groups in California. We defined immunohistochemical (IHC) surrogates for each breast cancer subtype among Chinese, Japanese, Filipina, Korean, Vietnamese, and South Asian patients diagnosed with incident, primary, invasive breast cancer between 2002 and 2007 in the California Cancer Registry as: hormone receptor-positive (HR+)/HER2- [estrogen receptor-positive (ER+) and/or progesterone receptor-positive (PR+), human epidermal growth factor receptor 2-negative (HER2-)], triple-negative (ER-, PR-, and HER2-), and HER2-positive (ER±, PR±, and HER2+). We calculated frequencies of breast cancer subtypes among Asian ethnic groups and evaluated their associations with clinical and demographic factors. Complete IHC data were available for 8,140 Asian women. Compared to non-Hispanic White women, Korean [odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.5-2.2], Filipina (OR = 1.3, 95% CI = 1.2-1.5), Vietnamese (OR = 1.3, 95% CI = 1.1-1.6), and Chinese (OR = 1.1, 95% CI = 1.0-1.3) women had a significantly increased risk of being diagnosed with HER2-positive breast cancer subtypes after adjusting for age, stage, grade, socioeconomic status, histology, diagnosis year, nativity, and hospital ownership status. We report a significant ethnic disparity in HER2-positive breast cancer in a large population-based cohort enriched for Asian-Americans. Given the poor prognosis and high treatment costs of HER2-positive breast cancer, our results have implications for healthcare resource utilization, cancer biology, and clinical care.

US incidence of breast cancer subtypes defined by joint hormone receptor and HER2 status.

PubMed

Howlader, Nadia; Altekruse, Sean F; Li, Christopher I; Chen, Vivien W; Clarke, Christina A; Ries, Lynn A G; Cronin, Kathleen A

2014-04-28

In 2010, Surveillance, Epidemiology, and End Results (SEER) registries began collecting human epidermal growth factor 2 (HER2) receptor status for breast cancer cases. Breast cancer subtypes defined by joint hormone receptor (HR; estrogen receptor [ER] and progesterone receptor [PR]) and HER2 status were assessed across the 28% of the US population that is covered by SEER registries. Age-specific incidence rates by subtype were calculated for non-Hispanic (NH) white, NH black, NH Asian Pacific Islander (API), and Hispanic women. Joint HR/HER2 status distributions by age, race/ethnicity, county-level poverty, registry, stage, Bloom-Richardson grade, tumor size, and nodal status were evaluated using multivariable adjusted polytomous logistic regression. All statistical tests were two-sided. Among case patients with known HR/HER2 status, 36810 (72.7%) were found to be HR(+)/HER2(-), 6193 (12.2%) were triple-negative (HR(-)/HER2(-)), 5240 (10.3%) were HR(+)/HER2(+), and 2328 (4.6%) were HR(-)/HER2(+); 6912 (12%) had unknown HR/HER2 status. NH white women had the highest incidence rate of the HR(+)/HER2(-) subtype, and NH black women had the highest rate of the triple-negative subtype. Compared with women with the HR(+)/HER2(-) subtype, triple-negative patients were more likely to be NH black and Hispanic; HR(+)/HER2(+) patients were more likely to be NH API; and HR(-)/HER2(+) patients were more likely to be NH black, NH API, and Hispanic. Patients with triple-negative, HR(+)/HER2(+), and HR(-)/HER2(+) breast cancer were 10% to 30% less likely to be diagnosed at older ages compared with HR(+)/HER2(-) patients and 6.4-fold to 20.0-fold more likely to present with high-grade disease. In the future, SEER data can be used to monitor clinical outcomes in women diagnosed with different molecular subtypes of breast cancer for a large portion (approximately 28%) of the US population. Published by Oxford University Press 2014.

In vitro effects of bethanechol on specimens of intestinal smooth muscle obtained from the duodenum and jejunum of healthy dairy cows

PubMed Central

Pfeiffer, Julia B. R.; Mevissen, Meike; Steiner, Adrian; Portier, Christopher J.; Meylan, Mireille

2009-01-01

Objective To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction. Sample Population Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows. Procedures Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (Amax), and area under the curve (AUC) were evaluated. Results Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M3 antagonists (more commonly for basal tone than for Amax and AUC). The M2 receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M3 receptor antagonists were generally more potent than the M2 receptor antagonists. In a protection experiment, an M3 receptor antagonist was less potent than when used in combination with an M2 receptor antagonist. Receptor antagonists for M1 and M4 did not affect contractility variables. Conclusions and Clinical Relevance Bethanechol acting on muscarinic receptor subtypes M2 and M3 may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows. PMID:17331022

NK2 tachykinin receptors and contraction of circular muscle of the human colon: characterization of the NK2 receptor subtype.

PubMed

Giuliani, S; Barbanti, G; Turini, D; Quartara, L; Rovero, P; Giachetti, A; Maggi, C A

1991-10-22

The contractile effect of substance P, neurokinin A, receptor selective agonists for tachykinin receptors and NK2 tachykinin receptor antagonists was investigated in mucosa-free circular strips of the human isolated colon. Neurokinin A and substance P produced concentration-dependent contractions which approached 80-90% of the maximal response to carbachol. Neurokinin A was about 370 times more potent than substance P. The action of neurokinin A and substance P was not modified by peptidase inhibitors (bestatin, captopril and thiorphan, 1 microM each). The NK2 receptor selective agonist, [beta-Ala8]neurokinin A-(4-10) closely mimicked the response to neurokinin A while NK1 and NK3 receptor selective agonists were active only at microM concentrations. The pseudopeptide, MDL 28,564, which is one of the most selective NK2 ligands available, behaved as a full agonist. Responses to [beta-Ala8]neurokinin A were antagonized by NK2 receptor selective antagonists, with the rank order of potency MEN 10,376 greater than L 659,877 much greater than R 396. These data indicate that NK2 tachykinin receptors play a dominant role in determining the contraction of the circular muscle of the human colon to peptides of this family. The NK2 receptor subtype responsible for this effect belongs to the same subtype (NK2A) previously identified in the rabbit pulmonary artery and guinea-pig bronchi.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+.

PubMed

Li, Mufeng; Silberberg, Shai D; Swartz, Kenton J

2013-09-03

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg(2+). Here we investigated the active forms of ATP and found that the action of MgATP(2-) and ATP(4-) differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP(2-) promotes opening with very low efficacy. In contrast, both free ATP and MgATP(2-) robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg(2+) to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP(2-) and weak regulation by Mg(2+). These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP(2-) and regulation by Mg(2+), and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons.

Subtype-specific control of P2X receptor channel signaling by ATP and Mg2+

PubMed Central

Li, Mufeng; Silberberg, Shai D.; Swartz, Kenton J.

2013-01-01

The identity and forms of activating ligands for ion channels are fundamental to their physiological roles in rapid electrical signaling. P2X receptor channels are ATP-activated cation channels that serve important roles in sensory signaling and inflammation, yet the active forms of the nucleotide are unknown. In physiological solutions, ATP is ionized and primarily found in complex with Mg2+. Here we investigated the active forms of ATP and found that the action of MgATP2− and ATP4− differs between subtypes of P2X receptors. The slowly desensitizing P2X2 receptor can be activated by free ATP, but MgATP2− promotes opening with very low efficacy. In contrast, both free ATP and MgATP2− robustly open the rapidly desensitizing P2X3 subtype. A further distinction between these two subtypes is the ability of Mg2+ to regulate P2X3 through a distinct allosteric mechanism. Importantly, heteromeric P2X2/3 channels present in sensory neurons exhibit a hybrid phenotype, characterized by robust activation by MgATP2− and weak regulation by Mg2+. These results reveal the existence of two classes of homomeric P2X receptors with differential sensitivity to MgATP2− and regulation by Mg2+, and demonstrate that both restraining mechanisms can be disengaged in heteromeric channels to form fast and sensitive ATP signaling pathways in sensory neurons. PMID:23959888

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

PubMed Central

Chess-Williams, R.; Chapple, C. R.; Verfurth, F.; Noble, A. J.; Couldwell, C. J.; Michel, M. C.

1996-01-01

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means. PMID:8937710

Peroxisome Proliferator-Activated Receptor Subtype- and Cell-Type-Specific Activation of Genomic Target Genes upon Adenoviral Transgene Delivery

PubMed Central

Nielsen, Ronni; Grøntved, Lars; Stunnenberg, Hendrik G.; Mandrup, Susanne

2006-01-01

Investigations of the molecular events involved in activation of genomic target genes by peroxisome proliferator-activated receptors (PPARs) have been hampered by the inability to establish a clean on/off state of the receptor in living cells. Here we show that the combination of adenoviral delivery and chromatin immunoprecipitation (ChIP) is ideal for dissecting these mechanisms. Adenoviral delivery of PPARs leads to a rapid and synchronous expression of the PPAR subtypes, establishment of transcriptional active complexes at genomic loci, and immediate activation of even silent target genes. We demonstrate that PPARγ2 possesses considerable ligand-dependent as well as independent transactivation potential and that agonists increase the occupancy of PPARγ2/retinoid X receptor at PPAR response elements. Intriguingly, by direct comparison of the PPARs (α, γ, and β/δ), we show that the subtypes have very different abilities to gain access to target sites and that in general the genomic occupancy correlates with the ability to activate the corresponding target gene. In addition, the specificity and potency of activation by PPAR subtypes are highly dependent on the cell type. Thus, PPAR subtype-specific activation of genomic target genes involves an intricate interplay between the properties of the subtype- and cell-type-specific settings at the individual target loci. PMID:16847324

Effect of sex steroid hormones on replication and transmission of major HIV subtypes.

PubMed

Ragupathy, Viswanath; Devadas, Krishnakumar; Tang, Shixing; Wood, Owen; Lee, Sherwin; Dastyer, Armeta; Wang, Xue; Dayton, Andrew; Hewlett, Indira

2013-11-01

The HIV epidemic is expanding worldwide with an increasing number of distinct viral subtypes and circulating recombinant forms (CRFs). Out of 34 million adults living with HIV and AIDS, women account for one half of all HIV-1 infections worldwide. These gender differences in HIV pathogenesis may be attributed to sex hormones. Little is known about the role of sex hormone effects on HIV Subtypes pathogenesis. The aim of our study was to determine sex hormone effects on replication and transmissibility of HIV subtypes. Peripheral blood mononuclear cells (PBMC) and monocyte derived dendritic cells (MDDC) from male and female donors were infected with HIV subtypes A-D and CRF02_AG, CRF01_AE, MN (lab adapted), Group-O, Group-N and HIV-2 at a concentration of 5ng/ml of p24 or p27. Virus production was evaluated by measuring p24 and p27 levels in culture supernatants. Similar experiments were carried out in the presence of physiological concentrations of sex steroid hormones. R5/X4 expressions measured by flow cytometry and transmissibility was evaluated by transfer of HIV from primary dendritic cells (DC) to autologous donor PBMC. Our results from primary PBMC and MDDC from male and female donors indicate in the absence of physiological concentrations of hormone treatment virus production was observed in three clusters; high replicating virus (subtype B and C), moderate replicative virus (subtype A, D, CRF01_AE, Group_N) and least replicative virus (strain MN). However, dose of sex steroid hormone treatment influenced HIV replication and transmission kinetics in PBMC, DCs and cell lines. Such effects were inconsistent between donors and HIV subtypes. Sex hormone effects on HIV entry receptors (CCR5/CXCR4) did not correlate with virus production. Subtypes B and C showed higher replication in PBMC from males and females and were transmitted more efficiently through DC to male and female PBMC compared with other HIV-1 subtypes, HIV-1 Group O and HIV-2. These findings are consistent with increased worldwide prevalence of subtype B and C compared to other subtypes. Sex steroid hormones had variable effect on replication or transmission of different subtypes. These findings suggest that subtype, gender and sex hormones may play a crucial role in the replication and transmission of HIV. Published by Elsevier Ltd.

Bradykinin-stimulated cyclooxygenase activity stimulates vas deferens epithelial anion secretion in vitro in swine and humans.

PubMed

Pierucci-Alves, Fernando; Schultz, Bruce D

2008-09-01

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (I SC; indicating net anion secretion), an effect that was 60%-93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase I SC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in I SC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion.

Bradykinin-Stimulated Cyclooxygenase Activity Stimulates Vas Deferens Epithelial Anion Secretion In Vitro in Swine and Humans1

PubMed Central

Pierucci-Alves, Fernando; Schultz, Bruce D.

2008-01-01

Epithelia lining the male reproductive duct modulate fertility by altering the luminal environment to which sperm are exposed. Although vas deferens epithelial cells reportedly express high levels of cyclooxygenases (Ptgs), and activation of bradykinin (BK) receptors can lead to upregulation of PTGS activity in epididymal epithelia, it remains unknown whether BKs and/or PTGSs have any role in modulating epithelial ion transport across vas deferens epithelia. Porcine and human vas deferens epithelial cell primary cultures and the PVD9902 cell line responded to lysylbradykinin with an increase in short circuit current (ISC; indicating net anion secretion), an effect that was 60%–93% reduced by indomethacin. The BK effect was inhibited by the B2 receptor subtype (BDKRB2) antagonist HOE140, whereas the B1 receptor subtype agonist des-Arg9-BK had no effect. BDKRB2 immunoreactivity was documented in most epithelial cells composing the native epithelium and on Western blots derived from cultured cells. Gene expression analysis revealed that the PTGS2 transcript is 20 times more abundant than its PTGS1 counterpart in cultured porcine vas deferens epithelia and that BDKRB2 mRNA is likewise highly expressed. Subsequent experiments revealed that prostaglandin E2, 1-OH prostaglandin E1 (prostaglandin E receptor 4 [PTGER4] agonist) and butaprost (PTGER2 agonist) increase ISC in a concentration-dependent manner, whereas sulprostone (mixed PTGER1 and PTGER3 agonist) produced no change in ISC. These results demonstrate that autacoids can affect epithelial cells to acutely modulate the luminal environment to which sperm are exposed in the vas deferens by enhancing PTGS activity, leading to the production of prostaglandins that act at PTGER4 and/or PTGER2 to induce or enhance anion secretion. PMID:18480467

Sigma receptors as potential therapeutic targets for neuroprotection.

PubMed

Nguyen, Linda; Kaushal, Nidhi; Robson, Matthew J; Matsumoto, Rae R

2014-11-15

Sigma receptors comprise a unique family of proteins that have been implicated in the pathophysiology and treatment of many central nervous system disorders, consistent with their high level of expression in the brain and spinal cord. Mounting evidence indicate that targeting sigma receptors may be particularly beneficial in a number of neurodegenerative conditions including Alzheimer׳s disease, Parkinson׳s disease, stroke, methamphetamine neurotoxicity, Huntington׳s disease, amyotrophic lateral sclerosis, and retinal degeneration. In this perspective, a brief overview is given on sigma receptors, followed by a focus on common mechanisms of neurodegeneration that appear amenable to modulation by sigma receptor ligands to convey neuroprotective effects and/or restorative functions. Within each of the major mechanisms discussed herein, the neuroprotective effects of sigma ligands are summarized, and when known, the specific sigma receptor subtype(s) involved are identified. Together, the literature suggests sigma receptors may provide a novel target for combatting neurodegenerative diseases through both neuronal and glial mechanisms. Copyright © 2014 Elsevier B.V. All rights reserved.

Incidence and risk factors for breast cancer subtypes in three distinct South-East Asian ethnic groups: Chinese, Malay and natives of Sarawak, Malaysia.

PubMed

Devi, C R Beena; Tang, Tieng Swee; Corbex, Marilys

2012-12-15

We determined the incidences of the estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) subtypes among breast cancer cases in Sarawak, Malaysia and their correlation with various risk factors in the three ethnic groups: Chinese, Malay and native. Subtype status was ascertained for 1,034 cases of female breast cancer (93% of all cases diagnosed since 2003), and the age-standardized incidence rates (ASRs) of each subtype were inferred. Case-case comparisons across subtypes were performed for reproductive risk factors. We found 48% luminal A (ER+/PR+/HER2-), 29% triple-negative (ER-/PR-/HER2-), 12% triple-positive (ER+/PR+/HER2+) and 11% HER2-overexpressing (ER-/PR-/HER2+) subtypes, with ASRs of 10.6, 6.0, 2.8 and 2.8 per 100,000, respectively. The proportions of subtypes and ASRs differed significantly by ethnic groups: HER2-positive cases were more frequent in Malays (29%; 95% CI [23;35]) than Chinese (22%; [19;26] and natives (21%; [16;26]); triple-negative cases were less frequent among Chinese (23%; [20;27]) than Malays (33%; [27;39]) and natives (37%; [31;43]). The results of the case-case comparison were in accordance with those observed in western case series. Some uncommon associations, such as between triple-negative subtype and older age at menopause (OR, 1.59; p < 0.05), were found. The triple-negative and HER2+ subtypes predominate in our region, with significant differences among ethnic groups. Our results support the idea that the risk factors for different subtypes vary markedly. Westernized populations are more likely to have factors that increase the risk for the luminal A type, while risk factors for the triple-negative type are more frequent in local populations. Copyright © 2012 UICC.

Age-Specific Incidence of Breast Cancer Subtypes: Understanding the Black–White Crossover

PubMed Central

2012-01-01

Background Breast cancer incidence is higher among black women than white women before age 40 years, but higher among white women than black women after age 40 years (black–white crossover). We used newly available population-based data to examine whether the age-specific incidences of breast cancer subtypes vary by race and ethnicity. Methods We classified 91908 invasive breast cancers diagnosed in California between January 1, 2006, and December 31, 2009, by subtype based on tumor expression of estrogen receptor (ER) and progesterone receptor (PR)—together referred to as hormone receptor (HR)—and human epidermal growth factor receptor 2 (HER2). Breast cancer subtypes were classified as ER or PR positive and HER2 negative (HR+/HER2−), ER or PR positive and HER2 positive (HR+/HER2+), ER and PR negative and HER2 positive (HR−/HER2+), and ER, PR, and HER2 negative (triple-negative). We calculated and compared age-specific incidence rates, incidence rate ratios, and 95% confidence intervals by subtype and race (black, white, Hispanic, and Asian). All P values are two-sided. Results We did not observe an age-related black–white crossover in incidence for any molecular subtype of breast cancer. Compared with white women, black women had statistically significantly higher rates of triple-negative breast cancer at all ages but statistically significantly lower rates of HR+/HER2− breast cancers after age 35 years (all P < .05). The age-specific incidence of HR+/HER2+ and HR−/HER2+ subtypes did not vary markedly between white and black women. Conclusions The black–white crossover in breast cancer incidence occurs only when all breast cancer subtypes are combined and relates largely to higher rates of triple-negative breast cancers and lower rates of HR+/HER2− breast cancers in black vs white women. PMID:22773826

Receptor mimicry by antibody F045–092 facilitates universal binding to the H3 subtype of influenza virus

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

Lee, Peter S.; Ohshima, Nobuko; Stanfield, Robyn L.

Influenza viruses present a significant health challenge each year, as in the H3N2 epidemic of 2012–2013. Here we describe an antibody, F045–092, that possesses broadly neutralizing activity against the entire H3 subtype and accommodates the natural variation and additional glycosylation in all strains tested from 1963 to 2011. Crystal structures of F045–092 in complex with HAs from 1975 and 2011 H3N2 viruses reveal the structural basis for its neutralization breadth through insertion of its 23-residue HCDR3 into the receptor-binding site that involves striking receptor mimicry. F045–092 extends its recognition to divergent subtypes, including H1, H2 and H13, using the enhancedmore » avidity of its IgG to overcome lower-affinity Fab binding, as observed with other antibodies that target the receptor-binding site. This unprecedented level of antibody cross-reactivity against the H3 subtype can potentially inform on development of a pan-H3 vaccine or small-molecule therapeutics.« less

Synthesis and serotonergic activity of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives: novel antagonists for the vascular 5-HT(1B)-like receptor.

PubMed

Moloney, G P; Martin, G R; Mathews, N; Milne, A; Hobbs, H; Dodsworth, S; Sang, P Y; Knight, C; Williams, M; Maxwell, M; Glen, R C

1999-07-15

The synthesis and vascular 5-HT(1B)-like receptor activity of a novel series of substituted 2, N-benzylcarboxamido-5-(2-ethyl-1-dioxoimidazolidinyl)-N, N-dimethyltryptamine derivatives are described. Modifications to the 5-ethylene-linked heterocycle and to substituents on the 2-benzylamide side chain have been explored. Several compounds were identified which exhibited affinity at the vascular 5-HT(1B)-like receptor of pK(B) > 7.0, up to 100-fold selectivity over alpha(1)-adrenoceptor affinity and 5-HT(2A) receptor affinity, and which exhibited a favorable pharmacokinetic profile. N-Benzyl-3-[2-(dimethylamino)ethyl]-5-[2-(4,4-dimethyl-2, 5-dioxo-1-imidazolidinyl)ethyl]-1H-indole-2-carboxamide (23) was identified as a highly potent, silent (as judged by the inability of angiotensin II to unmask 5-HT(1B)-like receptor-mediated agonist activity in the rabbit femoral artery), and competitive vascular 5-HT(1B)-like receptor antagonist with a plasma elimination half-life of approximately 4 h in dog plasma and with good oral bioavailability. The selectivity of compounds from this series for the vascular 5-HT(1B)-like receptors over other receptor subtypes is discussed as well as a proposed mode of binding to the receptor pharmacophore. It has been proposed that the aromatic ring of the 2, N-benzylcarboxamide group can occupy an aromatic binding site rather than the indole ring. The resulting conformation allows an amine-binding site to be occupied by the ethylamine nitrogen and a hydrogen-bonding site to be occupied by one of the hydantoin carbonyls. The electronic nature of the 2,N-benzylcarboxamide aromatic group as well as the size of substituents on this aromatic group is crucial for producing potent and selective antagonists. The structural requirement on the 3-ethylamine side chain incorporating the protonatable nitrogen is achieved by the bulky 2, N-benzylcarboxamide group and its close proximity to the 3-side chain.

Supervised Risk Predictor of Breast Cancer Based on Intrinsic Subtypes

PubMed Central

Parker, Joel S.; Mullins, Michael; Cheang, Maggie C.U.; Leung, Samuel; Voduc, David; Vickery, Tammi; Davies, Sherri; Fauron, Christiane; He, Xiaping; Hu, Zhiyuan; Quackenbush, John F.; Stijleman, Inge J.; Palazzo, Juan; Marron, J.S.; Nobel, Andrew B.; Mardis, Elaine; Nielsen, Torsten O.; Ellis, Matthew J.; Perou, Charles M.; Bernard, Philip S.

2009-01-01

Purpose To improve on current standards for breast cancer prognosis and prediction of chemotherapy benefit by developing a risk model that incorporates the gene expression–based “intrinsic” subtypes luminal A, luminal B, HER2-enriched, and basal-like. Methods A 50-gene subtype predictor was developed using microarray and quantitative reverse transcriptase polymerase chain reaction data from 189 prototype samples. Test sets from 761 patients (no systemic therapy) were evaluated for prognosis, and 133 patients were evaluated for prediction of pathologic complete response (pCR) to a taxane and anthracycline regimen. Results The intrinsic subtypes as discrete entities showed prognostic significance (P = 2.26E-12) and remained significant in multivariable analyses that incorporated standard parameters (estrogen receptor status, histologic grade, tumor size, and node status). A prognostic model for node-negative breast cancer was built using intrinsic subtype and clinical information. The C-index estimate for the combined model (subtype and tumor size) was a significant improvement on either the clinicopathologic model or subtype model alone. The intrinsic subtype model predicted neoadjuvant chemotherapy efficacy with a negative predictive value for pCR of 97%. Conclusion Diagnosis by intrinsic subtype adds significant prognostic and predictive information to standard parameters for patients with breast cancer. The prognostic properties of the continuous risk score will be of value for the management of node-negative breast cancers. The subtypes and risk score can also be used to assess the likelihood of efficacy from neoadjuvant chemotherapy. PMID:19204204

Selective 5-Hydroxytrytamine 2C Receptor Agonists Derived from the Lead Compound Tranylcypromine – Identification of Drugs with Antidepressant-Like Action

PubMed Central

Cho, Sung Jin; Jensen, Niels H.; Kurome, Toru; Kadari, Sudhakar; Manzano, Michael L.; Malberg, Jessica E.; Caldarone, Barbara; Roth, Bryan L.; Kozikowski, Alan P.

2009-01-01

We report here the design, synthesis, and pharmacological properties of a series of compounds related to tranylcypromine (9), which itself was discovered as a lead compound in a high-throughput screening campaign. Starting from 9, which shows modest activity as a 5-HT2C agonist, a series of 1-aminomethyl-2-phenylcyclopropanes was investigated as 5-HT2C agonists through iterative structural modifications. Key pharmacophore feature of this new class of ligands is a 2-aminomethyl-trans-cyclopropyl side chain attached to a substituted benzene ring. Among the tested compounds, several were potent and efficacious 5-HT2C receptor agonists with selectivity over both 5-HT2A and 5-HT2B receptors in functional assays. The most promising compound is 37 with 120- and 14-fold selectivity over 5-HT2A and 5-HT2B, respectively (EC50 = 585, 65, and 4.8 nM at the 2A, 2B, and 2C subtypes, respectively). In animal studies, compound 37 (10–60 mg/kg) decreased immobility time in the mouse forced swim test. PMID:19284718

Dm5-HT2B: Pharmacological Characterization of the Fifth Serotonin Receptor Subtype of Drosophila melanogaster

PubMed Central

Blenau, Wolfgang; Daniel, Stöppler; Balfanz, Sabine; Thamm, Markus; Baumann, Arnd

2017-01-01

Serotonin (5-hydroxytryptamine, 5-HT) is an important regulator of physiological and behavioral processes in both protostomes (e.g., insects) and deuterostomes (e.g., mammals). In insects, serotonin has been found to modulate the heart rate and to control secretory processes, development, circadian rhythms, aggressive behavior, as well as to contribute to learning and memory. Serotonin exerts its activity by binding to and activating specific membrane receptors. The clear majority of these receptors belong to the superfamily of G-protein-coupled receptors. In Drosophila melanogaster, a total of five genes have been identified coding for 5-HT receptors. From this family of proteins, four have been pharmacologically examined in greater detail, so far. While Dm5-HT1A, Dm5-HT1B, and Dm5-HT7 couple to cAMP signaling cascades, the Dm5-HT2A receptor leads to Ca2+ signaling in an inositol-1,4,5-trisphosphate-dependent manner. Based on sequence similarity to homologous genes in other insects, a fifth D. melanogaster gene was uncovered coding for a Dm5-HT2B receptor. Knowledge about this receptor’s pharmacological properties is very limited. This is quite surprising because Dm5-HT2B has been attributed to distinct physiological functions based on genetic interference with its gene expression. Mutations were described reducing the response of the larval heart to 5-HT, and specific knockdown of Dm5-HT2B mRNA in hemocytes resulted in a higher susceptibility of the flies to bacterial infection. To gain deeper understanding of Dm5-HT2B’s pharmacology, we evaluated the receptor’s response to a series of established 5-HT receptor agonists and antagonists in a functional cell-based assay. Metoclopramide and mianserin were identified as two potent antagonists that may allow pharmacological interference with Dm5-HT2B signaling in vitro and in vivo. PMID:28553207

(3H)WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

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

Norman, A.B.; Battaglia, G.; Creese, I.

1985-12-01

In the presence of a 30 nM prazosin mask, (/sup 3/H)-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ((/sup 3/H)WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for (/sup 3/H) WB4101 binding in cerebral cortex. We have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at (/sup 3/H)WB4101-binding sites in the presence of 30 nM prazosin and (/sup 3/H) lysergic acid diethylamide ((/sup 3/H)LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5more » mM MgSO4, the Bmax of (/sup 3/H)WB4101 is significantly lower than the Bmax of (/sup 3/H)LSD in various brain regions. WB4101 competition for (/sup 3/H) LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of (/sup 3/H)WB4101 binding derived from saturation experiments. This suggests that (/sup 3/H)WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by (/sup 3/H)LSD. The selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for (/sup 3/H)WB4101 but compete for multiple (/sup 3/H)LSD 5-HT1 binding sites. These data indicate that (/sup 3/H)WB4101 selectively labels the 5-HT1A serotonin receptor, whereas (/sup 3/H) LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of (/sup 3/H)WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of (/sup 3/H)WB4101 binding.« less

The α3β4* nicotinic ACh receptor subtype mediates physical dependence to morphine: mouse and human studies

PubMed Central

Muldoon, P P; Jackson, K J; Perez, E; Harenza, J L; Molas, S; Rais, B; Anwar, H; Zaveri, N T; Maldonado, R; Maskos, U; McIntosh, J M; Dierssen, M; Miles, M F; Chen, X; De Biasi, M; Damaj, M I

2014-01-01

BACKGROUND AND PURPOSE Recent data have indicated that α3β4* neuronal nicotinic (n) ACh receptors may play a role in morphine dependence. Here we investigated if nACh receptors modulate morphine physical withdrawal. EXPERIMENTAL APPROACHES To assess the role of α3β4* nACh receptors in morphine withdrawal, we used a genetic correlation approach using publically available datasets within the GeneNetwork web resource, genetic knockout and pharmacological tools. Male and female European-American (n = 2772) and African-American (n = 1309) subjects from the Study of Addiction: Genetics and Environment dataset were assessed for possible associations of polymorphisms in the 15q25 gene cluster and opioid dependence. KEY RESULTS BXD recombinant mouse lines demonstrated an increased expression of α3, β4 and α5 nACh receptor mRNA in the forebrain and midbrain, which significantly correlated with increased defecation in mice undergoing morphine withdrawal. Mice overexpressing the gene cluster CHRNA5/A3/B4 exhibited increased somatic signs of withdrawal. Furthermore, α5 and β4 nACh receptor knockout mice expressed decreased somatic withdrawal signs compared with their wild-type counterparts. Moreover, selective α3β4* nACh receptor antagonists, α-conotoxin AuIB and AT-1001, attenuated somatic signs of morphine withdrawal in a dose-related manner. In addition, two human datasets revealed a protective role for variants in the CHRNA3 gene, which codes for the α3 nACh receptor subunit, in opioid dependence and withdrawal. In contrast, we found that the α4β2* nACh receptor subtype is not involved in morphine somatic withdrawal signs. CONCLUSION AND IMPLICATIONS Overall, our findings suggest an important role for the α3β4* nACh receptor subtype in morphine physical dependence. PMID:24750073

The qEEG Signature of Selective NMDA NR2B Negative Allosteric Modulators; A Potential Translational Biomarker for Drug Development

PubMed Central

Keavy, Deborah; Bristow, Linda J.; Sivarao, Digavalli V.; Batchelder, Margaret; King, Dalton; Thangathirupathy, Srinivasan; Macor, John E.; Weed, Michael R.

2016-01-01

The antidepressant activity of the N-methyl-D-aspartate (NMDA) receptor channel blocker, ketamine, has led to the investigation of negative allosteric modulators (NAMs) selective for the NR2B receptor subtype. The clinical development of NR2B NAMs would benefit from a translational pharmacodynamic biomarker that demonstrates brain penetration and functional inhibition of NR2B receptors in preclinical species and humans. Quantitative electroencephalography (qEEG) is a translational measure that can be used to demonstrate pharmacodynamic effects across species. NMDA receptor channel blockers, such as ketamine and phencyclidine, increase the EEG gamma power band, which has been used as a pharmacodynamic biomarker in the development of NMDA receptor antagonists. However, detailed qEEG studies with ketamine or NR2B NAMs are lacking in nonhuman primates. The aim of the present study was to determine the effects on the qEEG power spectra of the NR2B NAMs traxoprodil (CP-101,606) and BMT-108908 in nonhuman primates, and to compare them to the NMDA receptor channel blockers, ketamine and lanicemine. Cynomolgus monkeys were surgically implanted with EEG radio-telemetry transmitters, and qEEG was measured after vehicle or drug administration. The relative power for a number of frequency bands was determined. Ketamine and lanicemine increased relative gamma power, whereas the NR2B NAMs traxoprodil and BMT-108908 had no effect. Robust decreases in beta power were elicited by ketamine, traxoprodil and BMT-108908; and these agents also produced decreases in alpha power and increases in delta power at the doses tested. These results suggest that measurement of power spectra in the beta and delta bands may represent a translational pharmacodynamic biomarker to demonstrate functional effects of NR2B NAMs. The results of these studies may help guide the selection of qEEG measures that can be incorporated into early clinical evaluation of NR2B NAMs in healthy humans. PMID:27035340

Structure-Activity Relationships of Constrained Phenylethylamine Ligands for the Serotonin 5-HT2 Receptors

PubMed Central

Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L.; Gloriam, David E.

2013-01-01

Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9–11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9–11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9–11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands. PMID:24244317

Structure-activity relationships of constrained phenylethylamine ligands for the serotonin 5-HT2 receptors.

PubMed

Isberg, Vignir; Paine, James; Leth-Petersen, Sebastian; Kristensen, Jesper L; Gloriam, David E

2013-01-01

Serotonergic ligands have proven effective drugs in the treatment of migraine, pain, obesity, and a wide range of psychiatric and neurological disorders. There is a clinical need for more highly 5-HT2 receptor subtype-selective ligands and the most attention has been given to the phenethylamine class. Conformationally constrained phenethylamine analogs have demonstrated that for optimal activity the free lone pair electrons of the 2-oxygen must be oriented syn and the 5-oxygen lone pairs anti relative to the ethylamine moiety. Also the ethyl linker has been constrained providing information about the bioactive conformation of the amine functionality. However, combined 1,2-constriction by cyclization has only been tested with one compound. Here, we present three new 1,2-cyclized phenylethylamines, 9-11, and describe their synthetic routes. Ligand docking in the 5-HT2B crystal structure showed that the 1,2-heterocyclized compounds can be accommodated in the binding site. Conformational analysis showed that 11 can only bind in a higher-energy conformation, which would explain its absent or low affinity. The amine and 2-oxygen interactions with D3.32 and S3.36, respectively, can form but shift the placement of the core scaffold. The constraints in 9-11 resulted in docking poses with the 4-bromine in closer vicinity to 5.46, which is polar only in the human 5-HT2A subtype, for which 9-11 have the lowest affinity. The new ligands, conformational analysis and docking expand the structure-activity relationships of constrained phenethylamines and contributes towards the development of 5-HT2 receptor subtype-selective ligands.

Update on melatonin receptors: IUPHAR Review 20.

PubMed

Jockers, Ralf; Delagrange, Philippe; Dubocovich, Margarita L; Markus, Regina P; Renault, Nicolas; Tosini, Gianluca; Cecon, Erika; Zlotos, Darius P

2016-09-01

Melatonin receptors are seven transmembrane-spanning proteins belonging to the GPCR superfamily. In mammals, two melatonin receptor subtypes exist - MT1 and MT2 - encoded by the MTNR1A and MTNR1B genes respectively. The current review provides an update on melatonin receptors by the corresponding subcommittee of the International Union of Basic and Clinical Pharmacology. We will highlight recent developments of melatonin receptor ligands, including radioligands, and give an update on the latest phenotyping results of melatonin receptor knockout mice. The current status and perspectives of the structure of melatonin receptor will be summarized. The physiological importance of melatonin receptor dimers and biologically important and type 2 diabetes-associated genetic variants of melatonin receptors will be discussed. The role of melatonin receptors in physiology and disease will be further exemplified by their functions in the immune system and the CNS. Finally, antioxidant and free radical scavenger properties of melatonin and its relation to melatonin receptors will be critically addressed. © 2016 The British Pharmacological Society.

Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2017-03-01

Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

Discrimination of putative M1 and M2 muscarinic receptor subtypes in rat brain by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ)

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

Norman, A.B.; Creese, I.

1986-03-01

The EC/sub 50/ of EEDQ for the inhibition of (/sup 3/H)(-)QNB binding in vitro was approximately 3 fold lower for homogenates of hippocampus than brainstem (containing predominantly putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes respectively). Furthermore, the time-dependent loss of (/sup 3/H)(-)QNB binding produced by 100 ..mu..M EEDQ was faster in homogenates of hippocampus than brainstem. Administration of EEDQ (20 mg/kg i.p.) irreversibly reduced the Bmax of (/sup 3/H)(-)QNB binding by 56% and 34% in hippocampus and brainstem respectively. Pirenzepine competition for the remaining (/sup 3/H)(-)QNB binding sites following in vitro and in vivo treatment with EEDQ revealedmore » a significant increase in the proportion of (/sup 3/H)(-)QNB binding sites having low affinity for pirenzepine (M/sub 2/ receptors), indicating that the high affinity pirenzepine binding sites (M/sub 1/ receptors) were selectively and irreversibly lost. Thus, EEDQ discriminates the same putative M/sub 1/ and M/sub 2/ muscarinic receptor subtypes that are discriminated by pirenzepine. The reduction of (/sup 3/H)(-)QNB binding could be prevented both in vitro and in vivo by atropine or scopolamine. These data may indicate differences in the accessibility of these putative receptor subtypes to EEDQ or, alternatively, differences in the availability of carboxyl groups able to interact with EEDQ at the ligand recognition site of M/sub 1/ and M/sub 2/ muscarinic receptors.« less

Synaptic GluN2A and GluN2B Containing NMDA Receptors within the Superficial Dorsal Horn Activated following Primary Afferent Stimulation

PubMed Central

MacDermott, Amy B.

2014-01-01

NMDA receptors are important elements in pain signaling in the spinal cord dorsal horn. They are heterotetramers, typically composed of two GluN1 and two of four GluN2 subunits: GluN2A-2D. Mice lacking some of the GluN2 subunits show deficits in pain transmission yet functional synaptic localization of these receptor subtypes in the dorsal horn has not been fully resolved. In this study, we have investigated the composition of synaptic NMDA receptors expressed in monosynaptic and polysynaptic pathways from peripheral sensory fibers to lamina I neurons in rats. We focused on substance P receptor-expressing (NK1R+) projection neurons, critical for expression of hyperalgesia and allodynia. EAB-318 and (R)-CPP, GluN2A/B antagonists, blocked both monosynaptic and polysynaptic NMDA EPSCs initiated by primary afferent activation by ∼90%. Physiological measurements exploiting the voltage dependence of monosynaptic EPSCs similarly indicated dominant expression of GluN2A/B types of synaptic NMDA receptors. In addition, at synapses between C fibers and NK1R+ neurons, NMDA receptor activation initiated a secondary, depolarizing current. Ifenprodil, a GluN2B antagonist, caused modest suppression of monosynaptic NMDA EPSC amplitudes, but had a widely variable, sometimes powerful, effect on polysynaptic responses following primary afferent stimulation when inhibitory inputs were blocked to mimic neuropathic pain. We conclude that GluN2B subunits are moderately expressed at primary afferent synapses on lamina I NK1R+ neurons, but play more important roles for polysynaptic NMDA EPSCs driven by primary afferents following disinhibition, supporting the view that the analgesic effect of the GluN2B antagonist on neuropathic pain is at least in part, within the spinal cord. PMID:25122884

In breast cancer subtypes steroid sulfatase (STS) is associated with less aggressive tumour characteristics.

PubMed

McNamara, Keely M; Guestini, Fouzia; Sauer, Torill; Touma, Joel; Bukholm, Ida Rashida; Lindstrøm, Jonas C; Sasano, Hironobu; Geisler, Jürgen

2018-05-01

The majority of breast cancer cases are steroid dependent neoplasms, with hormonal manipulation of either CYP19/aromatase or oestrogen receptor alpha axis being the most common therapy. Alternate pathways of steroid actions are documented, but their interconnections and correlations to BC subtypes and clinical outcome could be further explored. We evaluated selected steroid receptors (Androgen Receptor, Oestrogen Receptor alpha and Beta, Glucocorticoid Receptor) and oestrogen pathways (steroid sulfatase (STS), 17β-hydroxysteroid dehydrogenase 2 (17βHSD2) and aromatase) in a cohort of 139 BC cases from Norway. Using logistic and cox regression analysis, we examined interactions between these and clinical outcomes such as distant metastasis, local relapse and survival. Our principal finding is an impact of STS expression on the risk for distant metastasis (p<0.001) and local relapses (p <0.001), HER2 subtype (p<0.015), and survival (p<0.001). The suggestion of a beneficial effect of alternative oestrogen synthesis pathways was strengthened by inverted, but non-significant findings for 17βHSD2. Increased intratumoural metabolism of oestrogens through STS is associated with significantly lower incidence of relapse and/or distant metastasis and correspondingly improved prognosis. The enrichment of STS in the HER2 overexpressing subtype is intriguing, especially given the possible role of HER-2 over-expression in endocrine resistance.

Opioid receptor subtypes: fact or artifact?

PubMed

Dietis, N; Rowbotham, D J; Lambert, D G

2011-07-01

There is a vast amount of pharmacological evidence favouring the existence of multiple subtypes of opioid receptors. In addition to the primary classification of µ (mu: MOP), δ (delta: DOP), κ (kappa: KOP) receptors, and the nociceptin/orphanin FQ peptide receptor (NOP), various groups have further classified the pharmacological µ into µ(1-3), the δ into δ(1-2)/δ(complexed/non-complexed), and the κ into κ(1-3). From an anaesthetic perspective, the suggestions that µ(1) produced analgesia and µ(2) produced respiratory depression are particularly important. However, subsequent to the formal identification of the primary opioid receptors (MOP/DOP/KOP/NOP) by cloning and the use of this information to produce knockout animals, evidence for these additional subtypes is lacking. Indeed, knockout of a single gene (and hence receptor) results in a loss of all function associated with that receptor. In the case of MOP knockout, analgesia and respiratory depression is lost. This suggests that further sub-classification of the primary types is unwise. So how can the wealth of pharmacological data be reconciled with new molecular information? In addition to some simple misclassification (κ(3) is probably NOP), there are several possibilities which include: (i) alternate splicing of a common gene product, (ii) receptor dimerization, (iii) interaction of a common gene product with other receptors/signalling molecules, or (iv) a combination of (i)-(iii). Assigning variations in ligand activity (pharmacological subtypes) to one or more of these molecular suggestions represents an interesting challenge for future opioid research.

Activation of neurokinin NK(2) receptors by tachykinin peptides causes contraction of uterus in pregnant women near term.

PubMed

Patak, E N; Ziccone, S; Story, M E; Fleming, A J; Lilley, A; Pennefather, J N

2000-06-01

The aim of this study was firstly to elucidate whether the mammalian tachykinins substance P (SP), neurokinin A (NKA) and neurokinin B (NKB)-regulated contractility of myometrium obtained from near-term pregnant women, and secondly to investigate the receptor subtype(s) responsible. In the presence of peptidase inhibitors, i.e. thiorphan (3 micromol/l; endopeptidase 24.11 inhibitor), captopril (10 micromol/l; angiotensin converting enzyme inhibitor) and bestatin (10 micromol/l; aminopeptidase inhibitor); all three mammalian tachykinins elicited concentration-related contractions of isolated myometrial preparations. The rank order of agonist potency of the mammalian tachykinins in the presence of the peptidase inhibitors was NKA > SP = NKB, indicating that the contractile effects were mediated by activation of an NK(2) receptor. The NK(2) receptor-selective agonist, [Lys(5), MeLeu(9), Nle(10)]NKA(4-10), produced concentration-related contractile responses, while the respective NK(1) and NK(3) receptor-selective agonists, [Sar(9), Met(O(2))(11)]SP and [N-MePhe(7)]NKB, had no effect either in the absence or presence of the peptidase inhibitors. The NK(2) receptor-selective antagonist, SR48968, produced concentration-related rightward shift in the log concentration curve to [Lys(5), MeLeu(9), Nle(10)]NKA(4-10). This study shows that tachykinins elicit contractile effects on human myometrium obtained from pregnant women near term, and that these effects are mediated by an NK(2) receptor. An excitatory effect of the tachykinins on these preparations could indicate a physiological role for these peptides in enhancing contractility of the uterus in women at term.

Cockroach GABAB receptor subtypes: molecular characterization, pharmacological properties and tissue distribution.

PubMed

Blankenburg, S; Balfanz, S; Hayashi, Y; Shigenobu, S; Miura, T; Baumann, O; Baumann, A; Blenau, W

2015-01-01

γ-aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter in the central nervous system (CNS). Its effects are mediated by either ionotropic GABAA receptors or metabotropic GABAB receptors. GABAB receptors regulate, via Gi/o G-proteins, ion channels, and adenylyl cyclases. In humans, GABAB receptor subtypes are involved in the etiology of neurologic and psychiatric disorders. In arthropods, however, these members of the G-protein-coupled receptor family are only inadequately characterized. Interestingly, physiological data have revealed important functions of GABAB receptors in the American cockroach, Periplaneta americana. We have cloned cDNAs coding for putative GABAB receptor subtypes 1 and 2 of P. americana (PeaGB1 and PeaGB2). When both receptor proteins are co-expressed in mammalian cells, activation of the receptor heteromer with GABA leads to a dose-dependent decrease in cAMP production. The pharmacological profile differs from that of mammalian and Drosophila GABAB receptors. Western blot analyses with polyclonal antibodies have revealed the expression of PeaGB1 and PeaGB2 in the CNS of the American cockroach. In addition to the widespread distribution in the brain, PeaGB1 is expressed in salivary glands and male accessory glands. Notably, PeaGB1-like immunoreactivity has been detected in the GABAergic salivary neuron 2, suggesting that GABAB receptors act as autoreceptors in this neuron. Copyright © 2014 Elsevier Ltd. All rights reserved.

Differentiation of muscarinic cholinergic receptor subtypes in human cortex and pons - Implications for anti-motion sickness therapy

NASA Technical Reports Server (NTRS)

Mccarthy, Bruce G.; Peroutka, Stephen J.

1988-01-01

Radioligand binding studies were used to analyze muscarinic cholinergic receptor subtypes in human cortex and pons. Muscarinic cholinergic receptors were labeled by H-3-quinuclidinyl benzilate (H-3-QNB). Scopolamine was equipotent in both brain regions and did not discriminate subtypes of H-3-QNB binding. By contrast, the M1 selective antagonist pirenzepine was approximately 33-fold more potent in human cortex than pons. Carbachol, a putative M2 selective agonist, was more than 100-fold more potent in human pons than cortex. These results demonstrate that the human pons contains a relatively large proportion of carbachol-sensitive muscarinic cholinergic receptors. Drugs targeted to this subpopulation of muscarinic cholinergic receptors may prove to be effective anti-motion sickness agents with less side effects than scopolamine.

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.

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.

Relationships between immunophenotype, Ki-67 index, microvascular density, Ep-CAM/P-cadherin, and MMP-2 expression in early-stage invasive ductal breast cancer.

PubMed

Niemiec, Joanna A; Adamczyk, Agnieszka; Małecki, Krzysztof; Majchrzyk, Kaja; Ryś, Janusz

2012-12-01

There is still a lack of complete consensus on immunohistochemical surrogate markers for luminal A (LA) and luminal B (LB), HER2, and basal-like subtypes of breast carcinomas and their correlation with cancer cell adhesion and invasion-promoting factors. Therefore, early-stage invasive ductal breast cancer patients (N=209) were recruited to the study and divided into 4 subtypes, on the basis of the expression of the estrogen/progesterone receptor and HER2 (LA: 74.4% of cases; LB: 7.8%; HER2: 5.6%; and triple-negative phenotype: 12.2%). Regardless of the above-mentioned classification, we divided all carcinomas into 2 groups: carcinomas expressing at least 1 basal marker [cytokeratine (CK)5/6, CK5, vimentin, epidermal growth factor receptor, or aberrant CK8/18 expression-membranous or in <10% of cells] versus carcinomas negative for basal markers. Then we studied the relationships between the above subtypes (2 classifications) and (i) the expression of adhesion molecules (Ep-CAM, P-cadherin), (ii) matrix metalloproteinases (MMP)-2, (iii) the proliferation index (MIB-1 LI), and (iv) the microvascular density. We confirmed that triple-negative phenotypes are characterized by basal marker expression, a high tumor grade, and high MIB-1 LI. In this subtype, we found MMP-2 expression in stromal leukocytes less frequently. Both LA carcinomas and carcinomas negative for basal markers were more often negative for epithelial cell adhesion molecule (Ep-CAM) and P-cadherin. Moreover, we noted a higher mean value of microvascular density in CK5/6 and Ep-CAM-immunopositive tumors, carcinomas with aberrant CK8/18 expression, and carcinomas with no or strong expression of MMP-2 in stromal fibroblast-like cells. These results might suggest that mechanisms of stroma remodeling and carcinogenesis (Ep-CAM is the suggested marker of breast progenitors) may differ between breast cancer subtypes.

Differences in neurokinin receptor pharmacology between rat and guinea-pig superior cervical ganglia.

PubMed

Seabrook, G R; Main, M; Bowery, B; Wood, N; Hill, R G

1992-04-01

1. The depolarizations elicited by seven neurokinin receptor agonists were examined in both rat and guinea-pig superior cervical ganglia by use of grease-gap methodology in the presence of tetrodotoxin (0.1 microM). Responses were normalised with respect to 1 microM eledoisin. 2. The rank order of agonist potency in the rat ganglia was senktide greater than substance P greater than substance P methyl ester = eleidosin = Sar-Met-substance P greater than neurokinin B greater than neurokinin A, whereas in guinea-pig superior cervical ganglion (SCG) the rank order was senktide greater than Sar-Met-substance P greater than neurokinin B = eledoisin = substance P methyl ester. The concentration-effect curves for substance P and neurokinin A in guinea-pig ganglia were biphasic which precluded the determination of meaningful potency values. 3. The maximal depolarization achieved by subtype selective ligands was different between these two species. On rat and guinea-pig SCG, the NK3-selective ligand, senktide, produced a maximal depolarization of 27% and 274% respectively, whereas the NK1-selective ligand, substance P methyl ester, produced depolarizations of 77% and 64% respectively. 4. The depolarizations induced by substance P methyl ester and senktide in either species were unaffected by atropine (1 microM), suggesting a lack of involvement of presynaptic neurokinin receptors in the generation of the response. 5. The potency of substance P methyl ester, senktide, and neurokinin A were unaffected by pretreating ganglia with the peptidase inhibitors bacitracin (40 micrograms ml-1), leupeptin (4 micrograms ml-1), and chymostatin (2 micrograms ml-1). Similarly, these peptidase inhibitors had no effect on the maximal depolarizations achieved by any of these agonists.6. It is evident that rat and guinea-pig superior cervical ganglia possess both NK, and NK3 receptors, but that their net contribution to depolarizations are different between the two species. The depolarizations in guinea-pig SCG are mediated predominantly by an NK3 subtype and in rat SCG by an NK, receptor subtype.

Candidate Luminal B Breast Cancer Genes Identified by Genome, Gene Expression and DNA Methylation Profiling

PubMed Central

Addou-Klouche, Lynda; Finetti, Pascal; Saade, Marie-Rose; Manai, Marwa; Carbuccia, Nadine; Bekhouche, Ismahane; Letessier, Anne; Charafe-Jauffret, Emmanuelle; Jacquemier, Jocelyne; Spicuglia, Salvatore; de The, Hugues; Viens, Patrice; Bertucci, François; Birnbaum, Daniel; Chaffanet, Max

2014-01-01

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype. PMID:24416132

Taxonomy of breast cancer based on normal cell phenotype predicts outcome

PubMed Central

Santagata, Sandro; Thakkar, Ankita; Ergonul, Ayse; Wang, Bin; Woo, Terri; Hu, Rong; Harrell, J. Chuck; McNamara, George; Schwede, Matthew; Culhane, Aedin C.; Kindelberger, David; Rodig, Scott; Richardson, Andrea; Schnitt, Stuart J.; Tamimi, Rulla M.; Ince, Tan A.

2014-01-01

Accurate classification is essential for understanding the pathophysiology of a disease and can inform therapeutic choices. For hematopoietic malignancies, a classification scheme based on the phenotypic similarity between tumor cells and normal cells has been successfully used to define tumor subtypes; however, use of normal cell types as a reference by which to classify solid tumors has not been widely emulated, in part due to more limited understanding of epithelial cell differentiation compared with hematopoiesis. To provide a better definition of the subtypes of epithelial cells comprising the breast epithelium, we performed a systematic analysis of a large set of breast epithelial markers in more than 15,000 normal breast cells, which identified 11 differentiation states for normal luminal cells. We then applied information from this analysis to classify human breast tumors based on normal cell types into 4 major subtypes, HR0–HR3, which were differentiated by vitamin D, androgen, and estrogen hormone receptor (HR) expression. Examination of 3,157 human breast tumors revealed that these HR subtypes were distinct from the current classification scheme, which is based on estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Patient outcomes were best when tumors expressed all 3 hormone receptors (subtype HR3) and worst when they expressed none of the receptors (subtype HR0). Together, these data provide an ontological classification scheme associated with patient survival differences and provides actionable insights for treating breast tumors. PMID:24463450

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

Racial Variations in Prostate Cancer Molecular Subtypes and Androgen Receptor Signaling Reflect Anatomic Tumor Location.

PubMed

Faisal, Farzana A; Sundi, Debasish; Tosoian, Jeffrey J; Choeurng, Voleak; Alshalalfa, Mohammed; Ross, Ashley E; Klein, Eric; Den, Robert; Dicker, Adam; Erho, Nicholas; Davicioni, Elai; Lotan, Tamara L; Schaeffer, Edward M

2016-07-01

Prostate cancer (PCa) subtypes based on ETS gene expression have been described. Recent studies suggest there are racial differences in tumor location, with PCa located anteriorly more often among African-American (AA) compared to Caucasian-American (CA) men. In this retrospective analysis of a multi-institutional cohort treated by radical prostatectomy (179 CA, 121 AA), we evaluated associations among molecular subtype, race, anatomic tumor location, and androgen receptor (AR) signaling. Subtype (m-ERG(+), m-ETS(+), m-SPINK1(+), or triple-negative) was determined using distribution-based outlier analysis. AR signaling was investigated using gene expression profiling of canonical AR targets. m-ERG(+) was more common in CA than AA men (47% vs 22%, p<0.001). AA men were more likely to be m-SPINK1(+) (13% vs 7%; p=0.069) and triple-negative (50% vs 37%; p=0.043). Racial differences in molecular subtypes did not persist when tumors were analyzed by location, suggesting a biologically important relationship between tumor location and subtype. Accordingly, anterior tumor location was associated with higher Decipher scores and lower global AR signaling. This study demonstrates associations among patient race, prostate cancer molecular subtypes, and tumor location. Location-specific differences in androgen regulation may further underlie these relationships. Copyright © 2015. Published by Elsevier B.V.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors

PubMed Central

Armstrong, Lucas C.; Kirsch, Glenn E.; Fedorov, Nikolai B.; Wu, Caiyun; Kuryshev, Yuri A.; Sewell, Abby L.; Liu, Zhiqi; Motter, Arianne L.; Leggett, Carmine S.; Orr, Michael S.

2017-01-01

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and β-subunit composition. The α6β2β3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3β2β3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration–approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3β2β3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6β2β3 nicotinic receptors. PMID:28298165

Discovery and synthesis of a novel and selective drug-like P2X(1) antagonist.

PubMed

Jaime-Figueroa, S; Greenhouse, R; Padilla, F; Dillon, M P; Gever, J R; Ford, A P D W

2005-07-01

Although there is extensive literature to indicate that many different types of P2 purinoceptors are present in the lower urinary tract, the physiological role of these receptors in micturition is still uncertain. In part, this uncertainty has been caused by a lack of P2 subtype selective ligands. In this paper we report the discovery, gram scale synthesis, and binding results for 1, the first potent, drug-like, selective P2X(1) receptor antagonist described. Compound 1 was shown to be more than 30-fold selective over other purinergic receptor subtypes.

The purinergic receptor subtype P2Y2 mediates chemotaxis of neutrophils and fibroblasts in fibrotic lung disease

PubMed Central

Karmouty-Quintana, Harry; Cicko, Sanja; Ayata, Korcan; Zissel, Gernot; Goldmann, Torsten; Lungarella, Giuseppe; Ferrari, Davide; Di Virgilio, Francesco; Robaye, Bernard; Boeynaems, Jean-Marie; Blackburn, Michael R.; Idzko, Marco

2017-01-01

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with few available treatment options. Recently, the involvement of purinergic receptor subtypes in the pathogenesis of different lung diseases has been demonstrated. Here we investigated the role of the purinergic receptor subtype P2Y2 in the context of fibrotic lung diseases. The concentration of different nucleotides was measured in the broncho-alveolar lavage (BAL) fluid derived from IPF patients and animals with bleomycin-induced pulmonary fibrosis. In addition expression of P2Y2 receptors by different cell types was determined. To investigate the functional relevance of P2Y2 receptors for the pathogenesis of the disease the bleomycin model of pulmonary fibrosis was used. Finally, experiments were performed in pursuit of the involved mechanisms. Compared to healthy individuals or vehicle treated animals, extracellular nucleotide levels in the BAL fluid were increased in patients with IPF and in mice after bleomycin administration, paralleled by a functional up-regulation of P2Y2R expression. Both bleomycin-induced inflammation and fibrosis were reduced in P2Y2R-deficient compared to wild type animals. Mechanistic studies demonstrated that recruitment of neutrophils into the lungs, proliferation and migration of lung fibroblasts as well as IL6 production are key P2Y2R mediated processes. Our results clearly demonstrate the involvement of P2Y2R subtypes in the pathogenesis of fibrotic lung diseases in humans and mice and hence support the development of selective P2Y2R antagonists for the treatment of IPF. PMID:28415591

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis.

PubMed

Paul, A; Gunewardena, S; Stecklein, S R; Saha, B; Parelkar, N; Danley, M; Rajendran, G; Home, P; Ray, S; Jokar, I; Vielhauer, G A; Jensen, R A; Tawfik, O; Paul, S

2014-09-01

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype.

PKCλ/ι signaling promotes triple-negative breast cancer growth and metastasis

PubMed Central

Paul, A; Gunewardena, S; Stecklein, S R; Saha, B; Parelkar, N; Danley, M; Rajendran, G; Home, P; Ray, S; Jokar, I; Vielhauer, G A; Jensen, R A; Tawfik, O; Paul, S

2014-01-01

Triple-negative breast cancer (TNBC) is a distinct breast cancer subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor 2 (HER2/neu), and the patients with TNBC are often diagnosed with higher rates of recurrence and metastasis. Because of the absence of ER, PR and HER2/neu expressions, TNBC patients are insensitive to HER2-directed and endocrine therapies available for breast cancer treatment. Here, we report that expression of atypical protein kinase C isoform, PKCλ/ι, significantly increased and activated in all invasive breast cancer (invasive ductal carcinoma or IDC) subtypes including the TNBC subtype. Because of the lack of targeted therapies for TNBC, we choose to study PKCλ/ι signaling as a potential therapeutic target for TNBC. Our observations indicated that PKCλ/ι signaling is highly active during breast cancer invasive progression, and metastatic breast cancers, the advanced stages of breast cancer disease that developed more frequently in TNBC patients, are also characterized with high levels of PKCλ/ι expression and activation. Functional analysis in experimental mouse models revealed that depletion of PKCλ/ι significantly reduces TNBC growth as well as lung metastatic colonization. Furthermore, we have identified a PKCλ/ι-regulated gene signature consisting of 110 genes, which are significantly associated with indolent to invasive progression of human breast cancer and poor prognosis. Mechanistically, cytokines such as TGFβ and IL1β could activate PKCλ/ι signaling in TNBC cells and depletion of PKCλ/ι impairs NF-κB p65 (RelA) nuclear localization. We observed that cytokine-PKCλ/ι-RelA signaling axis, at least in part, involved in modulating gene expression to regulate invasion of TNBC cells. Overall, our results indicate that induction and activation of PKCλ/ι promote TNBC growth, invasion and metastasis. Thus, targeting PKCλ/ι signaling could be a therapeutic option for breast cancer, including the TNBC subtype. PMID:24786829

Greater absolute risk for all subtypes of breast cancer in the US than Malaysia.

PubMed

Horne, Hisani N; Beena Devi, C R; Sung, Hyuna; Tang, Tieng Swee; Rosenberg, Philip S; Hewitt, Stephen M; Sherman, Mark E; Anderson, William F; Yang, Xiaohong R

2015-01-01

Hormone receptor (HR) negative breast cancers are relatively more common in low-risk than high-risk countries and/or populations. However, the absolute variations between these different populations are not well established given the limited number of cancer registries with incidence rate data by breast cancer subtype. We, therefore, used two unique population-based resources with molecular data to compare incidence rates for the 'intrinsic' breast cancer subtypes between a low-risk Asian population in Malaysia and high-risk non-Hispanic white population in the National Cancer Institute's surveillance, epidemiology, and end results 18 registries database (SEER 18). The intrinsic breast cancer subtypes were recapitulated with the joint expression of the HRs (estrogen receptor and progesterone receptor) and human epidermal growth factor receptor-2 (HER2). Invasive breast cancer incidence rates overall were fivefold greater in SEER 18 than in Malaysia. The majority of breast cancers were HR-positive in SEER 18 and HR-negative in Malaysia. Notwithstanding the greater relative distribution for HR-negative cancers in Malaysia, there was a greater absolute risk for all subtypes in SEER 18; incidence rates were nearly 7-fold higher for HR-positive and 2-fold higher for HR-negative cancers in SEER 18. Despite the well-established relative breast cancer differences between low-risk and high-risk countries and/or populations, there was a greater absolute risk for HR-positive and HR-negative subtypes in the US than Malaysia. Additional analytical studies are sorely needed to determine the factors responsible for the elevated risk of all subtypes of breast cancer in high-risk countries like the United States.

The Breadth of Cross Sub-Type Neutralisation Activity of a Single Domain Antibody to Influenza Hemagglutinin Can Be Increased by Antibody Valency

PubMed Central

Hufton, Simon E.; Risley, Paul; Ball, Christina R.; Major, Diane; Engelhardt, Othmar G.; Poole, Stephen

2014-01-01

The response to the 2009 A(H1N1) influenza pandemic has highlighted the need for additional strategies for intervention which preclude the prior availability of the influenza strain. Here, 18 single domain VHH antibodies against the 2009 A(H1N1) hemagglutinin (HA) have been isolated from a immune alpaca phage displayed library. These antibodies have been grouped as having either (i) non-neutralising, (ii) H1N1 restricted neutralising or (iii) broad cross-subtype neutralising activity. The ability to neutralise different viral subtypes, including highly pathogenic avian influenza (H5N1), correlated with the absence of hemagglutination inhibition activity, loss of binding to HA at acid pH and the absence of binding to the head domain containing the receptor binding site. This data supports their binding to epitopes in the HA stem region and a mechanism of action other than blocking viral attachment to cell surface receptors. After conversion of cross-neutralising antibodies R1a-B6 and R1a-A5 into a bivalent format, no significant enhancement in neutralisation activity was seen against A(H1N1) and A(H5N1) viruses. However, bivalent R1a-B6 showed an 18 fold enhancement in potency against A(H9N2) virus and, surprisingly, gained the ability to neutralise an A(H2N2) virus. This demonstrates that cross-neutralising antibodies, which make lower affinity interactions with the membrane proximal stem region of more divergent HA sub-types, can be optimised by bivalency so increasing their breadth of anti-viral activity. The broad neutralising activity and favourable characteristics, such as high stability, simple engineering into bivalent molecules and low cost production make these single domain antibodies attractive candidates for diagnostics and immunotherapy of pandemic influenza. PMID:25084445

NMDA and AMPA receptors in the anterior cingulate cortex mediates visceral pain in visceral hypersensitivity rats.

PubMed

Zhou, Lin; Huang, Junjing; Gao, Jun; Zhang, Guanpo; Jiang, Jinjin

2014-02-01

Several studies have shown that N-methyl-D-aspartate (NMDA)-receptor activation in anterior cingulate cortex (ACC) neurons plays critical roles in modulating visceral pain responses in visceral hypersensitivity (VH) rats. However, there are few reports about the expressions of NMDA and α-amino-3-hydroxy-5-methyl-4-isox-azolepropionic-acid (AMPA) receptor subtypes in ACC of VH model rats at different time points. The current study was undertaken to investigate NR2A, NR2B and GluR2 expressions in ACC of VH rats that were induced by administration with 5% mustard oil. Our results indicated that NR2B, but not NR2A, was highly expressed in VH model group on day 15, 22, and 36 compared with normal group (p < 0.05). GluR2 expression was also higher in VH model group on day 15, 22, and 36 than that of normal group (p < 0.05). These findings suggested increased expression of NR2B and GluR2 might be key mechanisms for long-term synaptic plastic changes in VH rats. Copyright © 2014. Published by Elsevier Inc.

Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3

DTIC Science & Technology

2000-05-25

subsequent transmitter release. The rat hippocampal slice is a preparation richly endowed with ionotropic and metabotropic glutamate receptors ...M. Zhao and R. J. Wenthold (1996b). Ionotropic and metabotropic glutamate receptors show unique postsynaptic, presynaptic, and glial localizations in...epileptiform activity in the rat cortex. Neuroreport 3(10): 916-8. Shen, W. and M. M. Slaughter (1998). Metabotropic and ionotropic glutamate receptors

Pharmacological Properties and Discriminative Stimulus Effects of a Novel and Selective 5-HT2 Receptor Agonist AL-38022A [(S)-2-(8,9-dihydro-7H-pyrano[2,3-g]indazol-1-yl)-1-methylethylamine

PubMed Central

May, Jesse A.; Sharif, Najam A.; Chen, Hwang-Hsing; Liao, John C.; Kelly, Curtis R.; Glennon, Richard A.; Young, Richard; Li, Jun-Xu; Rice, Kenner C.; France, Charles P.

2013-01-01

AL-38022A is a novel synthetic serotonergic (5-HT) ligand that exhibited high affinity for each of the 5-HT2 receptor subtypes (Ki ≤ 2.2 nM), but a significantly lower (>100-fold less) affinity for other 5-HT receptors. In addition, AL-38022A displayed a very low affinity for a broad array of other receptors, neurotransmitter transport sites, ion channels, and second messenger elements, making it a relatively selective agent. AL-38022A potently stimulated functional responses via native and cloned rat (EC50 range: 1.9 – 22.5 nM) and human (EC50 range: 0.5 – 2.2 nM) 5-HT2 receptor subtypes including [Ca2+]i mobilization and tissue contractions with apparently similar potencies and intrinsic activities and was a full agonist at all 5-HT2 receptor subtypes. The CNS activity of AL-38022A was assessed by evaluating its discriminative stimulus effects in both a rat and a monkey drug discrimination paradigm using DOM as the training drug. AL-38022A fully generalized to the DOM stimulus in each of these studies; in monkeys MDL 100907 antagonized both DOM and AL-38022A. The pharmacological profile of AL-38022A suggests that it could be a useful tool in defining 5-HT2 receptor signaling and receptor characterization where 5-HT may function as a neurotransmitter. PMID:18718483

Modulation of Gardos channel activity by cytokines in sickle erythrocytes.

PubMed

Rivera, Alicia; Jarolim, Petr; Brugnara, Carlo

2002-01-01

It has recently been shown that the Gardos channel activity of mouse erythrocytes can be modified by endothelins, suggesting a functional linkage between endothelin receptors and the Gardos channel. Using (86)Rubidium ((86)Rb) influx, effects were estimated of proinflammatory molecules such as platelet activator factor (PAF), endothelin-1 (ET-1), interleukin-10 (IL-10), and regulated on activation normal T cells expressed and secreted (RANTES) on the Gardos channel activity in human normal and sickle red cells. It was found that PAF (EC(50): 15 +/- 7 nM), RANTES (EC(50), 9 +/- 6 ng/mL [1.2 +/- 0.8 nM]), IL-10 (EC(50), 11 +/- 8 ng/mL [204 +/- 148 nM]), and ET-1 (EC(50), 123 +/- 34 nM) induce a significant increase in Gardos channel activity-between 28% and 84%-over the control. In addition, these agents modify the Gardos channel affinity for internal Ca(++) (K(0.5)) by 2- to 6-fold. Biochemical evidence is provided for the presence of ET receptor subtype B in sickle and normal red cells. Furthermore, it was found that ET-1, PAF, RANTES, and IL-10 induce a significant increase in red cell density (P <.05). These data suggest that activation of the Gardos channel is functionally coupled to receptor motifs such as C-X-C (PAF), C-C (RANTES), and ET receptor subtype B. Thus, cell volume regulation or erythrocyte hydration states might be altered by activation of the Gardos channel by cytokines in vivo. The role of these mediators in promoting sickle cell dehydration in vivo is under investigation.

Post-translational regulation of P2X receptor channels: modulation by phospholipids

PubMed Central

Bernier, Louis-Philippe; Ase, Ariel R.; Séguéla, Philippe

2013-01-01

P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia. PMID:24324400

Cholecystokinin-8 inhibits methamphetamine-induced neurotoxicity via an anti-oxidative stress pathway.

PubMed

Wen, Di; An, Meiling; Gou, Hongyan; Liu, Xia; Liu, Li; Ma, Chunling; Cong, Bin

2016-12-01

As a powerful addictive psychostimulant drug, coupled with its neurotoxicity, methamphetamine (METH) abuse may lead to long-lasting abnormalities in brain structure and function. We found that pretreatment of cholecystokinin-8 (CCK-8) inhibited METH-induced brain cellular dopaminergic (DA) damage in the striatum and substantia nigra, and related behavioural deficits and hyperthermia. However, the mechanism of CCK-8 action on METH-induced toxicity is not clear. The aim of this study was to explore whether the possible protective effect of CCK-8 on METH-induced neurotoxicity involved anti-oxidative stress mechanisms. The subtypes of CCK receptors mediating the regulatory action of CCK-8 were also investigated. The present results revealed that CCK-8 dose-dependently inhibited METH-induced cytotoxic effect by activating the CCK2 receptor subtype in PC12 cells and CCK2 receptor stable transfected-HEK293 cells. Pre-treatment of CCK-8 before METH stimulation significantly attenuated the generation of reactive oxygen species and NADPH oxidase activation in PC12 cells. In conclusion, our study demonstrated a protective effect of CCK-8 on METH-induced neurotoxicity in vitro and suggested that a possible mechanism of this action was dependent on the activation of the CCK2 receptor to reduce the neurotoxicity and oxidative stress induced by METH stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Activation of lumbosacral 5-HT2C receptors induces bursts of rhythmic activity in sympathetic nerves to the vas deferens in male rats

PubMed Central

Stafford, Stuart A; Tang, Kim; Coote, John H

2006-01-01

We previously demonstrated that p-chloroamphetamine (PCA) intravenously (i.v.) evokes a specific patterned bursting response in the vas deferens nerve (VDN) of anaesthetised male rats that is associated with contraction of the vas deferens, and ejaculation and contraction of the bulbospongiosus muscles. The present study used selective 5-HT agonists to induce similar rhythmic bursting responses in the VDN in order to reveal the 5-HT receptor subtypes involved. The 5-HT2C receptor agonist (1.0 mg kg−1 Ro600175 i.v.) evoked the characteristic bursting pattern responses in the VDN. The 5-HT1A receptor agonist (1.0 mg kg−1 8-OH-DPAT i.v.) failed to elicit any responses. However, 8-OH-DPAT coadministered in combination with Ro600175 induced a potentiation of the responses. Responses were also evoked in rats with a mid-thoracic spinalisation, with a more predictable response being observed following the combination of agonists. This suggests an action of both agonists in the lumbosacral spinal cord. Responses were blocked by 0.5 mg kg−1 SB206553 i.v. (5-HT2B/C receptor antagonist) or 0.5 mg kg−1 WAY100635 i.v. (5-HT1A receptor antagonist), but not 0.1 or 1.0 mg kg−1 SB269970 i.v. (5-HT7 receptor antagonist). We suggest that activation of 5-HT2C and 5-HT1A receptor subtypes synergistically elicits contraction of the vas deferens through the activation of sympathetic preganglionic neurones in the spinal cord. These data support the idea of a proejaculatory action of 5-HT2C receptors in the lumbosacral spinal cord, suggesting a descending 5-HT excitatory pathway in addition to a 5-HT inhibitory pathway. An excitatory action of 8-OH-DPAT at lumbosacral sites is also evident. PMID:16799648

Activation of α7 nicotinic acetylcholine receptors persistently enhances hippocampal synaptic transmission and prevents Aß-mediated inhibition of LTP in the rat hippocampus.

PubMed

Ondrejcak, Tomas; Wang, Qinwen; Kew, James N C; Virley, David J; Upton, Neil; Anwyl, Roger; Rowan, Michael J

2012-02-29

Nicotinic acetylcholine receptors mediate fast cholinergic modulation of glutamatergic transmission and synaptic plasticity. Here we investigated the effects of subtype selective activation of the α7 nicotinic acetylcholine receptors on hippocampal transmission and the inhibition of synaptic long-term potentiation by the Alzheimer's disease associated amyloid ß-protein (Aß). The α7 nicotinic acetylcholine receptor agonist "compound A" ((R)-N-(1-azabicyclo[2.2.2]oct-3-yl)(5-(2-pyridyl))thiophene-2-carboxamide) induced a rapid-onset persistent enhancement of synaptic transmission in the dentate gyrus in vitro. Consistent with a requirement for activation of α7 nicotinic acetylcholine receptors, the type II α7-selective positive allosteric modulator PheTQS ((3aR, 4S, 9bS)-4-(4-methylphenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide) potentiated, and the antagonist methyllycaconitine (MLA) prevented the persistent enhancement. Systemic injection of the agonist also induced a similar MLA-sensitive persistent enhancement of synaptic transmission in the CA1 area in vivo. Remarkably, although compound A did not affect control long-term potentiation (LTP) in vitro, it prevented the inhibition of LTP by Aß1-42 and this effect was inhibited by MLA. These findings strongly indicate that activation of α7 nicotinic acetylcholine receptors is sufficient to persistently enhance hippocampal synaptic transmission and to overcome the inhibition of LTP by Aß. Copyright © 2011 Elsevier B.V. All rights reserved.

Similar Replicative Fitness Is Shared by the Subtype B and Unique BF Recombinant HIV-1 Isolates that Dominate the Epidemic in Argentina

PubMed Central

Rubio, Andrea E.; Abraha, Awet; Carpenter, Crystal A.; Troyer, Ryan M.; Reyes-Rodríguez, Ángel L.; Salomon, Horacio; Arts, Eric J.; Tebit, Denis M.

2014-01-01

The HIV-1 epidemic in South America is dominated by pure subtypes (mostly B and C) and more than 7 BF and BC recombinant forms. In Argentina, circulating recombinant forms (CRFs) comprised of subtypes B and F make up more than 50% of HIV infections. For this study, 28 HIV-1 primary isolates were obtained from patients in Buenos Aires, Argentina and initially classified into subtype B (n = 9, 32.1%), C (n = 1, 3.6%), and CRFs (n = 18, 64.3%) using partial pol and vpu-env sequences, which proved to be inconsistent and inaccurate for these phylogenetic analyses. Near full length genome sequences of these primary HIV-1 isolates revealed that nearly all intersubtype BF recombination sites were unique and countered previous “CRF” B/F classifications. The majority of these Argentinean HIV-1 isolates were CCR5-using but 4 had a dual/mixed tropism as predicted by both phenotypic and genotypic assays. Comparison of the replicative fitness of these BF primary HIV-1 isolates to circulating B, F, and C HIV-1 using pairwise competitions in peripheral blood mononuclear cells (PBMCs) indicated a similarity in fitness of these BF recombinants to subtypes B and F HIV-1 (of the same co-receptor usage) whereas subtype C HIV-1 was significantly less fit than all as previously reported. These results suggest that the multitude of BF HIV-1 strains present within the Argentinean population do not appear to have gained replicative fitness following recent B and F recombination events. PMID:24727861

Effect of neonatal handling on serotonin 1A sub-type receptors in the rat hippocampus.

PubMed

Stamatakis, A; Mantelas, A; Papaioannou, A; Pondiki, S; Fameli, M; Stylianopoulou, F

2006-06-19

Serotonin 1A sub-type receptors play an important role in the etiopathogenesis of depression, which is known to occur more often in females than males. Early experiences can be a predisposing factor for depression; however, the underlying cellular processes remain unknown. In an effort to address such issues, we employed neonatal handling, an experimental model of early experience, which has been previously shown to render females more vulnerable to display enhanced depression-like behavior in response to chronic stress, while it increases the ability of males to cope. In rat pre-pubertal (30 days of age) and adult (90 days) hippocampus, of both males and females, the effect of neonatal handling on serotonin 1A sub-type receptor mRNA and protein levels was determined by in situ hybridization and immunohistochemistry, respectively, while the number of binding sites was determined by in vitro autoradiography using [(3)H]8-hydroxy-2(di-n-propylamino)tetralin as the ligand. Our results revealed a significant sex difference in serotonin 1A sub-type receptor mRNA, protein and binding sites, with females having higher levels than males. Handling resulted in statistically significant decreased numbers of cells positive for serotonin 1A sub-type receptor mRNA or protein, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites in the area 4 of Ammon's horn and dentate gyrus of both pre-pubertal males and females. In adult animals the number of serotonin 1A sub-type receptor mRNA positive cells was increased as a result of handling in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of males, while it was decreased only in the area 4 of Ammon's horn of females. Furthermore, the number of serotonin sub-type 1A receptor immunopositive cells, as well as [(3)H]8-hydroxy-2(di-n-propylamino)tetralin binding sites was increased in the area 1 of Ammon's horn, area 4 of Ammon's horn and dentate gyrus of handled males, whereas it was decreased in these same brain areas in the handled females. We can thus infer that neonatal handling results in alterations in postsynaptic serotonergic neurotransmission, which may contribute to the sex dimorphic effects of handling as to the vulnerability toward depression-like behavior in response to chronic stressful stimuli.

Natural killer cell-mediated innate sieve effect on HIV-1: the impact of KIR/HLA polymorphism on HIV-1 subtype-specific acquisition in east Africa.

PubMed

Koehler, Rebecca N; Alter, Galit; Tovanabutra, Sodsai; Saathoff, Elmar; Arroyo, Miguel A; Walsh, Anne M; Sanders-Buell, Eric E; Maboko, Leonard; Hoelscher, Michael; Robb, Merlin L; Michael, Nelson L; McCutchan, Francine E; Kim, Jerome H; Kijak, Gustavo H

2013-10-15

Here we explore the association between killer cell immunoglobulin-like receptor (KIR)/HLA and human immunodeficiency virus type 1 (HIV-1) acquisition with different viral subtypes circulating in East Africa. In the prospective Cohort Development (CODE) cohort (Mbeya, Tanzania), carriers of KIR3DS1 and its putative ligand (HLA-A or HLA-B Bw4-80Ile alleles) showed increased HIV-1 acquisition risk (odds ratio [OR] = 3.46; 95% confidence interval [CI], 1.12-10.63; P = .04) and a trend for enrichment for subtype A and A-containing recombinants (78% vs. 46%; OR = 4.05; 95% CI, .91-28.30; P = .09) at the expense of subtype C (11% vs. 43%; OR = 0.17; 95% CI, .01-.97; P = .08). In vitro, only natural killer cells from KIR3DS1(+)/HLA-Bw4-80Ile(+) healthy donors showed a 2-fold increased capacity to inhibit replication of subtype C vs subtype A viruses (P = .01). These findings suggest the presence of an innate sieve effect and may inform HIV-1 vaccine development.

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

Kappa2 opioid receptor subtype binding requires the presence of the DOR-1 gene.

PubMed

Ansonoff, Michael A; Wen, Ting; Pintar, John E

2010-01-01

Over the past several years substantial evidence has documented that opioid receptor homo- and heterodimers form in cell lines expressing one or more of the opioid receptors. We used opioid receptor knockout mice to determine whether in vivo pharmacological characteristics of kappa1 and kappa2 opioid receptors changed following knockout of specific opioid receptors. Using displacement of the general opioid ligand diprenorphine, we observed that occupancy or knockout of the DOR-1 gene increases the binding density of kappa1 receptors and eliminates kappa2 receptors in crude membrane preparations while the total density of kappa opioid binding sites is unchanged. Further, the analgesic potency of U69,593 in cumulative dose response curves is enhanced in mice lacking the DOR-1 gene. These results demonstrate that the DOR-1 gene is required for the expression of the kappa2 opioid receptor subtype and are consistent with the possibility that a KOR-1/DOR-1 heterodimer mediates kappa2 pharmacology.

Immunohistochemical localization of alpha and beta adrenergic receptors in the human nasal turbinate.

PubMed

Shirasaki, Hideaki; Kanaizumi, Etsuko; Himi, Tetsuo

2016-06-01

Adrenergic receptors (ARs) include four general types (α1, α2, β1 and β2), which are found in different target tissues. α-AR agonists are commonly used for decongestant therapy of upper airway diseases. In order to clarify the roles of AR subtypes in the upper airways, we investigated the localization of these receptors by immunohistochemistry. Human turbinates were obtained after turbinectomy from 12 patients with nasal obstruction refractory to medical therapy. The specific cells expressing α- and β-AR proteins were identified by immunostaining using an anti-human AR subtype-specific antibodies (α1A-, α1D-, α2C- and β2-ARs) antibody. Immunohistochemical analysis revealed that immunoreactivities for α1D- and β2-ARs were densely distributed in submucosal glands. In contrast, immunoreactivities for α1A- and 2C-ARs were densely distributed in vascular smooth muscle. Our results suggested that adrenergic receptor (AR) subtypes had different roles in upper airway diseases, such as allergic rhinitis and nonallergic rhinitis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

5-HT2A receptor activation is necessary for CO2-induced arousal

PubMed Central

Smith, Haleigh R.; MacAskill, Amanda; Richerson, George B.

2015-01-01

Hypercapnia-induced arousal from sleep is an important protective mechanism pertinent to a number of diseases. Most notably among these are the sudden infant death syndrome, obstructive sleep apnea and sudden unexpected death in epilepsy. Serotonin (5-HT) plays a significant role in hypercapnia-induced arousal. The mechanism of 5-HT's role in this protective response is unknown. Here we sought to identify the specific 5-HT receptor subtype(s) involved in this response. Wild-type mice were pretreated with antagonists against 5-HT receptor subtypes, as well as antagonists against adrenergic, cholinergic, histaminergic, dopaminergic, and orexinergic receptors before challenge with inspired CO2 or hypoxia. Antagonists of 5-HT2A receptors dose-dependently blocked CO2-induced arousal. The 5-HT2C receptor antagonist, RS-102221, and the 5-HT1A receptor agonist, 8-OH-DPAT, attenuated but did not completely block CO2-induced arousal. Blockade of non-5-HT receptors did not affect CO2-induced arousal. None of these drugs had any effect on hypoxia-induced arousal. 5-HT2 receptor agonists were given to mice in which 5-HT neurons had been genetically eliminated during embryonic life (Lmx1bf/f/p) and which are known to lack CO2-induced arousal. Application of agonists to 5-HT2A, but not 5-HT2C, receptors, dose-dependently restored CO2-induced arousal in these mice. These data identify the 5-HT2A receptor as an important mediator of CO2-induced arousal and suggest that, while 5-HT neurons can be independently activated to drive CO2-induced arousal, in the absence of 5-HT neurons and endogenous 5-HT, 5-HT receptor activation can act in a permissive fashion to facilitate CO2-induced arousal via another as yet unidentified chemosensor system. PMID:25925320

Fractionating spatial memory with glutamate receptor subunit-knockout mice.

PubMed

Bannerman, David M

2009-12-01

In recent years, the contribution that different glutamate receptor subtypes and subunits make to spatial learning and memory has been studied extensively using genetically modified mice in which key proteins are knocked out. This has revealed dissociations between different aspects of spatial memory that were not previously apparent from lesion studies. For example, studies with GluA1 AMPAR [AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptor] subunit-knockout mice have revealed the presence of a GluA1-dependent, non-associative short-term memory mechanism that is important for performance on spatial working memory tasks, and a GluA1-independent, long-term associative memory mechanism which underlies performance on spatial reference memory tasks. Within this framework we have also studied the contributions of different GluN2-containing NMDARs [NMDA (N-methyl-D-aspartate) receptors] to spatial memory. Studies with GluN2 NMDAR mutants have revealed different contributions from GluN2A- and GluN2B-containing NMDARs to spatial learning. Furthermore, comparison of forebrain- and hippocampus-specific GluN2B-knockout mice has demonstrated that both hippocampal and extra-hippocampal NMDARs make important contributions to spatial memory performance.

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

Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

PubMed

Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

2004-01-01

In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

Characterization of endogenous calcium responses in neuronal cell lines.

PubMed

Vetter, Irina; Lewis, Richard J

2010-03-15

An increasing number of putative therapeutic targets have been identified in recent years for the treatment of neuronal pathophysiologies including pain, epilepsy, stroke and schizophrenia. Many of these targets signal through calcium (Ca(2+)), either by directly facilitating Ca(2+) influx through an ion channel, or through activation of G proteins that couple to intracellular Ca(2+) stores or voltage-gated Ca(2+) channels. Immortalized neuronal cell lines are widely used models to study neuropharmacology. However, systematic pharmacological characterization of the receptors and ion channels expressed in these cell lines is lacking. In this study, we systematically assessed endogenous Ca(2+) signaling in response to addition of agonists at potential therapeutic targets in a range of cell lines of neuronal origin (ND7/23, SH-SY5Y, 50B11, F11 and Neuro2A cells) as well as HEK293 cells, a cell line commonly used for over-expression of receptors and ion channels. This study revealed a remarkable diversity of endogenous Ca(2+) responses in these cell lines, with one or more cell lines responding to addition of trypsin, bradykinin, ATP, nicotine, acetylcholine, histamine and neurotensin. Subtype specificity of these responses was inferred from agonist potency and the effect of receptor subtype specific antagonist. Surprisingly, HEK293 and SH-SY5Y cells responded to the largest number of agonists with potential roles in neuronal signaling. These findings have implications for the heterologous expression of neuronal receptors and ion channels in these cell lines, and highlight the potential of neuron-derived cell lines for the study of a range of endogenously expressed receptors and ion channels that signal through Ca(2+). Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

Dynamic control of glutamatergic synaptic input in the spinal cord by muscarinic receptor subtypes defined using knockout mice.

PubMed

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-12-24

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M(2), M(3), and M(4)) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M(3)-single KO and M(1)/M(3) double-KO mice. In addition, the M(3) antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M(5)-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M(2)/M(4) double-KO mice, but not M(2)- or M(4)-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M(2)/M(4) antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M(2) and M(4) receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M(5) is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord.

Dynamic Control of Glutamatergic Synaptic Input in the Spinal Cord by Muscarinic Receptor Subtypes Defined Using Knockout Mice*

PubMed Central

Chen, Shao-Rui; Chen, Hong; Yuan, Wei-Xiu; Wess, Jürgen; Pan, Hui-Lin

2010-01-01

Activation of muscarinic acetylcholine receptors (mAChRs) in the spinal cord inhibits pain transmission. At least three mAChR subtypes (M2, M3, and M4) are present in the spinal dorsal horn. However, it is not clear how each mAChR subtype contributes to the regulation of glutamatergic input to dorsal horn neurons. We recorded spontaneous excitatory postsynaptic currents (sEPSCs) from lamina II neurons in spinal cord slices from wild-type (WT) and mAChR subtype knock-out (KO) mice. The mAChR agonist oxotremorine-M increased the frequency of glutamatergic sEPSCs in 68.2% neurons from WT mice and decreased the sEPSC frequency in 21.2% neurons. Oxotremorine-M also increased the sEPSC frequency in ∼50% neurons from M3-single KO and M1/M3 double-KO mice. In addition, the M3 antagonist J104129 did not block the stimulatory effect of oxotremorine-M in the majority of neurons from WT mice. Strikingly, in M5-single KO mice, oxotremorine-M increased sEPSCs in only 26.3% neurons, and J104129 abolished this effect. In M2/M4 double-KO mice, but not M2- or M4-single KO mice, oxotremorine-M inhibited sEPSCs in significantly fewer neurons compared with WT mice, and blocking group II/III metabotropic glutamate receptors abolished this effect. The M2/M4 antagonist himbacine either attenuated the inhibitory effect of oxotremorine-M or potentiated the stimulatory effect of oxotremorine-M in WT mice. Our study demonstrates that activation of the M2 and M4 receptor subtypes inhibits synaptic glutamate release to dorsal horn neurons. M5 is the predominant receptor subtype that potentiates glutamatergic synaptic transmission in the spinal cord. PMID:20940295

The presence and distribution of alpha adrenergic receptors in human renal pelvis and calyces.

PubMed

Karabacak, Osman Raif; Yilmazer, Demet; Ozturk, Ufuk; Sener, Nevzat Can; Saltas, Hakan; Karabacak, Yurdum; Alper, Murat

2013-10-01

In this study, we aimed to demonstrate the presence of Alpha (α) 1 receptors and subtypes in human pelvis and calyces, because an agent to facilitate kidney stone movement and help decrease pain may be an α 1 adrenergic blocker, as used in ureteral stones. Twenty patients who applied to our clinic for renal cell carcinoma were enrolled to the study. All patients underwent radical nephrectomy. After the specimens were removed, excisional biopsies were performed on healthy pelvises and calyces. Mean α-receptor stain rates in renal pelvis were 2.65 ± 0.74, 1.35 ± 0.81 and 2.9 ± 0.30 for α 1A, 1B and 1D, respectively. For calyces, the rates are 2.40 ± 0.82, 1.50 ± 0.76 and 2.75 ± 0.44 for α 1A, 1B and 1D, respectively (Fig. 1). When the staining patterns were compared, α 1A and 1D were expressed more in both pelvis and calyces than α 1B (p < 0.05). After the demonstration of α-adrenergic receptors in pelvis and calyces of human kidney, it may be helpful in coming up with new alternative treatments for patients suffering from kidney stones.

X-ray structure, symmetry and mechanism of an AMPA-subtype glutamate receptor

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

Sobolevsky, Alexander I.; Rosconi, Michael P.; Gouaux, Eric

2010-02-02

Ionotropic glutamate receptors mediate most excitatory neurotransmission in the central nervous system and function by opening a transmembrane ion channel upon binding of glutamate. Despite their crucial role in neurobiology, the architecture and atomic structure of an intact ionotropic glutamate receptor are unknown. Here we report the crystal structure of the {alpha}-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-sensitive, homotetrameric, rat GluA2 receptor at 3.6 {angstrom} resolution in complex with a competitive antagonist. The receptor harbours an overall axis of two-fold symmetry with the extracellular domains organized as pairs of local dimers and with the ion channel domain exhibiting four-fold symmetry. A symmetry mismatchmore » between the extracellular and ion channel domains is mediated by two pairs of conformationally distinct subunits, A/C and B/D. Therefore, the stereochemical manner in which the A/C subunits are coupled to the ion channel gate is different from the B/D subunits. Guided by the GluA2 structure and site-directed cysteine mutagenesis, we suggest that GluN1 and GluN2A NMDA (N-methyl-D-aspartate) receptors have a similar architecture, with subunits arranged in a 1-2-1-2 pattern. We exploit the GluA2 structure to develop mechanisms of ion channel activation, desensitization and inhibition by non-competitive antagonists and pore blockers.« less

In vitro contractile effects of neurokinin receptor blockade in the human ureter.

PubMed

Nakada, S Y; Jerde, T J; Bjorling, D E; Saban, R

2001-10-01

We identified the predominance of neurokinin-2 receptors and evaluated the inhibition of spontaneous contraction via the blockade of neurokinin-2 receptors in human ureteral segments. Excess ureteral segments from human subjects undergoing donor nephrectomy or reconstructive procedures were suspended in tissue baths containing Krebs buffer. After spontaneous contractions were recorded, tissues were incubated with 1 microM. solutions of phosphoramidon and captopril (to inhibit peptide degradation) and either the neurokinin-1 receptor antagonist CP 99,994, the neurokinin-2 receptor antagonist SR 48,968, the neurokinin-3 receptor antagonist SR 142,801 or dimethyl sulfoxide (control) for 1 hour. Contraction magnitude and frequency were again recorded and compared with spontaneous levels. Concentration-response curves to the tachykinins substance P, and neurokinins A and B were determined in the presence and absence of antagonists. Neurokinin A increased contractility at lower concentrations than substance P or neurokinin B (p <0.013). Neurokinin-2 receptor blockade produced a 100-fold rightward shift of the concentration-response curves (p <0.013), while neurokinins 1 and 3 receptor blockade had no effect. SR 48,968 significantly reduced contractility during the 1-hour incubation period, causing a 97% reduction in spontaneous rates compared with a 29% reduction in control tissues. CP 99,994 and SR 142,801 had no significant effect. Neurokinin-2 is the predominant receptor subtype responsible for tachykinin induced contraction of human ureteral smooth muscle. In vitro treatment with the neurokinin-2 antagonist SR 48,968 reduces the spontaneous contraction rate by 97% in vitro. Neurokinin-2 receptor antagonists may have clinical applications for ureteral disease.

Effects of norepinephrine on alpha-subtype receptors in the feline pulmonary vascular bed.

PubMed

Kaye, Alan D; Hoover, Jason M; Baber, Syed R; Ibrahim, Ikhlass N; Fields, Aaron M

2004-11-01

To test the hypothesis that norepinephrine induces a pressor response in the pulmonary vascular bed of the cat and identify the alpha-(1)adrenoceptor subtypes involved in the mediation or modulation of these effects. Prospective vehicle controlled study. University research laboratory. Intact chest preparation, adult mongrel cats. In separate experiments, the effects of 5-methyl-urapidil, a selective alpha-(1)A-subtype adrenoceptor antagonist, chloroethylclonidine, an alpha-(1)B-subtype and -(1)D-subtype adrenoceptor antagonist, and BMY 7378, the selective alpha-(1)D-subtype adrenoceptor antagonist, were investigated on pulmonary arterial responses to norepinephrine and other agonists in the pulmonary vascular bed of the cat. The systemic pressure and lobar arterial perfusion pressure were continuously monitored, electronically averaged, and permanently recorded. In the feline pulmonary vascular bed of the isolated left lower lobe, norepinephrine induced a dose-dependent vasoconstrictor response that was not significantly altered after administration of BMY 7378. However, the responses to norepinephrine were significantly attenuated following administration of 5-methyl-urapidil and chloroethylclonidine. The results of the present study suggest that norepinephrine has potent vasopressor activity in the pulmonary vascular bed of the cat and that this response may be mediated or modulated by both alpha-(1)A-subtype and -(1)B-subtype adrenoceptor sensitive pathways.

Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates

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

Srivastava, Indresh K.; Kan, Elaine; Sun Yide

2008-03-15

We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140{delta}V2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV{sub SF162P4} virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140{delta}V2TV1 (subtype C {delta}V2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C {delta}V2 trimer; however, we did not observe significant binding for the b12 mAb. Themore » molecular mass of subtype C {delta}V2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C {delta}V2 trimer binds to CD4 with an affinity comparable to o-gp140{delta}V2SF162 (subtype B {delta}V2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C {delta}V2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.« less

Muscarinic subtypes profile modulation within a series of new antagonists, bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine.

PubMed

Piergentili, Alessandro; Gentili, Francesco; Ghelfi, Francesca; Marucci, Gabriella; Pigini, Maria; Quaglia, Wilma; Giannella, Mario

2003-09-01

A set of new muscarinic antagonists, bridged bicyclic derivatives of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine (1), was synthesized and tested to evaluate their affinity and selectivity for M(1), M(2), M(3) and M(4) receptor subtypes. The conformational constraint of 1 in a bicyclic structure, and the variation in distance and stereochemistry of the active functions allowed us to modulate the selectivity of interaction with the M(1)-M(3) receptor subtypes. The most interesting compound was (cis,trans)-2-(2,2-diphenylethyl)-5-methyl-tetrahydro-[1,3]dioxolo[4,5-c]pyrrole oxalate (6), which is equipotent with Pirenzepine on rabbit vas deferens (M(1)-putative) but shows a better selectivity profile.

Erythropoiesis and Blood Pressure Are Regulated via AT1 Receptor by Distinctive Pathways.

PubMed

Kato, Hideki; Ishida, Junji; Matsusaka, Taiji; Ishimaru, Tomohiro; Tanimoto, Keiji; Sugiyama, Fumihiro; Yagami, Ken-Ichi; Nangaku, Masaomi; Fukamizu, Akiyoshi

2015-01-01

The renin-angiotensin system (RAS) plays a central role in blood pressure regulation. Although clinical and experimental studies have suggested that inhibition of RAS is associated with progression of anemia, little evidence is available to support this claim. Here we report that knockout mice that lack angiotensin II, including angiotensinogen and renin knockout mice, exhibit anemia. The anemia of angiotensinogen knockout mice was rescued by angiotensin II infusion, and rescue was completely blocked by simultaneous administration of AT1 receptor blocker. To genetically determine the responsible receptor subtype, we examined AT1a, AT1b, and AT2 knockout mice, but did not observe anemia in any of them. To investigate whether pharmacological AT1 receptor inhibition recapitulates the anemic phenotype, we administered AT1 receptor antagonist in hypotensive AT1a receptor knockout mice to inhibit the remaining AT1b receptor. In these animals, hematocrit levels barely decreased, but blood pressure further decreased to the level observed in angiotensinogen knockout mice. We then generated AT1a and AT1b double-knockout mice to completely ablate the AT1 receptors; the mice finally exhibited the anemic phenotype. These results provide clear evidence that although erythropoiesis and blood pressure are negatively controlled through the AT1 receptor inhibition in vivo, the pathways involved are complex and distinct, because erythropoiesis is more resistant to AT1 receptor inhibition than blood pressure control.

International Workshop on Structural and Functional Aspects of the Cholinergic Synapse Held in Jerusalem, Israel on 30 August-4 September 1987

DTIC Science & Technology

1987-09-04

agonist were compared to those of the ganglionic Ml-selective agonist, McN-A- 343 and to oxotremorine which has a relative high selectivity toward M2...lower than that of oxotremorine However AF102B was similar to McN-A-343 in displacing H-QNB from forebrain homogenate (rich in Ml receptor subtype

Dietary fat intake and development of specific breast cancer subtypes.

PubMed

Sieri, Sabina; Chiodini, Paolo; Agnoli, Claudia; Pala, Valeria; Berrino, Franco; Trichopoulou, Antonia; Benetou, Vassiliki; Vasilopoulou, Effie; Sánchez, María-José; Chirlaque, Maria-Dolores; Amiano, Pilar; Quirós, J Ramón; Ardanaz, Eva; Buckland, Genevieve; Masala, Giovanna; Panico, Salvatore; Grioni, Sara; Sacerdote, Carlotta; Tumino, Rosario; Boutron-Ruault, Marie-Christine; Clavel-Chapelon, Françoise; Fagherazzi, Guy; Peeters, Petra H M; van Gils, Carla H; Bueno-de-Mesquita, H Bas; van Kranen, Henk J; Key, Timothy J; Travis, Ruth C; Khaw, Kay Tee; Wareham, Nicholas J; Kaaks, Rudolf; Lukanova, Annekatrin; Boeing, Heiner; Schütze, Madlen; Sonestedt, Emily; Wirfält, Elisabeth; Sund, Malin; Andersson, Anne; Chajes, Veronique; Rinaldi, Sabina; Romieu, Isabelle; Weiderpass, Elisabete; Skeie, Guri; Dagrun, Engeset; Tjønneland, Anne; Halkjær, Jytte; Overvard, Kim; Merritt, Melissa A; Cox, David; Riboli, Elio; Krogh, Vittorio

2014-04-09

We prospectively evaluated fat intake as predictor of developing breast cancer (BC) subtypes defined by estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 receptor (HER2), in a large (n = 337327) heterogeneous cohort of women, with 10062 BC case patients after 11.5 years, estimating BC hazard ratios (HRs) by Cox proportional hazard modeling. High total and saturated fat were associated with greater risk of ER(+)PR(+) disease (HR = 1.20, 95% confidence interval [CI] = 1.00 to 1.45; HR = 1.28, 95% CI = 1.09 to 1.52; highest vs lowest quintiles) but not ER(-)PR(-) disease. High saturated fat was statistically significantly associated with greater risk of HER2(-) disease. High saturated fat intake particularly increases risk of receptor-positive disease, suggesting saturated fat involvement in the etiology of this BC subtype. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Characterization of a neurokinin B receptor site in rat brain using a highly selective radioligand.

PubMed

Laufer, R; Gilon, C; Chorev, M; Selinger, Z

1986-08-05

We have recently characterized a tachykinin receptor subtype (SP-N) whose preferred ligand is the mammalian neuropeptide, neurokinin B (Laufer, R., Wormser, U., Friedman, Z. Y., Gilon, C., Chorev, M., and Selinger, Z. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7444-7448). To investigate this novel tachykinin receptor, we have now prepared a radiolabeled peptide, N alpha-[( 125I]desamino-3-iodotyrosyl)-[Asp5,6, N-methyl-Phe8]substance P (5-11) heptapeptide (125I-BH-NH-Senktide), which selectively interacts with the SP-N receptor subtype. The binding of 125I-BH-NH-Senktide to rat cerebral cortex membranes was studied under conditions that minimized nonspecific binding. Unlike other tachykinin receptor probes, this radioligand is not degraded during the binding experiment. Binding of 125I-BH-NH-Senktide is reversible, saturable, and of high affinity (KD = 0.9 nM). The radioligand labels a single class of binding site (122 fmol binding sites/mg of protein), as indicated by a linear Scatchard plot and a Hill coefficient close to unity (nH = 1.05). The pharmacological specificity of this binding site corresponds to that of the neuronal SP-N receptor in guinea pig ileum myenteric plexus, which was determined by a functional bioassay. Among various rat brain regions, the highest binding was observed in the cerebral cortex, olfactory bulb, hypothalamus, and hippocampus. These results suggest the existence and specific distribution of a neurokinin B receptor site of the SP-N type in rat brain. 125I-BH-NH-Senktide is the first selective and potent probe for this receptor and is thus an important tool for further studies of its distribution, regulation, and functional role.

Sphingosine-1-Phosphate Receptor Subtype 3: A Novel Therapeutic Target of K-Ras Mutant Driven Non-Small Cell Lung Carcinoma

DTIC Science & Technology

2015-10-01

Breeding of LSL K-RasG12D transgenic mice (Projective: months 5-8; Actual: 100% completion) 2b. Nasal instillation of adenoviral particles carrying...1a. Regulatory review and approval of animal protocol (Projective: Months 1-2; Actual: 100% completion). 1b. Mice acquisition and breeding of...S1P3-/-:LSL-K-RasG12D and S1P3+/+:LSL-K-RasG12D bi- transgenic mice (Projective: Months 3-4; Actual: 100% completion). 1c. Nasal instillation of

Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons

PubMed Central

Lundius, Ebba Gregorsson; Sanchez-Alavez, Manuel; Ghochani, Yasmin; Klaus, Joseph; Tabarean, Iustin V.

2010-01-01

The preoptic area/anterior hypothalamus (PO/AH), a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase (ERK) and was not dependent on synaptic activity. Furthermore, a population of nonGABAergic neurons was depolarized and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca2+ release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked indicating that it represents a postsynaptic effect. Single-cell reverse transcription –PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons while H1 receptors were expressed in nonGABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons which express H1 and H3 receptor subtypes, respectively. PMID:20335473

Histamine influences body temperature by acting at H1 and H3 receptors on distinct populations of preoptic neurons.

PubMed

Lundius, Ebba Gregorsson; Sanchez-Alavez, Manuel; Ghochani, Yasmin; Klaus, Joseph; Tabarean, Iustin V

2010-03-24

The preoptic area/anterior hypothalamus, a region that contains neurons that control thermoregulation, is the main locus at which histamine affects body temperature. Here we report that histamine reduced the spontaneous firing rate of GABAergic preoptic neurons by activating H3 subtype histamine receptors. This effect involved a decrease in the level of phosphorylation of the extracellular signal-regulated kinase and was not dependent on synaptic activity. Furthermore, a population of non-GABAergic neurons was depolarized, and their firing rate was enhanced by histamine acting at H1 subtype receptors. In our experiments, activation of the H1R receptors was linked to the PLC pathway and Ca(2+) release from intracellular stores. This depolarization persisted in TTX or when fast synaptic potentials were blocked, indicating that it represents a postsynaptic effect. Single-cell reverse transcription-PCR analysis revealed expression of H3 receptors in a population of GABAergic neurons, while H1 receptors were expressed in non-GABAergic cells. Histamine applied in the median preoptic nucleus induced a robust, long-lasting hyperthermia effect that was mimicked by either H1 or H3 histamine receptor subtype-specific agonists. Our data indicate that histamine modulates the core body temperature by acting at two distinct populations of preoptic neurons that express H1 and H3 receptor subtypes, respectively.

Development of 2′-substituted (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine analogues as potent N-methyl-d-aspartic acid receptor agonists

PubMed Central

Risgaard, Rune; Nielsen, Simon D.; Hansen, Kasper B.; Jensen, Christina M.; Nielsen, Birgitte; Traynelis, Stephen F.; Clausen, Rasmus P.

2013-01-01

A series of 2′-substituted analogues of the selective NMDA receptor ligand (2S,1′R,2′S)-2-(carboxycyclopropyl)glycine ((S)-CCG-IV) have been designed, synthesized and pharmacologically characterized. The design was based on a docking study hypothesizing that substituents in the 2′-position would protrude into a region where differences among the NMDA receptor GluN2 subunits exist. Various synthetic routes were explored, and two different routes provided a series of alkyl-substituted analogues. Pharmacological characterization revealed that these compounds are NMDA receptor agonists and that potency decreases with increasing size of the alkyl groups. Variations in agonist activity are observed at the different recombinant NMDA receptor subtypes. This study demonstrates that it is possible to introduce substituents in the 2′-position of (S)-CCG-IV while maintaining agonist activity and that variation among NMDA receptor subtypes may be achieved by probing this region of the receptor. PMID:23614571

Lorcaserin and pimavanserin: emerging selectivity of serotonin receptor subtype–targeted drugs

PubMed Central

Meltzer, Herbert Y.; Roth, Bryan L.

2013-01-01

Serotonin (5-hydroxytryptamine, or 5-HT) receptors mediate a plethora of physiological phenomena in the brain and the periphery. Additionally, serotonergic dysfunction has been implicated in nearly every neuropsychiatric disorder. The effects of serotonin are mediated by fourteen GPCRs. Both the therapeutic actions and side effects of commonly prescribed drugs are frequently due to nonspecific actions on various 5-HT receptor subtypes. For more than 20 years, the search for clinically efficacious drugs that selectively target 5-HT receptor subtypes has been only occasionally successful. This review provides an overview of 5-HT receptor pharmacology and discusses two recent 5-HT receptor subtype–selective drugs, lorcaserin and pimavanserin, which target the 5HT2C and 5HT2A receptors and provide new treatments for obesity and Parkinson’s disease psychosis, respectively. PMID:24292660

Purinergic receptor immunoreactivity in the rostral ventromedial medulla.

PubMed

Close, L N; Cetas, J S; Heinricher, M M; Selden, N R

2009-01-23

The rostral ventromedial medulla (RVM) has long been recognized to play a pivotal role in nociceptive modulation. Pro-nociception within the RVM is associated with a distinct functional class of neurons, ON-cells that begin to discharge immediately before nocifensive reflexes. Anti-nociceptive function within the RVM, including the analgesic response to opiates, is associated with another distinct class, OFF-cells, which pause immediately prior to nocifensive reflexes. A third class of RVM neurons, NEUTRAL-cells, does not alter firing in association with nocifensive reflexes. ON-, OFF- and NEUTRAL-cells show differential responsiveness to various behaviorally relevant neuromodulators, including purinergic ligands. Iontophoresis of semi-selective P2X ligands, which are associated with nociceptive transmission in the spinal cord and dorsal root ganglia, preferentially activate ON-cells. By contrast, P2Y ligands activate OFF-cells and P1 ligands suppress the firing of NEUTRAL cells. The current study investigates the distribution of P2X, P2Y and P1 receptor immunoreactivity in RVM neurons of Sprague-Dawley rats. Co-localization with tryptophan hydroxylase (TPH), a well-established marker for serotonergic neurons was also studied. Immunoreactivity for the four purinergic receptor subtypes examined was abundant in all anatomical subdivisions of the RVM. By contrast, TPH-immunoreactivity was restricted to a relatively small subset of RVM neurons concentrated in the nucleus raphe magnus and pallidus, as expected. There was a significant degree of co-localization of each purinergic receptor subtype with TPH-immunoreactivity. This co-localization was most pronounced for P2Y1 receptor immunoreactivity, although this was the least abundant among the different purinergic receptor subtypes examined. Immunoreactivity for multiple purinergic receptor subtypes was often co-localized in single neurons. These results confirm the physiological finding that purinergic receptors are widely expressed in the RVM. Purinergic neurotransmission in this region may play an important role in nociception and/or nociceptive modulation, as at other levels of the neuraxis.

Oxidized Low-Density Lipoprotein Suppresses Expression of Prostaglandin E Receptor Subtype EP3 in Human THP-1 Macrophages

PubMed Central

Sui, Xuxia; Liu, Yanmin; Li, Qi; Liu, Gefei; Song, Xuhong; Su, Zhongjing; Chang, Xiaolan; Zhou, Yingbi; Liang, Bin; Huang, Dongyang

2014-01-01

EP3, one of four prostaglandin E2 (PGE2) receptors, is significantly lower in atherosclerotic plaques than in normal arteries and is localized predominantly in macrophages of the plaque shoulder region. However, mechanisms behind this EP3 expression pattern are still unknown. We investigated the underlying mechanism of EP3 expression in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophages with oxidized low-density lipoprotein (oxLDL) treatment. We found that oxLDL decreased EP3 expression, in a dose-dependent manner, at both the mRNA and protein levels. Moreover, oxLDL inhibited nuclear factor-κB (NF-κB)-dependent transcription of the EP3 gene by the activation of peroxisome proliferator-activated receptor-γ (PPAR-γ). Finally, chromatin immunoprecipitation revealed decreased binding of NF-κB to the EP3 promoter with oxLDL and PPAR-γ agonist treatment. Our results show that oxLDL suppresses EP3 expression by activation of PPAR-γ and subsequent inhibition of NF-κB in macrophages. These results suggest that down-regulation of EP3 expression by oxLDL is associated with impairment of EP3-mediated anti-inflammatory effects, and that EP3 receptor activity may exert a beneficial effect on atherosclerosis. PMID:25333975

Molecular cloning and characterization of chicken prostaglandin E receptor subtypes 2 and 4 (EP2 and EP4).

PubMed

Kwok, Amy Ho Yan; Wang, Yajun; Wang, Crystal Ying; Leung, Frederick C

2008-06-01

Prostaglandin E(2) (PGE(2)) is an important chemical mediator responsible for regulation of many vital physiological processes. Four receptor subtypes have been identified to mediate its biological actions. Among these subtypes, prostaglandin E receptor subtypes 2 and 4 (EP(2) and EP(4)), both coupled to cAMP-protein kinase A (cAMP-PKA) signaling pathway, are proposed to play crucial roles under both physiological and pathological conditions. Though both receptors were extensively studied in mammals, little is known about their functionality and expression in non-mammalian species including chicken. In present study, the full-length cDNAs for chicken EP(2) and EP(4) receptors were first cloned from adult chicken ovary and testis, respectively. Chicken EP(2) is 356 amino acids in length and shows high amino acid identity to that of human (61%), mouse (63%), and rat (61%). On the other hand, the full-length cDNA of EP(4) gene encodes a precursor of 475 amino acids with a high degree of amino acid identity to that of mammals, including human (87%), mouse (86%), rat (84%), dog (85%), and cattle (83%), and a comparatively lower sequence identity to zebrafish (52%). RT-PCR assays revealed that EP(2) mRNA was expressed in all tissues examined including the oviduct, while EP(4) expression was detected only in a few tissues. Using the pGL3-CRE-luciferase reporter system, we also demonstrated that PGE(2) could induce luciferase activity in DF-1 cells expressing EP(2) and EP(4) in dose-dependent manners (EC(50): <1 nM), confirming that both receptors could be activated by PGE(2) and functionally coupled to the cAMP-PKA signaling pathway. Together, our study establishes a molecular basis to understand the physiological roles of PGE(2) in target tissues of chicken.

G Protein-Coupled Kinin Receptors and Immunity Against Pathogens.

PubMed

Scharfstein, Julio; Ramos, Pablo I P; Barral-Netto, Manoel

2017-01-01

For decades, immunologists have considered the complement system as a paradigm of a proteolytic cascade that, acting cooperatively with the immune system, enhances host defense against infectious organisms. In recent years, advances made in thrombosis research disclosed a functional link between activated neutrophils, monocytes, and platelet-driven thrombogenesis. Forging a physical barrier, the fibrin scaffolds generated by synergism between the extrinsic and intrinsic (contact) pathways of coagulation entrap microbes within microvessels, limiting the systemic spread of infection while enhancing the clearance of pathogens by activated leukocytes. Insight from mice models of thrombosis linked fibrin formation via the intrinsic pathway to the autoactivation of factor XII (FXII) by negatively charged "contact" substances, such as platelet-derived polyphosphates and DNA from neutrophil extracellular traps. Following cleavage by FXIIa, activated plasma kallikrein (PK) initiates inflammation by liberating the nonapeptide bradykinin (BK) from an internal domain of high molecular weight kininogen (HK). Acting as a paracrine mediator, BK induces vasodilation and increases microvascular permeability via activation of endothelial B2R, a constitutively expressed subtype of kinin receptor. During infection, neutrophil-driven extravasation of plasma fuels inflammation via extravascular activation of the kallikrein-kinin system (KKS). Whether liberated by plasma-borne PK, tissue kallikrein, and/or microbial-derived proteases, the short-lived kinins activate immature dendritic cells via B2R, thus linking the infection-associated innate immunity/inflammation to the adaptive arm of immunity. As inflammation persists, a GPI-linked carboxypeptidase M removes the C-terminal arginine from the primary kinin, converting the B2R agonist into a high-affinity ligand for B1R, a GPCR subtype that is transcriptionally upregulated in injured/inflamed tissues. As reviewed here, lessons taken from studies of kinin receptor function in experimental infections have shed light on the complex proteolytic circuits that, acting at the endothelial interface, reciprocally couple immunity to the proinflammatory KKS. © 2017 Elsevier Inc. All rights reserved.

The metabotropic P2Y4 receptor participates in the commitment to differentiation and cell death of human neuroblastoma SH-SY5Y cells.

PubMed

Cavaliere, Fabio; Nestola, Valeria; Amadio, Susanna; D'Ambrosi, Nadia; Angelini, Daniela F; Sancesario, Giuseppe; Bernardi, Giorgio; Volonté, Cinzia

2005-02-01

Extracellular nucleotides exert a variety of biological actions through different subtypes of P2 receptors. Here we characterized in the human neuroblastoma SH-SY5Y cells the simultaneous presence of various P2 receptors, belonging to the P2X ionotropic and P2Y metabotropic families. Western blot analysis detected the P2X1,2,4,5,6,7 and P2Y1,2,4,6, but not the P2X3 and P2Y12 receptors. We then investigated which biological effects were mediated by the P2Y4 subtype and its physiological pyrimidine agonist UTP. We found that neuronal differentiation of the SH-SY5Y cells with dibutiryl-cAMP increased the expression of the P2Y4 protein and that UTP itself was able to positively interfere with neuritogenesis. Moreover, transient transfection and activation of P2Y4 also facilitated neuritogenesis in SH-SY5Y cells, as detected by morphological phase contrast analysis and confocal examination of neurofilament proteins NFL. This was concurrent with increased transcription of immediate-early genes linked to differentiation such as cdk-5 and NeuroD6, and activity of AP-1 transcription family members such as c-fos, fos-B, and jun-D. Nevertheless, a prolonged activation of the P2Y4 receptor by UTP also induced cell death, both in naive, differentiated, and P2Y4-transfected SH-SY5Y cells, as measured by direct count of intact nuclei and cytofluorimetric analysis of damaged DNA. Taken together, our data indicate that the high expression and activation of the P2Y4 receptor participates in the neuronal differentiation and commitment to death of SH-SY5Y cells.

Heteromultimerization modulates P2X receptor functions through participating extracellular and C-terminal subdomains.

PubMed

Koshimizu, Taka-aki; Ueno, Susumu; Tanoue, Akito; Yanagihara, Nobuyuki; Stojilkovic, Stanko S; Tsujimoto, Gozoh

2002-12-06

P2X purinergic receptors (P2XRs) differ among themselves with respect to their ligand preferences and channel kinetics during activation, desensitization, and recovery. However, the contributions of distinct receptor subdomains to the subtype-specific behavior have been incompletely characterized. Here we show that homomeric receptors having the extracellular domain of the P2X(3) subunit in the P2X(2a)-based backbone (P2X(2a)/X(3)ex) mimicked two intrinsic functions of P2X(3)R, sensitivity to alphabeta-methylene ATP and ecto-ATPase-dependent recovery from endogenous desensitization; these two functions were localized to the N- and C-terminal halves of the P2X(3) extracellular loop, respectively. The chimeric P2X(2a)R/X(3)ex receptors also desensitized with accelerated rates compared with native P2X(2a)R, and the introduction of P2X(2) C-terminal splicing into the chimeric subunit (P2X(2b)/X(3)ex) further increased the rate of desensitization. Physical and functional heteromerization of native P2X(2a) and P2X(2b) subunits was also demonstrated. In heteromeric receptors, the ectodomain of P2X(3) was a structural determinant for ligand selectivity and recovery from desensitization, and the C terminus of P2X(2) was an important factor for the desensitization rate. Furthermore, [gamma-(32)P]8-azido ATP, a photoreactive agonist, was effectively cross-linked to P2X(3) subunit in homomeric receptors but not in heteromeric P2X(2) + P2X(3)Rs. These results indicate that heteromeric receptors formed by distinct P2XR subunits develop new functions resulting from integrative effects of the participating extracellular and C-terminal subdomains.

4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes.

PubMed

Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte; Bräuner-Osborne, Hans

2005-01-01

4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies, and the series of 4-alkyl-HIBO analogues have been extended in this paper in the search for versatile agents. Pharmacological characterization of five new analogues, branched and unbranched 4-alkyl-HIBO analogues, have been carried out. The present compounds are all weak antagonists at Group I mGluRs (mGluR1 and 5) presenting only small differences in potencies (Ki values ranging from 89 to 670 microM). Affinities were studied at native and cloned iGluRs, and the compounds described show preference for the AMPA receptor subtypes GluR1 and 2 over GluR3 and 4. However, compared to previous 4-alkyl-HIBO analogues, these compounds show a remarkably high affinity for the Kain preferring subtype GluR5. The observed GluR5 affinities were either similar or higher compared to their GluR1 and 2 affinity. Isopropyl-HIBO showed the highest affinity for GluR5 (Ki=0.16 microM), and represents a unique compound with high affinity towards the three subtypes GluR1, 2 and 5. In general, these compounds represent new selectivity profiles compared to previously reported Glu receptor analogues.

Antagonism at the NR2B subunit of NMDA receptors induces increased connectivity of the prefrontal and subcortical regions regulating reward behavior.

PubMed

Gass, Natalia; Becker, Robert; Sack, Markus; Schwarz, Adam J; Reinwald, Jonathan; Cosa-Linan, Alejandro; Zheng, Lei; von Hohenberg, Christian Clemm; Inta, Dragos; Meyer-Lindenberg, Andreas; Weber-Fahr, Wolfgang; Gass, Peter; Sartorius, Alexander

2018-04-01

Evidence indicates that ketamine's rapid antidepressant efficacy likely results from its antagonism of NR2B-subunit-containing NMDA receptors (NMDAR). Since ketamine equally blocks NR2A- and NR2B-containing NMDAR, and has affinity to other receptors, NR2B-selective drugs might have improved therapeutic efficiency and side effect profile. We aimed to compare the effects of (S)-ketamine and two different types of NR2B-selective antagonists on functional brain networks in rats, in order to find common circuits, where their effects intersect, and that might explain their antidepressant action. The experimental design comprised four parallel groups of rats (N = 37), each receiving (S)-Ketamine, CP-101,606, Ro 25-6981 or saline. After compound injection, we acquired resting-state functional magnetic resonance imaging time series. We used graph theoretical approach to calculate brain network properties. Ketamine and CP-101,606 diminished the global clustering coefficient and small-worldness index. At the nodal level, all compounds induced increased connectivity of the regions mediating reward and cognitive aspects of emotional processing, such as ventromedial prefrontal cortex, septal nuclei, and nucleus accumbens. The dorsal hippocampus and regions involved in sensory processing and aversion, such as superior and inferior colliculi, exhibited an opposite effect. The effects common to ketamine and NR2B-selective compounds were localized to the same brain regions as those reported in depression, but in the opposite direction. The upregulation of the reward circuitry might partially underlie the antidepressant and anti-anhedonic effects of the antagonists and could potentially serve as a translational imaging phenotype for testing putative antidepressants, especially those targeting the NR2B receptor subtype.

Molecular subtype and tumor characteristics of breast cancer metastases as assessed by gene expression significantly influence patient post-relapse survival

PubMed Central

Tobin, N. P.; Harrell, J. C.; Lövrot, J.; Egyhazi Brage, S.; Frostvik Stolt, M.; Carlsson, L.; Einbeigi, Z.; Linderholm, B.; Loman, N.; Malmberg, M.; Walz, T.; Fernö, M.; Perou, C. M.; Bergh, J.; Hatschek, T.; Lindström, L. S.; Hatschek, Thomas; Fernö, Mårten; Lindström, Linda Sofie; Hedenfalk, Ingrid; Brandberg, Yvonne; Carstensen, John; Egyhazy, Suzanne; Stolt, Marianne Frostvik; Skoog, Lambert; Hellström, Mats; Maliniemi, Maarit; Svensson, Helene; Åström, Gunnar; Bergh, Jonas; Bjöhle, Judith; Lidbrink, Elisabet; Rotstein, Sam; Wallberg, Birgitta; Einbeigi, Zakaria; Carlsson, Per; Linderholm, Barbro; Walz, Thomas; Loman, Niklas; Malmström, Per; Söderberg, Martin; Malmberg, Martin; Carlsson, Lena; Umeå; Lindh, Birgitta; Sundqvist, Marie; Malmberg, Lena

2015-01-01

Background We and others have recently shown that tumor characteristics are altered throughout tumor progression. These findings emphasize the need for re-examination of tumor characteristics at relapse and have led to recommendations from ESMO and the Swedish Breast Cancer group. Here, we aim to determine whether tumor characteristics and molecular subtypes in breast cancer metastases confer clinically relevant prognostic information for patients. Patients and methods The translational aspect of the Swedish multicenter randomized trial called TEX included 111 patients with at least one biopsy from a morphologically confirmed locoregional or distant breast cancer metastasis diagnosed from December 2002 until June 2007. All patients had detailed clinical information, complete follow-up, and metastasis gene expression information (Affymetrix array GPL10379). We assessed the previously published gene expression modules describing biological processes [proliferation, apoptosis, human epidermal receptor 2 (HER2) and estrogen (ER) signaling, tumor invasion, immune response, and angiogenesis] and pathways (Ras, MAPK, PTEN, AKT-MTOR, PI3KCA, IGF1, Src, Myc, E2F3, and β-catenin) and the intrinsic subtypes (PAM50). Furthermore, by contrasting genes expressed in the metastases in relation to survival, we derived a poor metastasis survival signature. Results A significant reduction in post-relapse breast cancer-specific survival was associated with low-ER receptor signaling and apoptosis gene module scores, and high AKT-MTOR, Ras, and β-catenin module scores. Similarly, intrinsic subtyping of the metastases provided statistically significant post-relapse survival information with the worst survival outcome in the basal-like [hazard ratio (HR) 3.7; 95% confidence interval (CI) 1.3–10.9] and HER2-enriched (HR 4.4; 95% CI 1.5–12.8) subtypes compared with the luminal A subtype. Overall, 25% of the metastases were basal-like, 32% HER2-enriched, 10% luminal A, 28% luminal B, and 5% normal-like. Conclusions We show that tumor characteristics and molecular subtypes of breast cancer metastases significantly influence post-relapse patient survival, emphasizing that molecular investigations at relapse provide prognostic and clinically relevant information. ClinicalTrials.gov This is the translational part of the Swedish multicenter and randomized trial TEX, clinicaltrials.gov identifier nct01433614 (http://www.clinicaltrials.gov/ct2/show/nct01433614). PMID:25361981

GABAB receptor attenuation of GABAA currents in neurons of the mammalian central nervous system.

PubMed

Shen, Wen; Nan, Changlong; Nelson, Peter T; Ripps, Harris; Slaughter, Malcolm M

2017-03-01

Ionotropic receptors are tightly regulated by second messenger systems and are often present along with their metabotropic counterparts on a neuron's plasma membrane. This leads to the hypothesis that the two receptor subtypes can interact, and indeed this has been observed in excitatory glutamate and inhibitory GABA receptors. In both systems the metabotropic pathway augments the ionotropic receptor response. However, we have found that the metabotropic GABA B receptor can suppress the ionotropic GABA A receptor current, in both the in vitro mouse retina and in human amygdala membrane fractions. Expression of amygdala membrane microdomains in Xenopus oocytes by microtransplantation produced functional ionotropic and metabotropic GABA receptors. Most GABA A receptors had properties of α -subunit containing receptors, with ~5% having ρ -subunit properties. Only GABA A receptors with α -subunit-like properties were regulated by GABA B receptors. In mouse retinal ganglion cells, where only α -subunit-containing GABA A receptors are expressed, GABA B receptors suppressed GABA A receptor currents. This suppression was blocked by GABA B receptor antagonists, G-protein inhibitors, and GABA B receptor antibodies. Based on the kinetic differences between metabotropic and ionotropic receptors, their interaction would suppress repeated, rapid GABAergic inhibition. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Two unusual hepatitis C virus subtypes, 2j and 2q, in Spain: Identification by nested-PCR and sequencing of a NS5B region.

PubMed

Margall, N; March, F; Español, M; Torras, X; Gallego, A; Coll, P

2015-10-01

Many studies have reported the use of the NS5B gene to subtype hepatitis C virus (HCV). Other HCV genes, such as HCV-5' UTR, Core (C) and E1, have also been used. In some studies, NS5B have been used together with 5'-UTR or C genes to improve genotyping results obtained using commercial procedures. Only two studies in Spain have compared molecular techniques versus commercial procedures regarding the efficacy of HCV subtyping. The aim of this study was to determine whether nested PCR and sequencing of a NS5B region was more reliable than commercial procedures to subtype HCV. We analyzed the results of HCV genotyping in [726] serum specimens collected from 2001 to 2013. From 2001 to 2011, we used PCR and INNO-LiPA hybridization or its new version Versant HCV Genotype 2.0 assay (471 samples). From 2012 to 2013, we used nested PCR and sequencing of a NS5B region (255 cases). This method used two pairs of primers to amplify the RNA of the sample converted to DNA by retrotranscription. The amplification product of 270 base pairs was further sequenced. To identify the subtype, the sequences obtained were compared to those in the international database: http://hcv.lanl.gov./content/sequence/, HCV/ToolsOutline.html and Geno2pheno[hcv] http://hcv.bioinf.mpi-inf.mpg.de/index.php. Nested PCR of a NS5B region and sequencing identified all but one subtype (0.4%, 1/255), differentiated all 1a subtypes from 1b subtypes, and characterized all HCV 2-4 subtypes. This approach also distinguished two subtypes, 2j and 2q, that had rarely been detected previously in Spain. However, commercial procedures failed to subtype 12.7% (60/471) of samples and to genotype 0.6% of specimens (3/471). Nested PCR and sequencing of a NS5B region improved the subtyping of HCV in comparison with classical procedures and identified two rare subtypes in Spain: 2j and 2q. However, full length genome sequencing is recommended to confirm HCV 2j and 2q subtypes. Copyright © 2015. Published by Elsevier B.V.

Opposing functions of spinal M2, M3, and M4 receptor subtypes in regulation of GABAergic inputs to dorsal horn neurons revealed by muscarinic receptor knockout mice.

PubMed

Zhang, Hong-Mei; Chen, Shao-Rui; Matsui, Minoru; Gautam, Dinesh; Wess, Jürgen; Pan, Hui-Lin

2006-03-01

Spinal muscarinic acetylcholine receptors (mAChRs) play an important role in the regulation of nociception. To determine the role of individual mAChR subtypes in control of synaptic GABA release, spontaneous inhibitory postsynaptic currents (sIPSCs) and miniature IPSCs (mIPSCs) were recorded in lamina II neurons using whole-cell recordings in spinal cord slices of wild-type and mAChR subtype knockout (KO) mice. The mAChR agonist oxotremorine-M (3-10 microM) dose-dependently decreased the frequency of GABAergic sIPSCs and mIPSCs in wild-type mice. However, in the presence of the M2 and M4 subtype-preferring antagonist himbacine, oxotremorine-M caused a large increase in the sIPSC frequency. In M3 KO and M1/M3 double-KO mice, oxotremorine-M produced a consistent decrease in the frequency of sIPSCs, and this effect was abolished by himbacine. We were surprised to find that in M2/M4 double-KO mice, oxotremorine-M consistently increased the frequency of sIPSCs and mIPSCs in all neurons tested, and this effect was completely abolished by 4-diphenylacetoxy-N-methylpiperidine methiodide, an M3 subtype-preferring antagonist. In M2 or M4 single-KO mice, oxotremorine-M produced a variable effect on sIPSCs; it increased the frequency of sIPSCs in some cells but decreased the sIPSC frequency in other neurons. Taken together, these data strongly suggest that activation of the M3 subtype increases synaptic GABA release in the spinal dorsal horn of mice. In contrast, stimulation of presynaptic M2 and M4 subtypes predominantly attenuates GABAergic inputs to dorsal horn neurons in mice, an action that is opposite to the role of M2 and M4 subtypes in the spinal cord of rats.

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

PubMed

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

1999-10-22

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

Benzodiazepine-induced anxiolysis and reduction of conditioned fear are mediated by distinct GABAA receptor subtypes in mice

PubMed Central

Smith, Kiersten S.; Engin, Elif; Meloni, Edward G.; Rudolph, Uwe

2012-01-01

GABAA receptor modulating drugs such as benzodiazepines (BZs) have been used to treat anxiety disorders for over five decades. In order to determine whether the same or different GABAA receptor subtypes are necessary for the anxiolytic-like action of BZs in unconditioned anxiety and conditioned fear models, we investigated the role of different GABAA receptor subtypes by challenging wild type, α1(H101R), α2(H101R) and α3(H126R) mice bred on the C57BL/6J background with diazepam or chlordiazepoxide in the elevated plus maze and the fear-potentiated startle paradigms. Both drugs significantly increased open arm exploration in the elevated plus maze in wild type, α1(H101R) and α3(H126R), but this effect was abolished in α2(H101R) mice; these were expected results based on previous published results. In contrast, while administration of diazepam and chlordiazepoxide significantly attenuated fear-potentiated startle (FPS) in wild type mice and α3(H126R) mice, the fear-reducing effects of these drugs were absent in both α1(H101R) and α2(H101R) point mutants, indicating that both α1- and α2-containing GABAA receptors are necessary for BZs to exert their effects on conditioned fear responses.. Our findings illustrate both an overlap and a divergence between the GABAA receptor subtype requirements for the impact of BZs, specifically that both α1- and α2-containing GABAA receptors are necessary for BZs to reduce conditioned fear whereas only α2-containing GABAA receptors are needed for BZ-induced anxiolysis in unconditioned tests of anxiety. This raises the possibility that GABAergic pharmacological interventions for specific anxiety disorders can be differentially tailored. PMID:22465203

Functional expression of ionotropic purinergic receptors on mouse taste bud cells.

PubMed

Hayato, Ryotaro; Ohtubo, Yoshitaka; Yoshii, Kiyonori

2007-10-15

Neurotransmitter receptors on taste bud cells (TBCs) and taste nerve fibres are likely to contribute to taste transduction by mediating the interaction among TBCs and that between TBCs and taste nerve fibres. We investigated the functional expression of P2 receptor subtypes on TBCs of mouse fungiform papillae. Electrophysiological studies showed that 100 microm ATP applied to their basolateral membranes either depolarized or hyperpolarized a few cells per taste bud. Ca(2+) imaging showed that similarly applied 1 mum ATP, 30 microm BzATP (a P2X(7) agonist), or 1 microm 2MeSATP (a P2Y(1) and P2Y(11) agonist) increased intracellular Ca(2+) concentration, but 100 microm UTP (a P2Y(2) and P2Y(4) agonist) and alpha,beta-meATP (a P2X agonist except for P2X(2), P2X(4) and P2X(7)) did not. RT-PCR suggested the expression of P2X(2), P2X(4), P2X(7), P2Y(1), P2Y(13) and P2Y(14) among the seven P2X subtypes and seven P2Y subtypes examined. Immunohistostaining confirmed the expression of P2X(2). The exposure of the basolateral membranes to 3 mm ATP for 30 min caused the uptake of Lucifer Yellow CH in a few TBCs per taste bud. This was antagonized by 100 microm PPADS (a non-selective P2 blocker) and 1 microm KN-62 (a P2X(7) blocker). These results showed for the first time the functional expression of P2X(2) and P2X(7) on TBCs. The roles of P2 receptor subtypes in the taste transduction, and the renewal of TBCs, are discussed.

Interactions Between Vitamin D and Breast Cancer

DTIC Science & Technology

2009-09-01

ERBB2 GRB7 B EGFR MKI67 AURKA BIRC5 CCND1 CCNA1 TP53 MMP11 CTSL2 BAX BCL2 VDR C CYP24A1 CYP27B1 HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF D...BIRC5 CCND1 CCNA1 TP53 MMP11 CTSL2 BAX BCL2 VDR G CYP24A1 CYP27B1 HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF H CDKN1A IGFBP3 SPP1 AR PTHLH...NAD) C08 Hs.199248 NM_000958 PTGER4 Prostaglandin E receptor 4 (subtype EP4) C09 Hs.497822 NM_007207 DUSP10 Dual specificity phosphatase 10 C10

Intrinsic Subtype and Therapeutic Response Among HER2-Positive Breast Tumors from the NCCTG (Alliance) N9831 Trial

PubMed Central

Perez, Edith A.; Ballman, Karla V.; Mashadi-Hossein, Afshin; Tenner, Kathleen S.; Kachergus, Jennifer M.; Norton, Nadine; Necela, Brian M.; Carr, Jennifer M.; Ferree, Sean; Perou, Charles M.; Baehner, Frederick; Cheang, Maggie Chon U.

2017-01-01

Background: Genomic data from human epidermal growth factor receptor 2–positive (HER2+) tumors were analyzed to assess the association between intrinsic subtype and clinical outcome in a large, well-annotated patient cohort. Methods: Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna algorithm on the NanoString platform to define intrinsic subtype, risk of recurrence scores, and risk categories for 1392 HER2+ tumors. Subtypes were evaluated for recurrence-free survival (RFS) using Kaplan-Meier and Cox model analysis following adjuvant chemotherapy (n = 484) or chemotherapy plus trastuzumab (n = 908). All statistical tests were two-sided. Results: Patients with HER2+ tumors from N9831 were primarily scored as HER2-enriched (72.1%). These individuals received statistically significant benefit from trastuzumab (hazard ratio [HR] = 0.68, 95% confidence interval [CI] = 0.52 to 0.89, P = .005), as did the patients (291 of 1392) with luminal-type tumors (HR = 0.52, 95% CI = 0.32 to 0.85, P = .01). Patients with basal-like tumors (97 of 1392) did not have statistically significantly better RFS when treated with trastuzumab and chemotherapy compared with chemotherapy alone (HR = 1.06, 95% CI = 0.53 to 2.13, P = .87). Conclusions: The majority of clinically defined HER2-positive tumors were classified as HER2-enriched or luminal using the Prosigna algorithm. Intrinsic subtype alone cannot replace conventional histopathological evaluation of HER2 status because many tumors that are classified as luminal A or luminal B will benefit from adjuvant trastuzumab if that subtype is accompanied by HER2 overexpression. However, among tumors that overexpress HER2, we speculate that assessment of intrinsic subtype may influence treatment, particularly with respect to evaluating alternative therapeutic approaches for that subset of HER2-positive tumors of the basal-like subtype. PMID:27794124

Intrinsic Subtype and Therapeutic Response Among HER2-Positive Breaty st Tumors from the NCCTG (Alliance) N9831 Trial.

PubMed

Perez, Edith A; Ballman, Karla V; Mashadi-Hossein, Afshin; Tenner, Kathleen S; Kachergus, Jennifer M; Norton, Nadine; Necela, Brian M; Carr, Jennifer M; Ferree, Sean; Perou, Charles M; Baehner, Frederick; Cheang, Maggie Chon U; Thompson, E Aubrey

2017-02-01

Genomic data from human epidermal growth factor receptor 2-positive (HER2+) tumors were analyzed to assess the association between intrinsic subtype and clinical outcome in a large, well-annotated patient cohort. Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna algorithm on the NanoString platform to define intrinsic subtype, risk of recurrence scores, and risk categories for 1392 HER2+ tumors. Subtypes were evaluated for recurrence-free survival (RFS) using Kaplan-Meier and Cox model analysis following adjuvant chemotherapy (n = 484) or chemotherapy plus trastuzumab (n = 908). All statistical tests were two-sided. Patients with HER2+ tumors from N9831 were primarily scored as HER2-enriched (72.1%). These individuals received statistically significant benefit from trastuzumab (hazard ratio [HR] = 0.68, 95% confidence interval [CI] = 0.52 to 0.89, P = .005), as did the patients (291 of 1392) with luminal-type tumors (HR = 0.52, 95% CI = 0.32 to 0.85, P = .01). Patients with basal-like tumors (97 of 1392) did not have statistically significantly better RFS when treated with trastuzumab and chemotherapy compared with chemotherapy alone (HR = 1.06, 95% CI = 0.53 to 2.13, P = .87). The majority of clinically defined HER2-positive tumors were classified as HER2-enriched or luminal using the Prosigna algorithm. Intrinsic subtype alone cannot replace conventional histopathological evaluation of HER2 status because many tumors that are classified as luminal A or luminal B will benefit from adjuvant trastuzumab if that subtype is accompanied by HER2 overexpression. However, among tumors that overexpress HER2, we speculate that assessment of intrinsic subtype may influence treatment, particularly with respect to evaluating alternative therapeutic approaches for that subset of HER2-positive tumors of the basal-like subtype. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar.

PubMed

Motomura, K; Kusagawa, S; Kato, K; Nohtomi, K; Lwin, H H; Tun, K M; Thwe, M; Oo, K Y; Lwin, S; Kyaw, O; Zaw, M; Nagai, Y; Takebe, Y

2000-11-20

We have previously shown that HIV-1 env subtypes B' (a Thai-B cluster within subtype B) and E (CRF01_AE) are distributed in Yangon, the capital city of Myanmar. However, HIV strains from the rest of country have not yet been genetically characterized. In the present study, we determined env (C2/V3) and gag (p17) subtypes of 25 specimens from central Myanmar (Mandalay). Phylogenetic analyses identified 5 subtype C (20%), in addition to 10 CRF01_AE (40%) and 4 subtype B' (16%). Interestingly, the remaining six specimens (24%) showed discordance between gag and env subtypes; three gag subtype B'/env subtype C, one gag subtype B'/env subtype E, one gag subtype C/env subtype B', and one gag subtype C/env subtype E. These discordant specimens were found frequently among injecting drug users (4 of 12, 33%) and female commercial sex workers (2 of 8, 25%) engaging in high-risk behaviors. The recombinant nature of these HIV-1 strains was verified in three specimens, indicating the presence of new forms of HIV-1 intersubtype C/B' and C/B'/E recombinants with different recombination breakpoints. The data suggest that multiple subtypes of B', C, and CRF01_AE are cocirculating in central Myanmar, leading to the evolution of new forms of intersubtype recombinants among the risk populations exhibiting one of the highest HIV infection rates in the region.

Selective regulation of nuclear orphan receptors 4A by adenosine receptor subtypes in human mast cells

PubMed Central

Zhang, Li; Paine, Catherine

2010-01-01

Nuclear orphan receptors 4A (NR4A) are early responsive genes that belong to the superfamily of hormone receptors and comprise NR4A1, NR4A2 and NR4A3. They have been associated to transcriptional activation of multiple genes involved in inflammation, apoptosis and cell cycle control. Here, we establish a link between NR4As and adenosine, a paradoxical inflammatory molecule that can contribute to persistence of inflammation or mediate inflammatory shutdown. Transcriptomics screening of the human mast cell-line HMC-1 revealed a sharp induction of transcriptionally active NR4A2 and NR4A3 by the adenosine analogue NECA. The concomitant treatment of NECA and the adenosine receptor A2A (A2AAR) selective antagonist SCH-58261 exaggerated this effect, suggesting that upregulation of these factors in mast cells is mediated by other AR subtypes (A2B and A3) and that A2AAR activation counteracts NR4A2 and NR4A3 induction. In agreement with this, A2AAR-silencing amplified NR4A induction by NECA. Interestingly, a similar A2AAR modulatory effect was observed on ERK1/2 phosphorylation because A2AAR blockage exacerbated NECA-mediated phosphorylation of ERK1/2. In addition, PKC or MEK1/2 inhibition prevented ERK1/2 phosphorylation and antagonized AR-mediated induction of NR4A2 and NR4A3, suggesting the involvement of these kinases in AR to NR4A signaling. Finally, we observed that selective A2AAR activation with CGS-21680 blocked PMA-induced ERK1/2 phosphorylation and modulated the overexpression of functional nuclear orphan receptors 4A. Taken together, these results establish a novel PKC/ERK/nuclear orphan receptors 4A axis for adenosinergic signaling in mast cells, which can be modulated by A2AAR activation, not only in the context of adenosine but of other mast cell activating stimuli as well. PMID:21234122

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild-Type Mice Treated with a Novel Avpr1b Receptor Antagonist

PubMed Central

Roper, J A; Craighead, M; O’Carroll, A-M; Lolait, S J

2010-01-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. PMID:20846299

Attenuated stress response to acute restraint and forced swimming stress in arginine vasopressin 1b receptor subtype (Avpr1b) receptor knockout mice and wild-type mice treated with a novel Avpr1b receptor antagonist.

PubMed

Roper, J A; Craighead, M; O'Carroll, A-M; Lolait, S J

2010-11-01

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin-releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic-pituitary-adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild-type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress-induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild-type mice that had been pre-treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint-stressed knockout male mice, and in Avpr1b-antagonist-treated male wild-type mice. By contrast, plasma CORT levels were not reduced in acutely restraint-stressed female knockout animals, or in female wild-type animals pre-treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic-pituitary-adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors. © 2010 The Authors. Journal of Neuroendocrinology © 2010 Blackwell Publishing Ltd.

Differential expression of appetite-regulating genes in avian models of anorexia and obesity.

PubMed

Yi, J; Yuan, J; Gilbert, E R; Siegel, P B; Cline, M A

2017-08-01

Chickens from lines that have been selected for low (LWS) or high (HWS) juvenile body weight for more than 57 generations provide a unique model by which to research appetite regulation. The LWS display different severities of anorexia, whereas all HWS become obese. In the present study, we measured mRNA abundance of various factors in appetite-associated nuclei in the hypothalamus. The lateral hypothalamus (LHA), paraventricular nucleus (PVN), ventromedial hypothalamus (VMH), dorsomedial nucleus (DMN) and arcuate nucleus (ARC) were collected from 5 day-old chicks that were fasted for 180 minutes or provided with continuous access to food. Fasting increased neuropeptide Y receptor subtype 1 (NPYR1) mRNA in the LHA and c-Fos in the VMH, at the same time as decreasing c-Fos in the LHA, neuropeptide Y receptor subtype 5 and ghrelin in the PVN, and neuropeptide Y receptor subtype 2 in the ARC. Fasting increased melanocortin receptor subtype 3 (MC3R) expression in the DMN and NPY in the ARC of LWS but not HWS chicks. Expression of NPY was greater in LWS than HWS in the DMN. neuropeptide Y receptor subtype 5 mRNA was greater in LWS than HWS in the LHA, PVN and ARC. Expression of orexin was greater in LWS than HWS in the LHA. There was greater expression of NPYR1, melanocortin receptor subtype 4 and cocaine- and amphetamine-regulated transcript in HWS than LWS and mesotocin in LWS than HWS in the PVN. In the ARC, agouti-related peptide and MC3R were greater in LWS than HWS and, in the VMH, orexin receptor 2 and leptin receptor were greater in LWS than HWS. Greater mesotocin in the PVN, orexin in the LHA and ORXR2 in the VMH of LWS may contribute to their increased sympathetic tone and anorexic phenotype. The results of the present study also suggest that an increased hypothalamic anorexigenic tone in the LWS over-rides orexigenic factors such as NPY and AgRP that were more highly expressed in LWS than HWS in several nuclei. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Nonconventional three-finger toxin BMLCL from krait Bungarus multicinctus venom with high affinity interacts with nicotinic acetylcholine receptors.

PubMed

Utkin, Yu N; Kasheverov, I E; Kudryavtsev, D S; Andreeva, T V; Starkov, V G; Ziganshin, R H; Kuznetsov, D V; Anh, Hoang Ngoc; Thao, Nguyen Thi Thanh; Khoa, Nguyen Cuu; Tsetlin, V I

2015-01-01

Nonconventional three-finger toxin BMLCL was isolated from B. multicinctus venom, and its interaction with different subtypes of nicotinic acetylcholine receptor (nAChR) was studied. It was found that BMLCL is able to interact with high efficiency with both α7 and muscle type nAChRs.

Full-Length Characterization of Hepatitis C Virus Subtype 3a Reveals Novel Hypervariable Regions under Positive Selection during Acute Infection▿

PubMed Central

Humphreys, Isla; Fleming, Vicki; Fabris, Paolo; Parker, Joe; Schulenberg, Bodo; Brown, Anthony; Demetriou, Charis; Gaudieri, Silvana; Pfafferott, Katja; Lucas, Michaela; Collier, Jane; Huang, Kuan-Hsiang Gary; Pybus, Oliver G.; Klenerman, Paul; Barnes, Eleanor

2009-01-01

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies. PMID:19740991

Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3

PubMed Central

Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Campos-Martínez, Gisselle A.; Meizoso-Huesca, Aldo; García-Sáinz, J. Adolfo

2015-01-01

Results The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1–3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1–3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes. Conclusion Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes. PMID:26473723

The orthosteric GABAA receptor ligand Thio-4-PIOL displays distinctly different functional properties at synaptic and extrasynaptic receptors

PubMed Central

Hoestgaard-Jensen, K; O'Connor, R M; Dalby, N O; Simonsen, C; Finger, B C; Golubeva, A; Hammer, H; Bergmann, M L; Kristiansen, U; Krogsgaard-Larsen, P; Bräuner-Osborne, H; Ebert, B; Frølund, B; Cryan, J F; Jensen, A A

2013-01-01

BACKGROUND AND PURPOSE Explorations into the heterogeneous population of native GABA type A receptors (GABAARs) and the physiological functions governed by the multiple GABAAR subtypes have for decades been hampered by the lack of subtype-selective ligands. EXPERIMENTAL APPROACH The functional properties of the orthosteric GABAA receptor ligand 5-(4-piperidyl)-3-isothiazolol (Thio-4-PIOL) have been investigated in vitro, ex vivo and in vivo. KEY RESULTS Thio-4-PIOL displayed substantial partial agonist activity at the human extrasynaptic GABAAR subtypes expressed in Xenopus oocytes, eliciting maximal responses of up to ∼30% of that of GABA at α5β3γ2S, α4β3δ and α6β3δ and somewhat lower efficacies at the corresponding α5β2γ2S, α4β2δ and α6β2δ subtypes (maximal responses of 4–12%). In contrast, it was an extremely low efficacious agonist at the α1β3γ2S, α1β2γ2S, α2β2γ2S, α2β3γ2S, α3β2γ2S and α3β3γ2S GABAARs (maximal responses of 0–4%). In concordance with its agonism at extrasynaptic GABAARs and its de facto antagonism at the synaptic receptors, Thio-4-PIOL elicited robust tonic currents in electrophysiological recordings on slices from rat CA1 hippocampus and ventrobasal thalamus and antagonized phasic currents in hippocampal neurons. Finally, the observed effects of Thio-4-PIOL in rat tests of anxiety, locomotion, nociception and spatial memory were overall in good agreement with its in vitro and ex vivo properties. CONCLUSION AND IMPLICATIONS The diverse signalling characteristics of Thio-4-PIOL at GABAARs represent one of the few examples of a functionally subtype-selective orthosteric GABAAR ligand reported to date. We propose that Thio-4-PIOL could be a useful pharmacological tool in future studies exploring the physiological roles of native synaptic and extrasynaptic GABAARs. PMID:23957253

MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.

PubMed

McNew, Kelsey L; Whipple, William J; Mehta, Anita K; Grant, Trevor J; Ray, Leah; Kenny, Connor; Singh, Anurag

2016-12-01

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G 1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G 2 -M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G 2 -M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204-16. ©2016 AACR. ©2016 American Association for Cancer Research.

Enhancement of Adipocyte Browning by Angiotensin II Type 1 Receptor Blockade.

PubMed

Tsukuda, Kana; Mogi, Masaki; Iwanami, Jun; Kanno, Harumi; Nakaoka, Hirotomo; Wang, Xiao-Li; Bai, Hui-Yu; Shan, Bao-Shuai; Kukida, Masayoshi; Higaki, Akinori; Yamauchi, Toshifumi; Min, Li-Juan; Horiuchi, Masatsugu

2016-01-01

Browning of white adipose tissue (WAT) has been highlighted as a new possible therapeutic target for obesity, diabetes and lipid metabolic disorders, because WAT browning could increase energy expenditure and reduce adiposity. The new clusters of adipocytes that emerge with WAT browning have been named 'beige' or 'brite' adipocytes. Recent reports have indicated that the renin-angiotensin system (RAS) plays a role in various aspects of adipose tissue physiology and dysfunction. The biological effects of angiotensin II, a major component of RAS, are mediated by two receptor subtypes, angiotensin II type 1 receptor (AT1R) and type 2 receptor (AT2R). However, the functional roles of angiotensin II receptor subtypes in WAT browning have not been defined. Therefore, we examined whether deletion of angiotensin II receptor subtypes (AT1aR and AT2R) may affect white-to-beige fat conversion in vivo. AT1a receptor knockout (AT1aKO) mice exhibited increased appearance of multilocular lipid droplets and upregulation of thermogenic gene expression in inguinal white adipose tissue (iWAT) compared to wild-type (WT) mice. AT2 receptor-deleted mice did not show miniaturization of lipid droplets or alteration of thermogenic gene expression levels in iWAT. An in vitro experiment using adipose tissue-derived stem cells showed that deletion of the AT1a receptor resulted in suppression of adipocyte differentiation, with reduction in expression of thermogenic genes. These results indicate that deletion of the AT1a receptor might have some effects on the process of browning of WAT and that blockade of the AT1 receptor could be a therapeutic target for the treatment of metabolic disorders.

Molecular characterization of five steroid receptors from pengze crucian carp and their expression profiles of juveniles in response to 17α-ethinylestradiol and 17α-methyltestosterone.

PubMed

Zheng, Yao; Wang, Lihong; Li, Meng; Liang, Hongwei; Qin, Fang; Liu, Shaozhen; Wang, Houpeng; Wu, Tingting; Zhang, Yingying; Wang, Zaizhao

2013-09-15

Pengze crucian carp (Carassius auratus var. pengze, Pcc), a triploid gynogenetic fish, was used in this study to investigate the cross-talk between EDCs and steroid receptors. The full-length cDNAs of five steroid receptors (esr1, er alpha2, esr2a, esr2b, ar) and partial cDNA of vtg B were isolated. The tissue distributions of these genes were analyzed in adult fish by qRT-PCR. Then the expression profiles of five steroid receptors (esrs and ar) and vtg B were detected in the juveniles exposed to 17α-ethinylestradiol (EE2, 0.1, 1 and 10ng/L) and 17α-methyltestosterone (MT, 50μg/L) for 4weeks. The results demonstrated that esrs, ar, and vtg B were predominantly expressed in liver of adult fish. However, among these detected genes, esr1 and er alpha2 mRNAs are sensitive biomarkers in response to EE2 at 0.1, 1, and 10ng/L for 1 and 2weeks compared to esr2a, esr2b, ar, and vtg B in the juveniles of mono-female gynogenetic fish. Totally, the subtypes of esrs show biphasic responses to EE2 exposures for 4weeks, and most of the EE2 exposures at 0.1, 1, and 10ng/L for 1, 2, 3 and 4weeks did not induce the mRNA expressions of vtg B. However, 1-, 2-, and 4-week 50μg/L MT all significantly stimulated vtg B transcripts. Further investigations are needed to elucidate the mechanism underlying the insensitivity or down-regulation of vtg B mRNA in response to EE2 in juvenile Pcc. Copyright © 2013 Elsevier Inc. All rights reserved.

The Role of Adenosine Receptors in Psychostimulant Addiction

PubMed Central

Ballesteros-Yáñez, Inmaculada; Castillo, Carlos A.; Merighi, Stefania; Gessi, Stefania

2018-01-01

Adenosine receptors (AR) are a family of G-protein coupled receptors, comprised of four members, named A1, A2A, A2B, and A3 receptors, found widely distributed in almost all human body tissues and organs. To date, they are known to participate in a large variety of physiopathological responses, which include vasodilation, pain, and inflammation. In particular, in the central nervous system (CNS), adenosine acts as a neuromodulator, exerting different functions depending on the type of AR and consequent cellular signaling involved. In terms of molecular pathways and second messengers involved, A1 and A3 receptors inhibit adenylyl cyclase (AC), through Gi/o proteins, while A2A and A2B receptors stimulate it through Gs proteins. In the CNS, A1 receptors are widely distributed in the cortex, hippocampus, and cerebellum, A2A receptors are localized mainly in the striatum and olfactory bulb, while A2B and A3 receptors are found at low levels of expression. In addition, AR are able to form heteromers, both among themselves (e.g., A1/A2A), as well as with other subtypes (e.g., A2A/D2), opening a whole range of possibilities in the field of the pharmacology of AR. Nowadays, we know that adenosine, by acting on adenosine A1 and A2A receptors, is known to antagonistically modulate dopaminergic neurotransmission and therefore reward systems, being A1 receptors colocalized in heteromeric complexes with D1 receptors, and A2A receptors with D2 receptors. This review documents the present state of knowledge of the contribution of AR, particularly A1 and A2A, to psychostimulants-mediated effects, including locomotor activity, discrimination, seeking and reward, and discuss their therapeutic relevance to psychostimulant addiction. Studies presented in this review reinforce the potential of A1 agonists as an effective strategy to counteract psychostimulant-induced effects. Furthermore, different experimental data support the hypothesis that A2A/D2 heterodimers are partly responsible for the psychomotor and reinforcing effects of psychostimulant drugs, such as cocaine and amphetamine, and the stimulation of A2A receptor is proposed as a potential therapeutic target for the treatment of drug addiction. The overall analysis of presented data provide evidence that excitatory modulation of A1 and A2A receptors constitute promising tools to counteract psychostimulants addiction. PMID:29375384

Overexpression of human NR2B receptor subunit in LMAN causes stuttering and song sequence changes in adult zebra finches.

PubMed

Chakraborty, Mukta; Chen, Liang-Fu; Fridel, Emma E; Klein, Marguerita E; Senft, Rebecca A; Sarkar, Abhra; Jarvis, Erich D

2017-04-21

Zebra finches (Taeniopygia guttata) learn to produce songs in a manner reminiscent of spoken language development in humans. One candidate gene implicated in influencing learning is the N-methyl-D-aspartate (NMDA) subtype 2B glutamate receptor (NR2B). Consistent with this idea, NR2B levels are high in the song learning nucleus LMAN (lateral magnocellular nucleus of the anterior nidopallium) during juvenile vocal learning, and decreases to low levels in adults after learning is complete and the song becomes more stereotyped. To test for the role of NR2B in generating song plasticity, we manipulated NR2B expression in LMAN of adult male zebra finches by increasing its protein levels to those found in juvenile birds, using a lentivirus containing the full-length coding sequence of the human NR2B subunit. We found that increased NR2B expression in adult LMAN induced increases in song sequence diversity and slower song tempo more similar to juvenile songs, but also increased syllable repetitions similar to stuttering. We did not observe these effects in control birds with overexpression of NR2B outside of LMAN or with the green fluorescent protein (GFP) in LMAN. Our results suggest that low NR2B subunit expression in adult LMAN is important in conserving features of stereotyped adult courtship song.

The change in muscarinic receptor subtypes in different brain regions of rats treated with fluoxetine or propranolol in a model of post-traumatic stress disorder.

PubMed

Aykaç, Aslı; Aydın, Banu; Cabadak, Hülya; Gören, M Zafer

2012-06-15

This study shows the possible contribution of muscarinic receptors in the pathophysiology of post-traumatic stress disorder. Sprague-Dawley rats of both sexes were exposed to dirty cat litter (trauma) for 10 min and the protocol was repeated 1 week later with a trauma reminder (clean litter). The rats also received intraperitoneal fluoxetine (2.5, 5 or 10 mg/kg/day), propranolol (10 mg/kg/day) or saline for 7 days between two exposure sessions. Functional behavioral experiments were performed using elevated plus maze, following exposure to trauma reminder. Western blot analyses for M(1), M(2), M(3), M(4) and M(5) receptor proteins were employed in the homogenates of the hippocampus, the frontal cortex and the amygdaloid complex. The anxiety indices increased from 0.63±0.02 to 0.89±0.04 in rats exposed to the trauma reminder. The freezing times were also recorded as 47±6 and 133±12 s, in control and test animals respectively. Fluoxetine or propranolol treatments restored the increases in the anxiety indices and the freezing times. Female rats had higher anxiety indices compared to males. Western blot data showed increases in M(2) and M(5) expression in the frontal cortex. Expression of M(1) receptors increased and M(4) subtype decreased in the hippocampus. In the amygdaloid complex of rats, we also detected a down-regulation of M(4) receptors. Fluoxetine and propranolol only corrected the changes occurred in the frontal cortex. These results may imply that muscarinic receptors are involved in this experimental model of post-traumatic stress disorder. Copyright © 2012 Elsevier B.V. All rights reserved.

An exogenous retrovirus isolated from koalas with malignant neoplasias in a US zoo.

PubMed

Xu, Wenqin; Stadler, Cynthia K; Gorman, Kristen; Jensen, Nathaniel; Kim, David; Zheng, HaoQiang; Tang, Shaohua; Switzer, William M; Pye, Geoffrey W; Eiden, Maribeth V

2013-07-09

Leukemia and lymphoma account for more than 60% of deaths in captive koalas (Phascolarctos cinereus) in northeastern Australia. Although the endogenizing gammaretrovirus koala endogenous retrovirus (KoRV) was isolated from these koalas, KoRV has not been definitively associated with leukemogenesis. We performed KoRV screening in koalas from the San Diego Zoo, maintained for more than 45 y with very limited outbreeding, and the Los Angeles Zoo, maintained by continuously assimilating captive-born Australian koalas. San Diego Zoo koalas are currently free of malignant neoplasias and were infected with only endogenous KoRV, which we now term subtype "KoRV-A," whereas Los Angeles Zoo koalas with lymphomas/leukemias are infected in addition to KoRV-A by a unique KoRV we term subtype "KoRV-B." KoRV-B is most divergent in the envelope protein and uses a host receptor distinct from KoRV-A. KoRV-B also has duplicated enhancer regions in the LTR associated with increased pathology in gammaretroviruses. Whereas KoRV-A uses the sodium-dependent phosphate transporter 1 (PiT1) as a receptor, KoRV-B employs a different receptor, the thiamine transporter 1 (THTR1), to infect cells. KoRV-B is transmitted from dam to offspring through de novo infection, rather than via genetic inheritance like KoRV-A. Detection of KoRV-B in native Australian koalas should provide a history, and a mode for remediation, of leukemia/lymphoma currently endemic in this population.

The Genetic Diversity and Evolution of HIV-1 Subtype B Epidemic in Puerto Rico.

PubMed

López, Pablo; Rivera-Amill, Vanessa; Rodríguez, Nayra; Vargas, Freddie; Yamamura, Yasuhiro

2015-12-23

HIV-1 epidemics in Caribbean countries, including Puerto Rico, have been reported to be almost exclusively associated with the subtype B virus (HIV-1B). However, while HIV infections associated with other clades have been only sporadically reported, no organized data exist to accurately assess the prevalence of non-subtype B HIV-1 infection. We analyzed the nucleotide sequence data of the HIV pol gene associated with HIV isolates from Puerto Rican patients. The sequences (n = 945) were obtained from our "HIV Genotyping" test file, which has been generated over a period of 14 years (2001-2014). REGA subtyping tool found the following subtypes: B (90%), B-like (3%), B/D recombinant (6%), and D/B recombinant (0.6%). Though there were fewer cases, the following subtypes were also found (in the given proportions): A1B (0.3%), BF1 (0.2%), subtype A (01-AE) (0.1%), subtype A (A2) (0.1%), subtype F (12BF) (0.1%), CRF-39 BF-like (0.1%), and others (0.1%). Some of the recombinants were identified as early as 2001. Although the HIV epidemic in Puerto Rico is primarily associated with HIV-1B virus, our analysis uncovered the presence of other subtypes. There was no indication of subtype C, which has been predominantly associated with heterosexual transmission in other parts of the world.

Modulation of Long-Term Potentiation and Epileptiform Activity in the Rat Dentate Gyrus by the Group II Metabotropic Glutamate Receptor Subtype mGluR3

DTIC Science & Technology

2000-05-25

preparation richly endowed with ionotropic and metabotropic glutamate receptors , including mGluR3 (Shigemoto et al., 1997). NAAG is concentrated in...Zhao and R. J. Wenthold (1996b). Ionotropic and metabotropic glutamate receptors show unique postsynaptic, presynaptic, and glial localizations in...epileptiform activity in the rat cortex. Neuroreport 3(10): 916-8. Shen, W. and M. M. Slaughter (1998). Metabotropic and ionotropic glutamate receptors

Challenges in the development of dopamine D2- and D3-selective radiotracers for PET imaging studies.

PubMed

Mach, Robert H; Luedtke, Robert R

2018-03-01

The dopamine D2-like receptors (ie, D2/3 receptors) have been the most extensively studied CNS receptor with Positron Emission Tomography (PET). The 3 different radiotracers that have been used in these studies are [ 11 C]raclopride, [ 18 F]fallypride, and [ 11 C]PHNO. Because these radiotracers have a high affinity for both dopamine D2 and D3 receptors, the density of dopamine receptors in the CNS is reported as the D2/3 binding potential, which reflects a measure of the density of both receptor subtypes. Although the development of D2- and D3-selective PET radiotracers has been an active area of research for many years, this by and large presents an unmet need in the area of translational PET imaging studies. This article discusses some of the challenges that have inhibited progress in this area of research and the current status of the development of subtype selective radiotracers for imaging D3 and D2 dopamine receptors with PET. Copyright © 2017 John Wiley & Sons, Ltd.

The effects of the selective 5-HT(2C) receptor antagonist SB 242084 on learned helplessness in male Fischer 344 rats.

PubMed

Strong, Paul V; Greenwood, Benjamin N; Fleshner, Monika

2009-05-01

Rats exposed to an uncontrollable stressor demonstrate a constellation of behaviors such as exaggerated freezing and deficits in shuttle box escape learning. These behaviors in rats have been called learned helplessness and have been argued to model human stress-related mood disorders. Learned helplessness is thought to be caused by hyperactivation of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) and a subsequent exaggerated release of 5-HT in DRN projection sites. Blocking 5-HT(2C) receptors in the face of an increase in serotonin can alleviate anxiety behaviors in some animal models. However, specific 5-HT receptor subtypes involved in learned helplessness remain unknown. The current experiments tested the hypothesis that 5-HT(2C) receptor activation is necessary and sufficient for the expression of learned helplessness. The selective 5-HT(2C) receptor antagonist SB 242084 (1.0 mg/kg) administered i.p. to adult male Fischer 344 rats prior to shuttle box behavioral testing, but not before stress, blocked stress-induced deficits in escape learning but had no effect on the exaggerated shock-elicited freezing. The selective 5-HT(2C) receptor agonist CP-809101 was sufficient to produce learned helplessness-like behaviors in the absence of prior stress and these effects were blocked by pretreatment with SB 242084. Results implicate the 5-HT(2C) receptor subtype in mediating the shuttle box escape deficits produced by exposure to uncontrollable stress and suggest that different postsynaptic 5-HT receptor subtypes underlie the different learned helplessness behaviors.

Directly Observable Behavioral Effects of Lorcaserin in Rats.

PubMed

Serafine, Katherine M; Rice, Kenner C; France, Charles P

2015-12-01

(1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (lorcaserin) is approved by the United States Food and Drug Administration for treating obesity, and its therapeutic effects are thought to result from agonist activity at serotonin (5-HT)2C receptors. Lorcaserin has affinity for other 5-HT receptor subtypes, although its activity at those subtypes is not fully described. The current study compared the behavioral effects of lorcaserin (0.0032-32.0 mg/kg) to the effects of other 5-HT receptor selective agonists in rats (n = 8). The 5-HT2C receptor selective agonist 1-(3-chlorophenyl)piperazine (mCPP, 0.032-1.0 mg/kg) and lorcaserin induced yawning which was attenuated by the 5-HT2C receptor selective antagonist 6-chloro-5-methyl-N-(6-[(2-methylpyridin-3-yl)oxy]pydidin-3-yl)indoline-1-carboxamide (1.0 mg/kg). The 5-HT2A receptor selective agonist 2,5-dimethoxy-4-methylamphetamine (0.1-3.2 mg/kg) induced head twitching, which was attenuated by the 5-HT2A receptor selective antagonist R-(+)-2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL 100907, 0.01 mg/kg), lorcaserin (3.2 mg/kg), and mCPP (3.2 mg/kg). In rats pretreated with MDL 100907 (1.0 mg/kg), lorcaserin also induced head twitching. At larger doses, lorcaserin produced forepaw treading, which was attenuated by the 5-HT1A receptor selective antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (0.178 mg/kg). While the behavioral effects of lorcaserin in rats are consistent with it having agonist activity at 5-HT2C receptors, these data suggest that at larger doses it also has agonist activity at 5-HT2A and possibly 5-HT1A receptors. Mounting evidence suggests that 5-HT2C receptor agonists might be effective for treating drug abuse. A more complete description of the activity of lorcaserin at 5-HT receptor subtypes will facilitate a better understanding of the mechanisms that mediate its therapeutic effects. U.S. Government work not protected by U.S. copyright.

Directly Observable Behavioral Effects of Lorcaserin in Rats

PubMed Central

Serafine, Katherine M.; Rice, Kenner C.

2015-01-01

(1R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine (lorcaserin) is approved by the United States Food and Drug Administration for treating obesity, and its therapeutic effects are thought to result from agonist activity at serotonin (5-HT)2C receptors. Lorcaserin has affinity for other 5-HT receptor subtypes, although its activity at those subtypes is not fully described. The current study compared the behavioral effects of lorcaserin (0.0032–32.0 mg/kg) to the effects of other 5-HT receptor selective agonists in rats (n = 8). The 5-HT2C receptor selective agonist 1-(3-chlorophenyl)piperazine (mCPP, 0.032–1.0 mg/kg) and lorcaserin induced yawning which was attenuated by the 5-HT2C receptor selective antagonist 6-chloro-5-methyl-N-(6-[(2-methylpyridin-3-yl)oxy]pydidin-3-yl)indoline-1-carboxamide (1.0 mg/kg). The 5-HT2A receptor selective agonist 2,5-dimethoxy-4-methylamphetamine (0.1–3.2 mg/kg) induced head twitching, which was attenuated by the 5-HT2A receptor selective antagonist R-(+)-2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol] (MDL 100907, 0.01 mg/kg), lorcaserin (3.2 mg/kg), and mCPP (3.2 mg/kg). In rats pretreated with MDL 100907 (1.0 mg/kg), lorcaserin also induced head twitching. At larger doses, lorcaserin produced forepaw treading, which was attenuated by the 5-HT1A receptor selective antagonist N-(2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl)-N-(2-pyridyl)cyclohexanecarboxamide (0.178 mg/kg). While the behavioral effects of lorcaserin in rats are consistent with it having agonist activity at 5-HT2C receptors, these data suggest that at larger doses it also has agonist activity at 5-HT2A and possibly 5-HT1A receptors. Mounting evidence suggests that 5-HT2C receptor agonists might be effective for treating drug abuse. A more complete description of the activity of lorcaserin at 5-HT receptor subtypes will facilitate a better understanding of the mechanisms that mediate its therapeutic effects. PMID:26384326

Expression of messenger molecules and receptors in rat and human sphenopalatine ganglion indicating therapeutic targets.

PubMed

Steinberg, Anna; Frederiksen, Simona D; Blixt, Frank W; Warfvinge, Karin; Edvinsson, Lars

2016-12-01

Migraine and Cluster Headache (CH) are two primary headaches with severe disease burden. The disease expression and the mechanisms involved are poorly known. In some attacks of migraine and in most attacks of CH, there is a release of vasoactive intestinal peptide (VIP) originating from parasympathetic cranial ganglia such as the sphenopalatine ganglion (SPG). Patients suffering from these diseases are often deprived of effective drugs. The aim of the study was to examine the localization of the botulinum toxin receptor element synaptic vesicle glycoprotein 2A (SV-2A) and the vesicular docking protein synaptosomal-associated protein 25 (SNAP25) in human and rat SPG. Additionally the expression of the neurotransmitters pituitary adenylate cyclase activating polypeptide (PACAP-38), nitric oxide synthase (nNOS), VIP and 5-hydroxttryptamine subtype receptors (5-HT1B,1D,1F) were examined. SPG from adult male rats and from humans, the later removed at autopsy, were prepared for immunohistochemistry using specific antibodies against neurotransmitters, 5-HT1B,1D,1F receptors, and botulinum toxin receptor elements. We found that the selected neurotransmitters and 5-HT receptors were expressed in rat and human SPG. In addition, we found SV2-A and SNAP25 expression in both rat and human SPG. We report that all three 5-HT receptors studied occur in neurons and satellite glial cells (SGCs) of the SPG. 5-HT1B receptors were in addition found in the walls of intraganglionic blood vessels. Recent focus on the SPG has emphasized the role of parasympathetic mechanisms in the pathophysiology of mainly CH. The development of next generation's drugs and treatment of cranial parasympathetic symptoms, mediated through the SPG, can be modulated by treatment with BoNT-A and 5-HT receptor agonists.

Magnetic resonance imaging in breast cancer treated with neoadjuvant chemotherapy: radiologic-pathologic correlation of the response and disease-free survival depending on molecular subtype.

PubMed

Cruz Ciria, S; Jiménez Aragón, F; García Mur, C; Esteban Cuesta, H; Gros Bañeres, B

2014-01-01

To evaluate the radiologic and pathologic responses to neoadjuvant chemotherapy and their correlation in the molecular subtypes of breast cancer and to analyze their impact in disease-free survival. We included 205 patients with breast cancer treated with neoadjuvant chemotherapy. We evaluated the radiologic response by comparing MRI images acquired before and after chemotherapy. The pathologic response was classified on the Miller and Payne scale. For each subtype (HER2+, TN, luminal A, luminal B HER2-, and luminal B HER2+), we used the χ(2) test, Student's t-test, ANOVA, and Kendall's Tau-b to evaluate the radiologic response and the pathologic response, the radiologic-pathologic correlation, and the disease-free survival. The subtypes HER2+ (62.1%) and TN (45.2%) had higher rates of complete radiologic response. The pathologic response was 65.5% in the HER2+ subtype, 38.1% in the TN subtype, 2.6% in the luminal A subtype, 8.2% in the luminal B HER2- subtype, and 31% in the luminal B HER2+ subtype. The rate of radiologic-pathologic correlation was significant in all subtypes, higher in TN and HER2 (Tau-b coefficients 0.805 and 0.717, respectively). Disease-free survival was higher in HER2+ (91.9±3.3 months) and lower in TN (69.5±6.3 months), with significant differences between the cases with poor and good radiologic responses (P=.040). Survival was greater in cases with good radiologic response, except in cases with luminal A subtype. MRI can be a useful tool that provides information about the evolution of breast cancer treated with neoadjuvant chemotherapy, which varies with the immunohistochemical subtype. Copyright © 2012 SERAM. Published by Elsevier Espana. All rights reserved.

Opioid receptor subtypes mediating the noise-induced decreases in high-affinity choline uptake in the rat brain.

PubMed

Lai, H; Carino, M A

1992-07-01

Acute (20 min) exposure to 100-dB white noise elicits a naltrexone-sensitive decrease in sodium-dependent high-affinity choline uptake in the frontal cortex and hippocampus of the rat. In the present study, the subtypes of opioid receptors involved were investigated by pretreating rats with microinjection of specific opioid-receptor antagonists into the lateral cerebroventricle before noise exposure. We found that the noise-induced decrease in high-affinity choline uptake in the hippocampus was blocked by pretreatment with either mu-, delta-, or kappa-opioid-receptor antagonists, whereas the effect of noise on frontal cortical high-affinity choline uptake was blocked by a mu- and delta- but not by a kappa-antagonist. These data further confirm the role of endogenous opioids in mediating the effects of noise on central cholinergic activity and indicate that different neural mechanisms are involved in the effects of noise on the frontal cortical and hippocampal cholinergic systems.

Investigation of the antidyskinetic site of action of metabotropic and ionotropic glutamate receptor antagonists. Intracerebral infusions in 6-hydroxydopamine-lesioned rats with levodopa-induced dyskinesia.

PubMed

Maranis, Sotirios; Stamatis, Dimitrios; Tsironis, Christos; Konitsiotis, Spiridon

2012-05-15

Long-term levodopa replacement therapy in Parkinson's disease is confounded by abnormal involuntary movements, known as levodopa induced dyskinesia (LID). Dysfunctional glutamatergic neurotransmission has been implicated in the pathogenesis of LID making metabotropic and ionotropic glutamate receptors attractive novel therapeutic targets. The objective of the present study was to investigate the antidyskinetic site of action of different glutamate receptor antagonists in the brain. For that purpose, metabotropic glutamate subtype 5 (3-((2-Methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride, MTEP), NMDA NR2B selective ((aR,bS)-a-(4-Hydroxyphenyl)-b-methyl-4-(phenylmethyl)-1-piperidinepropanol maleate, Ro 25-6981) and AMPA (2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide disodium salt, NBQX) receptor antagonists or saline were administered by intracerebral infusion in the caudate-putamen (CPu), the substantia nigra zona reticulata (SNr) or the subthalamic nucleus (STN) of 6-hydroxydopamine-lesioned rats exhibiting LID. Dyskinesia was assessed with the modified version of the rat Abnormal Involuntary Movements scale (AIMS). Ro 25-6981 and to a lesser extent NBQX improved dyskinesia (82% and 19% reduction in AIM score respectively) after infusion in the caudate-putamen. None of the three drugs managed to noticeably reduce AIM score after infusion in the SNr. MTEP was the only drug that produced a reduction in AIM score (48%) when infused in STN. In conclusion, while the striatum proved important in the antidyskinetic action of NMDA and AMPA receptor antagonists, the results of this study highlight also the importance of the metabotropic glutamate receptors that reside in the STN as therapeutic targets in the treatment of LID. Copyright © 2012 Elsevier B.V. All rights reserved.

Characterization of "mini-nucleotides" as P2X receptor agonists in rat cardiomyocyte cultures. An integrated synthetic, biochemical, and theoretical study.

PubMed

Fischer, B; Yefidoff, R; Major, D T; Rutman-Halili, I; Shneyvays, V; Zinman, T; Jacobson, K A; Shainberg, A

1999-07-15

The design and synthesis of "mini-nucleotides", based on a xanthine-alkyl phosphate scaffold, are described. The physiological effects of the new compounds were evaluated in rat cardiac cell culture regarding Ca(2+) elevation and contractility. The results indicate biochemical and physiological profiles similar to those of ATP, although at higher concentrations. The biological target molecules of these "mini-nucleotides" were identified by using selective P2-R and A(1)-R antagonists and P2-R subtype selective agonists. On the basis of these results and of experiments in Ca(2+) free medium, in which [Ca(2+)](i) elevation was not observed, we concluded that interaction of the analogues is likely with P2X receptor subtypes, which causes Ca(2+) influx. Theoretical calculations analyzing electronic effects within the series of xanthine-alkyl phosphates were performed on reduced models at quantum mechanical levels. Calculated dipole moment vectors, electrostatic potential maps, and volume parameters suggest an explanation for the activity or inactivity of the synthesized derivatives and predict a putative binding site environment for the active agonists. Xanthine-alkyl phosphate analogues proved to be selective agents for activation of P2X-R subtypes, whereas ATP activated all P2-R subtypes in cardiac cells. Therefore, these analogues may serve as prototypes of selective drugs aiming at cardiac disorders mediated through P2X receptors.

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.

Bone Turnover Status: Classification Model and Clinical Implications

PubMed Central

Fisher, Alexander; Fisher, Leon; Srikusalanukul, Wichat; Smith, Paul N

2018-01-01

Aim: To develop a practical model for classification bone turnover status and evaluate its clinical usefulness. Methods: Our classification of bone turnover status is based on internationally recommended biomarkers of both bone formation (N-terminal propeptide of type1 procollagen, P1NP) and bone resorption (beta C-terminal cross-linked telopeptide of type I collagen, bCTX), using the cutoffs proposed as therapeutic targets. The relationships between turnover subtypes and clinical characteristic were assessed in1223 hospitalised orthogeriatric patients (846 women, 377 men; mean age 78.1±9.50 years): 451(36.9%) subjects with hip fracture (HF), 396(32.4%) with other non-vertebral (non-HF) fractures (HF) and 376 (30.7%) patients without fractures. Resalts: Six subtypes of bone turnover status were identified: 1 - normal turnover (P1NP>32 μg/L, bCTX≤0.250 μg/L and P1NP/bCTX>100.0[(median value]); 2- low bone formation (P1NP ≤32 μg/L), normal bone resorption (bCTX≤0.250 μg/L) and P1NP/bCTX>100.0 (subtype2A) or P1NP/bCTX<100.0 (subtype 2B); 3- low bone formation, high bone resorption (bCTX>0.250 μg/L) and P1NP/bCTX<100.0; 4- high bone turnover (both markers elevated ) and P1NP/bCTX>100.0 (subtype 4A) or P1NP/bCTX<100.0 (subtype 4B). Compared to subtypes 1 and 2A, subtype 2B was strongly associated with nonvertebral fractures (odds ratio [OR] 2.0), especially HF (OR 3.2), age>75 years and hyperparathyroidism. Hypoalbuminaemia and not using osteoporotic therapy were two independent indicators common for subtypes 3, 4A and 4B; these three subtypes were associated with in-hospital mortality. Subtype 3 was associated with fractures (OR 1.7, for HF OR 2.4), age>75 years, chronic heart failure (CHF), anaemia, and history of malignancy, and predicted post-operative myocardial injury, high inflammatory response and length of hospital stay (LOS) above10 days. Subtype 4A was associated with chronic kidney disease (CKD), anaemia, history of malignancy and walking aids use and predicted LOS>20 days, but was not discriminative for fractures. Subtype 4B was associated with fractures (OR 2.1, for HF OR 2.5), age>75 years, CKD and indicated risks of myocardial injury, high inflammatory response and LOS>10 days. Conclusions: We proposed a classification model of bone turnover status and demonstrated that in orthogeriatric patients altered subtypes are closely related to presence of nonvertebral fractures, comorbidities and poorer in-hospital outcomes. However, further research is needed to establish optimal cut points of various biomarkers and improve the classification model. PMID:29511368

Potentiation of GluN2C/D NMDA receptor subtypes in the amygdala facilitates the retention of fear and extinction learning in mice.

PubMed

Ogden, Kevin K; Khatri, Alpa; Traynelis, Stephen F; Heldt, Scott A

2014-02-01

NMDA receptors are glutamate receptor ion channels that contribute to synaptic plasticity and are important for many forms of learning and memory. In the amygdala, NMDA receptors are critical for the acquisition, retention, and extinction of classically conditioned fear responses. Although the GluN2B subunit has been implicated in both the acquisition and extinction of conditioned fear, GluN2C-knockout mice show reduced conditioned fear responses. Moreover, D-cycloserine (DCS), which facilitates fear extinction, selectively enhances the activity of GluN2C-containing NMDA receptors. To further define the contribution of GluN2C receptors to fear learning, we infused the GluN2C/GluN2D-selective potentiator CIQ bilaterally into the basolateral amygdala (3, 10, or 30 μg/side) following either fear conditioning or fear extinction training. CIQ both increased the expression of conditioned fear 24 h later and enhanced the extinction of the previously conditioned fear response. These results support a critical role for GluN2C receptors in the amygdala in the consolidation of learned fear responses and suggest that increased activity of GluN2C receptors may underlie the therapeutic actions of DCS.

The T160A hemagglutinin substitution affects not only receptor binding property but also transmissibility of H5N1 clade 2.3.4 avian influenza virus in guinea pigs.

PubMed

Gu, Min; Li, Qunhui; Gao, Ruyi; He, Dongchang; Xu, Yunpeng; Xu, Haixu; Xu, Lijun; Wang, Xiaoquan; Hu, Jiao; Liu, Xiaowen; Hu, Shunlin; Peng, Daxin; Jiao, Xinan; Liu, Xiufan

2017-02-06

We generated and characterized site-directed HA mutants on the genetic backbone of H5N1 clade 2.3.4 virus preferentially binding to α-2,3 receptors in order to identify the key determinants in hemagglutinin rendering the dual affinity to both α-2,3 (avian-type) and α-2,6 (human-type) linked sialic acid receptors of the current clade 2.3.4.4 H5NX subtype avian influenza reassortants. The results show that the T160A substitution resulted in the loss of a glycosylation site at 158N and led not only to enhanced binding specificity for human-type receptors but also transmissibility among guinea pigs, which could be considered as an important molecular marker for assessing pandemic potential of H5 subtype avian influenza isolates.

Genetically divergent strains of feline immunodeficiency virus from the domestic cat (Felis catus) and the African lion (Panthera leo) share usage of CD134 and CXCR4 as entry receptors.

PubMed

McEwan, William A; McMonagle, Elizabeth L; Logan, Nicola; Serra, Rodrigo C; Kat, Pieter; Vandewoude, Sue; Hosie, Margaret J; Willett, Brian J

2008-11-01

The env open reading frames of African lion (Panthera leo) lentivirus (feline immunodeficiency virus [FIV(Ple)]) subtypes B and E from geographically distinct regions of Africa suggest two distinct ancestries, with FIV(Ple)-E sharing a common ancestor with the domestic cat (Felis catus) lentivirus (FIV(Fca)). Here we demonstrate that FIV(Ple)-E and FIV(Fca) share the use of CD134 (OX40) and CXCR4 as a primary receptor and coreceptor, respectively, and that both lion CD134 and CXCR4 are functional receptors for FIV(Ple)-E. The shared usage of CD134 and CXCR4 by FIV(Fca) and FIV(Ple)-E may have implications for in vivo cell tropism and the pathogenicity of the E subtype among free-ranging lion populations.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway.

PubMed

Guo, Zhiying; Zhao, Ming; Howard, Erin W; Zhao, Qingxia; Parris, Amanda B; Ma, Zhikun; Yang, Xiaohe

2017-09-01

Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25-75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro . Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway

PubMed Central

Guo, Zhiying; Zhao, Ming; Howard, Erin W.; Zhao, Qingxia; Parris, Amanda B.; Ma, Zhikun; Yang, Xiaohe

2017-01-01

Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro. Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics. PMID:28947975

Breast cancer mortality in African-American and non-Hispanic white women by molecular subtype and stage at diagnosis: a population-based study

PubMed Central

Tao, Li; Gomez, Scarlett Lin; Keegan, Theresa HM; Kurian, Allison W.; Clarke, Christina A.

2015-01-01

Background Higher breast cancer mortality rates for African-American than non-Hispanic white women are well documented; however, it remains uncertain if this disparity occurs in disease subgroups defined by tumor molecular markers and stage at diagnosis. We examined racial differences in outcome according to subtype and stage in a diverse, population-based series of 103,498 patients. Methods We obtained data for all invasive breast cancers diagnosed 1/1/2005-12/31/2012 and followed through 12/31/2012 among 93,760 non-Hispanic white and 9,738 African-American women in California. Molecular subtypes were categorized according to tumor expression of hormone receptor (HR, based on estrogen and progesterone receptors) and human epidermal growth factor receptor 2 (HER2). Cox proportional hazards models were used to calculate hazard ratios (HR) and 95% confidence intervals (CI) for breast cancer-specific mortality. Results After adjustment for patient, tumor and treatment characteristics, outcomes were comparable by race for Stage I or IV cancer regardless of subtype, and HR+/HER2+ or HR-/HER2+ cancer regardless of stage. We found substantially higher hazards of breast cancer death among African-American women with Stage II/III HR+/HER2- (HR, 1.31, 95% CI, 1.03-1.65, and HR, 1.39, 95% CI, 1.10-1.75, respectively) and Stage III triple-negative cancers relative to whites. Conclusions There are substantial racial/ethnic disparities among patients with Stages II/III HR+/HER2- and Stage III triple-negative breast cancers but not for other subtype and stage. Impact These data provide insights to assess barriers to targeted treatment (e.g. trastuzumab or endocrine therapy) of particular subtypes of breast cancer among African-American patients. PMID:25969506

Breast Cancer Mortality in African-American and Non-Hispanic White Women by Molecular Subtype and Stage at Diagnosis: A Population-Based Study.

PubMed

Tao, Li; Gomez, Scarlett Lin; Keegan, Theresa H M; Kurian, Allison W; Clarke, Christina A

2015-07-01

Higher breast cancer mortality rates for African-American than non-Hispanic White women are well documented; however, it remains uncertain if this disparity occurs in disease subgroups defined by tumor molecular markers and stage at diagnosis. We examined racial differences in outcome according to subtype and stage in a diverse, population-based series of 103,498 patients. We obtained data for all invasive breast cancers diagnosed between January 1, 2005, and December 31, 2012, and followed through December 31, 2012, among 93,760 non-Hispanic White and 9,738 African-American women in California. Molecular subtypes were categorized according to tumor expression of hormone receptor (HR, based on estrogen and progesterone receptors) and human epidermal growth factor receptor 2 (HER2). Cox proportional hazards models were used to calculate relative hazard (RH) and 95% confidence intervals (CI) for breast cancer-specific mortality. After adjustment for patient, tumor, and treatment characteristics, outcomes were comparable by race for stage I or IV cancer regardless of subtype, and HR(+)/HER2(+) or HR(-)/HER2(+) cancer regardless of stage. We found substantially higher hazards of breast cancer death among African-American women with stage II/III HR(+)/HER2(-) (RH, 1.31; 95% CI, 1.03-1.65; and RH, 1.39; 95% CI, 1.10-1.75, respectively) and stage III triple-negative cancers relative to Whites. There are substantial racial/ethnic disparities among patients with stages II/III HR(+)/HER2(-) and stage III triple-negative breast cancers but not for other subtype and stage. These data provide insights to assess barriers to targeted treatment (e.g., trastuzumab or endocrine therapy) of particular subtypes of breast cancer among African-American patients. ©2015 American Association for Cancer Research.

Receptor-Defined Subtypes of Breast Cancer in Indigenous Populations in Africa: A Systematic Review and Meta-Analysis

PubMed Central

Eng, Amanda; McCormack, Valerie; dos-Santos-Silva, Isabel

2014-01-01

Background Breast cancer is the most common female cancer in Africa. Receptor-defined subtypes are a major determinant of treatment options and disease outcomes but there is considerable uncertainty regarding the frequency of poor prognosis estrogen receptor (ER) negative subtypes in Africa. We systematically reviewed publications reporting on the frequency of breast cancer receptor-defined subtypes in indigenous populations in Africa. Methods and Findings Medline, Embase, and Global Health were searched for studies published between 1st January 1980 and 15th April 2014. Reported proportions of ER positive (ER+), progesterone receptor positive (PR+), and human epidermal growth factor receptor-2 positive (HER2+) disease were extracted and 95% CI calculated. Random effects meta-analyses were used to pool estimates. Fifty-four studies from North Africa (n = 12,284 women with breast cancer) and 26 from sub-Saharan Africa (n = 4,737) were eligible. There was marked between-study heterogeneity in the ER+ estimates in both regions (I2>90%), with the majority reporting proportions between 0.40 and 0.80 in North Africa and between 0.20 and 0.70 in sub-Saharan Africa. Similarly, large between-study heterogeneity was observed for PR+ and HER2+ estimates (I2>80%, in all instances). Meta-regression analyses showed that the proportion of ER+ disease was 10% (4%–17%) lower for studies based on archived tumor blocks rather than prospectively collected specimens, and 9% (2%–17%) lower for those with ≥40% versus those with <40% grade 3 tumors. For prospectively collected samples, the pooled proportions for ER+ and triple negative tumors were 0.59 (0.56–0.62) and 0.21 (0.17–0.25), respectively, regardless of region. Limitations of the study include the lack of standardized procedures across the various studies; the low methodological quality of many studies in terms of the representativeness of their case series and the quality of the procedures for collection, fixation, and receptor testing; and the possibility that women with breast cancer may have contributed to more than one study. Conclusions The published data from the more appropriate prospectively measured specimens are consistent with the majority of breast cancers in Africa being ER+. As no single subtype dominates in the continent availability of receptor testing should be a priority, especially for young women with early stage disease where appropriate receptor-specific treatment modalities offer the greatest potential for reducing years of life lost. Please see later in the article for the Editors' Summary PMID:25202974

Role of Dopamine Receptors Subtypes, D1-Like and D2-Like, within the Nucleus Accumbens Subregions, Core and Shell, on Memory Consolidation in the One-Trial Inhibitory Avoidance Task

ERIC Educational Resources Information Center

Manago, Francesca; Castellano, Claudio; Oliverio, Alberto; Mele, Andrea; De Leonibus, Elvira

2009-01-01

Recent evidence demonstrated that dopamine within the nucleus accumbens mediates consolidation of both associative and nonassociative memories. However, the specific contribution of the nucleus accumbens subregions, core and shell, and of D1 and D2 receptors subtypes has not been yet clarified. The aim of this study was, therefore, to directly…

Enhancement effects of nicotine on neurogenic relaxation responses in the corpus cavernosum in rabbits: the role of nicotinic acetylcholine receptor subtypes.

PubMed

Ozturk Fincan, Gokce Sevim; Vural, Ismail Mert; Ercan, Zeynep Sevim; Sarioglu, Yusuf

2010-02-10

Nicotine acts as an agonist of nicotinic acetylcholine receptors, which belong to a superfamily of neurotransmitter-gated ion channels. We previously demonstrated that nicotine increases the electrical field stimulation (EFS)-evoked nitrergic relaxation responses via activation of nicotinic acetylcholine receptors. The aim of the present study is to investigate the subtypes of nicotinic acetylcholine receptors in rabbit corpus cavernosum. EFS-evoked relaxation responses were recorded from corpus cavernosum strips obtained from rabbits with an isometric force displacement transducers. Effects of nicotine on EFS-evoked relaxations were examined in pre-contracted tissues. Then the effect of nicotine on the EFS-evoked relaxations was examined in the presence of hexamethonium, dihydro-beta-erythroidine, mecamylamine or alpha-bungarotoxin. In our study, nicotine (3 x 10(-5), 10(-4)) transiently increased nitrergic relaxations induced by EFS in the rabbit isolated corpus cavernosum. While hexamethonium and mecamylamine near totally inhibited or abolished the neurorelaxation response to nicotine (3 x 10(-5)) on EFS, dihydro-beta-erythroidine and alpha-bungarotoxin partially inhibited these responses. These findings demonstrated that the alpha3-beta4, alpha4-beta2 and alpha7 subunits of nicotinic acetylcholine receptors play role on the nicotine-induced augmentation in EFS-evoked relaxation responses in rabbit corpus cavernosum. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Membrane Receptor-Induced Changes of the Protein Kinases A and C Activity May Play a Leading Role in Promoting Developmental Synapse Elimination at the Neuromuscular Junction.

PubMed

Tomàs, Josep M; Garcia, Neus; Lanuza, Maria A; Nadal, Laura; Tomàs, Marta; Hurtado, Erica; Simó, Anna; Cilleros, Víctor

2017-01-01

Synapses that are overproduced during histogenesis in the nervous system are eventually lost and connectivity is refined. Membrane receptor signaling leads to activity-dependent mutual influence and competition between axons directly or with the involvement of the postsynaptic cell and the associated glial cell/s. Presynaptic muscarinic acetylcholine (ACh) receptors (subtypes mAChR; M 1 , M 2 and M 4 ), adenosine receptors (AR; A 1 and A 2A ) and the tropomyosin-related kinase B receptor (TrkB), among others, all cooperate in synapse elimination. Between these receptors there are several synergistic, antagonic and modulatory relations that clearly affect synapse elimination. Metabotropic receptors converge in a limited repertoire of intracellular effector kinases, particularly serine protein kinases A and C (PKA and PKC), to phosphorylate protein targets and bring about structural and functional changes leading to axon loss. In most cells A 1 , M 1 and TrkB operate mainly by stimulating PKC whereas A 2A , M 2 and M 4 inhibit PKA. We hypothesize that a membrane receptor-induced shifting in the protein kinases A and C activity (inhibition of PKA and/or stimulation of PKC) in some nerve endings may play an important role in promoting developmental synapse elimination at the neuromuscular junction (NMJ). This hypothesis is supported by: (i) the tonic effect (shown by using selective inhibitors) of several membrane receptors that accelerates axon loss between postnatal days P5-P9; (ii) the synergistic, antagonic and modulatory effects (shown by paired inhibition) of the receptors on axonal loss; (iii) the fact that the coupling of these receptors activates/inhibits the intracellular serine kinases; and (iv) the increase of the PKA activity, the reduction of the PKC activity or, in most cases, both situations simultaneously that presumably occurs in all the situations of singly and paired inhibition of the mAChR, AR and TrkB receptors. The use of transgenic animals and various combinations of selective and specific PKA and PKC inhibitors could help to elucidate the role of these kinases in synapse maturation.

Clinical efficacy of raltegravir against B and non-B subtype HIV-1 in phase III clinical studies.

PubMed

Rockstroh, Jürgen K; Teppler, Hedy; Zhao, Jing; Sklar, Peter; Miller, Michael D; Harvey, Charlotte M; Strohmaier, Kim M; Leavitt, Randi Y; Nguyen, Bach-Yen T

2011-07-17

We evaluated the long-term efficacy of raltegravir according to HIV-1 subtype (B and non-B) using data from three phase III studies in treatment-experienced (BENCHMRK-1 and 2) and treatment-naive (STARTMRK) HIV-infected patients. HIV-1 subtypes were identified from baseline plasma specimens using genotypic data of the PhenoSense GT test (Monogram Biosciences, South San Francisco, California, USA). Non-B subtypes were combined for the current analyses due to small numbers of each specific subtype. An observed failure approach was used (only discontinuations due to lack of efficacy were treated as failures). Resistance evaluation was performed in patients with documented virologic failure. Seven hundred and forty-three patients received raltegravir and 519 received comparator (efavirenz in STARTMRK; optimized background therapy in BENCHMRK). Non-B subtype virus (A, A/C, A/D, A/G, A1, AE, AG, B/G, BF, C, D, D/F, F, F1, G, and complex) was isolated at baseline in 98 (13%) raltegravir recipients and 62 (12%) comparator recipients. Subtypes AE and C were most common, isolated in 41 and 43 patients, respectively. The proportion of raltegravir recipients achieving HIV RNA less than 50 copies/ml was similar between non-B and B subtypes (STARTMRK: 94.5 vs. 88.7%; BENCHMRK-1 and 2: 66.7 vs. 60.7%); change in CD4 cell count also was similar between non-B and B subtypes (STARTMRK: 243 vs. 221 cells/μl; BENCHMRK-1 and 2: 121 vs. 144 cells/μl). Phenotypic resistance to raltegravir in non-B virus was associated with integrase mutations observed previously in subtype B virus. In phase III studies in treatment-naive and treatment-experienced patients, raltegravir showed comparable and potent clinical efficacy against B and non-B HIV-1 subtypes.

Characterization and distribution of natriuretic peptide receptors in the rat uterus.

PubMed

Dos Reis, A M; Fujio, N; Dam, T V; Mukaddam-Daher, S; Jankowski, M; Tremblay, J; Gutkowska, J

1995-10-01

Atrial natriuretic peptide (ANP) receptors were characterized in rat uterus. The binding of [125I]ANP to uterine membranes was completely competed for by increasing concentrations of unlabeled ANP (Kd = 0.39 nM) and brain natriuretic peptide (Kd = 1.24 nM) and partially by C-type natriuretic peptide (CNP; Kd = 80.4 nM), but not by C-ANF. Also, [125I]Tyr-CNP bound to uterine membranes was completely competed by unlabeled CNP (Kd = 1.12 nM). Cross-linking of [125I]ANP to uterine membranes revealed the presence of one band of 130 kilodaltons, corresponding to the guanylyl cyclase (GC-A and/or GC-B) subtypes of natriuretic peptide receptors. The presence of messenger RNA coding for genes of both GC-A and GC-B receptors was shown by quantitative reverse transcriptase polymerase chain reaction. Furthermore, ANP and, to a lesser degree, CNP stimulated the production of cGMP in rat uterus. Autoradiographic studies localized the highest binding of [125I]ANP in the endometrium, whereas [125I]Tyr-CNP binding was distributed in the endometrium as well as in the myometrium. These results demonstrate that rat uterine ANP receptors are of the guanylyl cyclase-coupled subtypes. The uterus is a target of natriuretic peptides where ANP induces its biological effects through the production of cGMP.

Age/race differences in HER2 testing and in incidence rates for breast cancer triple subtypes: a population-based study and first report.

PubMed

Lund, Mary Jo; Butler, Ebonee N; Hair, Brionna Y; Ward, Kevin C; Andrews, Judy H; Oprea-Ilies, Gabriella; Bayakly, A Rana; O'Regan, Ruth M; Vertino, Paula M; Eley, J William

2010-06-01

Although US year 2000 guidelines recommended characterizing breast cancers by human epidermal growth factor receptor 2 (HER2), national cancer registries do not collect HER2, rendering a population-based understanding of HER2 and clinical "triple subtypes" (estrogen receptor [ER] / progesterone receptor [PR] / HER2) largely unknown. We document the population-based prevalence of HER2 testing / status, triple subtypes and present the first report of subtype incidence rates. Medical records were searched for HER2 on 1842 metropolitan Atlanta females diagnosed with breast cancer during 2003-2004. HER2 testing/status and triple subtypes were analyzed by age, race/ethnicity, tumor factors, socioeconomic status, and treatment. Age-adjusted incidence rates were calculated. Over 90% of cases received HER2 testing: 12.6% were positive, 71.7% negative, and 15.7% unknown. HER2 testing compliance was significantly better for women who were younger, of Caucasian or African-American descent, or diagnosed with early stage disease. Incidence rates (per 100,000) were 21.1 for HER2+ tumors and 27.8 for triple-negative tumors, the latter differing by race (36.3 and 19.4 for black and white women, respectively). HER2 recommendations are not uniformly adhered to. Incidence rates for breast cancer triple subtypes differ by age/race. As biologic knowledge is translated into the clinical setting eg, HER2 as a biomarker, it will be incumbent upon national cancer registries to report this information. Incidence rates cautiously extrapolate to an annual burden of 3000 and 17,000 HER2+ tumors for black and white women, respectively, and triple-negative tumors among 5000 and 16,000 respectively. Testing, rate, and burden variations warrant population-based in-depth exploration and clinical translation. (c) 2010 American Cancer Society.

Botulinum neurotoxin D-C uses synaptotagmin I and II as receptors, and human synaptotagmin II is not an effective receptor for type B, D-C and G toxins.

PubMed

Peng, Lisheng; Berntsson, Ronnie P-A; Tepp, William H; Pitkin, Rose M; Johnson, Eric A; Stenmark, Pål; Dong, Min

2012-07-01

Botulinum neurotoxins (BoNTs) are classified into seven types (A-G), but multiple subtype and mosaic toxins exist. These subtype and mosaic toxins share a high sequence identity, and presumably the same receptors and substrates with their parental toxins. Here, we report that a mosaic toxin, type D-C (BoNT/D-C), uses different receptors from its parental toxin BoNT/C. BoNT/D-C, but not BoNT/C, binds directly to the luminal domains of synaptic vesicle proteins synaptotagmin (Syt) I and II, and requires expression of SytI/II to enter neurons. The SytII luminal fragment containing the toxin-binding site can block the entry of BoNT/D-C into neurons and reduce its toxicity in vivo in mice. We also found that gangliosides increase binding of BoNT/D-C to SytI/II and enhance the ability of the SytII luminal fragment to block BoNT/D-C entry into neurons. These data establish SytI/II, in conjunction with gangliosides, as the receptors for BoNT/D-C, and indicate that BoNT/D-C is functionally distinct from BoNT/C. We further found that BoNT/D-C recognizes the same binding site on SytI/II where BoNT/B and G also bind, but utilizes a receptor-binding interface that is distinct from BoNT/B and G. Finally, we also report that human and chimpanzee SytII has diminished binding and function as the receptor for BoNT/B, D-C and G owing to a single residue change from rodent SytII within the toxin binding site, potentially reducing the potency of these BoNTs in humans and chimpanzees.

Male breast cancer according to tumor subtype and race: a population-based study.

PubMed

Chavez-Macgregor, Mariana; Clarke, Christina A; Lichtensztajn, Daphne; Hortobagyi, Gabriel N; Giordano, Sharon H

2013-05-01

Breast cancer occurs rarely in men. To the authors' knowledge, no population-based estimates of the incidence of human epidermal growth factor receptor 2 (HER2)-positive breast cancer or of the distribution of breast cancer subtypes among male breast cancer patients have been published to date. Therefore, the objective of the current study was to explore breast tumor subtype distribution by race/ethnicity among men in the large, ethnically diverse population of California. This study included men who were diagnosed with invasive breast cancer between 2005 and 2009 with known estrogen receptor (ER) and progesterone receptor (PR) (together, hormone receptor [HR]) status and HER2 status reported to the California Cancer Registry. Among the men with HR-positive tumors, survival probabilities between groups were compared using log-rank tests. Six hundred six patients were included. The median age at diagnosis was 68 years. Four hundred ninety-four men (81.5%) had HR-positive tumors (defined as ER-positive and/or PR-positive and HER2-negative). Ninety men (14.9%) had HER2-positive tumors, and 22 (3.6%) had triple receptor-negative (TN) tumors. Among the patients with HR-positive tumors, non-Hispanic black men and Hispanic men were more likely to have PR-negative tumors than non-Hispanic white men. No statistically significant differences in survival were observed according to tumor subtype (P = .08). Differences in survival according to race/ethnicity were observed among all patients (P = .087) and among those with HR-positive tumors (P = .0170), and non-Hispanic black men had poorer outcomes. In this large, representative cohort of men with breast cancer, the distribution of tumor subtypes was different from that reported for women and varied by patient race/ethnicity. Non-Hispanic black men were more likely to have TN tumors and ER-positive/PR-negative tumors than white men. Copyright © 2013 American Cancer Society.

Molecular evolution of hepatitis C virus in China: A nationwide study.

PubMed

Huang, K; Chen, J; Xu, R; Jiang, X; Ma, X; Jia, M; Wang, M; Huang, J; Liao, Q; Shan, Z; Dailey, C; Song, X; Lu, L; Li, C; Rong, X; Zhang, M; Fu, Y

2018-03-01

The evolutionary and epidemic history and the regional differences of hepatitis C virus (HCV) are complex and remain unclear in the vast territory China. Here we recruited 1540 HCV-RNA positive patients sampled in 29 provinces across whole China, which is the largest sample capacity and the most comprehensive geographic coverage of China to our knowledge. 1b, 2a, 3b, 6a and 3a were the major subtypes in China. 1b was the most predominant subtype which presented in every province. The second most predominant subtype, 2a, appeared to concentrate in the north of China. Subtypes 3a and 3b were mainly found in the Southwest region, while 6a was restricted in the South region. We further estimated the origins of the dominating subtypes and discovered for the first time that a Chinese-specific transmission pattern for some strains of subtype 2a which was restricted in north China, and Chinese subtype 3b originated from Thailand. Copyright © 2018 Elsevier Inc. All rights reserved.

Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

PubMed

Lee, Jonathan K; Garbe, James C; Vrba, Lukas; Miyano, Masaru; Futscher, Bernard W; Stampfer, Martha R; LaBarge, Mark A

2015-01-01

Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

D2 dopaminergic and 5-HT1A serotonergic activity of 2-(1-naphthyl)ethyl- and 2-(2-naphthyl)ethyl amines.

PubMed

Šukalović, V; Roglić, G; Husinec, S; Kostić-Rajaćić, S; Andrić, D; Šoškić, Vukić

2003-11-01

Several tertiary 2-phenylethyl, 2-(1-naphthyl)ethyl and 2-(2-naphthyl)ethyl amines were synthesized and their binding affinities for dopamine D(1), D(2) and serotonin 5-HT(1A) receptors evaluated in radioligand binding assays. All compounds were inactive in D(1) dopamine radioligand binding assay. The 2-(1-naphthyl)ethyl analogues expressed a low but significant binding affinity for the D(2) and moderate one for the 5-HT(1A) receptor subtypes. Most of the remaining compounds expressed binding affinity at the 5-HT(1A) receptor subtype but were inactive in D(2) receptor binding assay. Based on these results and considering the chemical characteristics of the compounds synthesized and evaluated for dopaminergic and serotonergic activity throughout the present study it can be concluded that hydrophobic type of interaction (stacking or edge-to-face) plays a significant role in the formation of receptor-ligand complexes of 2-(1-naphthyl)ethyl amines. This structural motive can be applied to design and synthesize new, more potent dopaminergic/serotonergic ligands by slight chemical modifications.

Synergistic Action of Presynaptic Muscarinic Acetylcholine Receptors and Adenosine Receptors in Developmental Axonal Competition at the Neuromuscular Junction.

PubMed

Nadal, Laura; Garcia, Neus; Hurtado, Erica; Simó, Anna; Tomàs, Marta; Lanuza, Maria Angel; Cilleros, Victor; Tomàs, Josep Maria

2016-01-01

The development of the nervous system involves the initial overproduction of synapses, which promotes connectivity. Hebbian competition between axons with different activities leads to the loss of roughly half of the overproduced elements and this refines connectivity. We used quantitative immunohistochemistry to investigate, in the postnatal day 7 (P7) to P9 neuromuscular junctions, the involvement of muscarinic receptors (muscarinic acetylcholine autoreceptors and the M1, M2, and M4 subtypes) and adenosine receptors (A1 and A2A subtypes) in the control of axonal elimination after the mouse levator auris longus muscle had been exposed to selective antagonists in vivo. In a previous study we analyzed the role of each of the individual receptors. Here we investigate the additive or occlusive effects of their inhibitors and thus the existence of synergistic activity between the receptors. The main results show that the A2A, M1, M4, and A1 receptors (in this order of ability) delayed axonal elimination at P7. M4 produces some occlusion of the M1 pathway and some addition to the A1 pathway, which suggests that they cooperate. M2 receptors may modulate (by allowing a permissive action) the other receptors, mainly M4 and A1. The continued action of these receptors (now including M2 but not M4) finally promotes axonal loss at P9. All 4 receptors (M2, M1, A1, and A2A, in this order of ability) are necessary. The M4 receptor (which in itself does not affect axon loss) seems to modulate the other receptors. We found a synergistic action between the M1, A1, and A2A receptors, which show an additive effect, whereas the potent M2 effect is largely independent of the other receptors (though can be modulated by M4). At P9, there is a full mutual dependence between the A1 and A2A receptors in regulating axon loss. In summary, postnatal axonal elimination is a regulated multireceptor mechanism that involves the cooperation of several muscarinic and adenosine receptor subtypes. © 2017 S. Karger AG, Basel.

NMDA receptor subunits and associated signaling molecules mediating antidepressant-related effects of NMDA-GluN2B antagonism

PubMed Central

Kiselycznyk, Carly; Jury, Nicholas; Halladay, Lindsay; Nakazawa, Kazu; Mishina, Masayoshi; Sprengel, Rolf; Grant, Seth G.N.; Svenningsson, Per; Holmes, Andrew

2015-01-01

Drugs targeting the glutamate N-methyl-D-aspartate receptor (NMDAR) may be efficacious for treating mood disorders, as exemplified by the rapid antidepressant effects produced by single administration of the NMDAR antagonist ketamine. Though the precise mechanisms underlying the antidepressant-related effects of NMDAR antagonism remain unclear, recent studies implicate specific NMDAR subunits, including GluN2A and GluN2B, as well as the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) subunit glutamate receptor interacting molecule, PSD-95. Here, integrating mutant and pharmacological in mice, we investigated the contribution of these subunits and molecules to antidepressant-related behaviors and the antidepressant-related effects of the GluN2B blocker, Ro 25-6981. We found that global deletion of GluA1 or PSD-95 reduced forced swim test (FST) immobility, mimicking the antidepressant-related effect produced by systemically administered Ro 25-6981 in C57BL/6J mice. Moreover, the FST antidepressant-like effects of systemic Ro 25-6981 were intact in mutants with global GluA1 deletion or GluN1 deletion in forebrain interneurons, but were absent in mutants constitutively lacking GluN2A or PSD-95. Next, we found that microinfusing Ro 25-6981 into the medial prefrontal cortex (mPFC), but not basolateral amygdala, of C57BL/6J mice was sufficient to produce an antidepressant-like effect. Together, these findings extend and refine current understanding of the mechanisms mediating antidepressant-like effects produced by NMDAR-GluN2B antagonists, and may inform the development of a novel class of medications for treating depression that target the GluN2B subtype of NMDAR. PMID:25800971

Emergence of canine parvovirus subtype 2b (CPV-2b) infections in Australian dogs.

PubMed

Clark, Nicholas J; Seddon, Jennifer M; Kyaw-Tanner, Myat; Al-Alawneh, John; Harper, Gavin; McDonagh, Phillip; Meers, Joanne

2018-03-01

Tracing the temporal dynamics of pathogens is crucial for developing strategies to detect and limit disease emergence. Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs around the globe. Previous work in Australia reported that the majority of cases were associated with the CPV-2a subtype, an unexpected finding since CPV-2a was rapidly replaced by another subtype (CPV-2b) in many countries. Using a nine-year dataset of CPV-2 infections from 396 dogs sampled across Australia, we assessed the population dynamics and molecular epidemiology of circulating CPV-2 subtypes. Bayesian phylogenetic Skygrid models and logistic regressions were used to trace the temporal dynamics of CPV-2 infections in dogs sampled from 2007 to 2016. Phylogenetic models indicated that CPV-2a likely emerged in Australia between 1973 and 1988, while CPV-2b likely emerged between 1985 and 1998. Sequences from both subtypes were found in dogs across continental Australia and Tasmania, with no apparent effect of climate variability on subtype occurrence. Both variant subtypes exhibited a classical disease emergence pattern of relatively high rates of evolution during early emergence followed by subsequent decreases in evolutionary rates over time. However, the CPV-2b subtype maintained higher mutation rates than CPV-2a and continued to expand, resulting in an increase in the probability that dogs will carry this subtype over time. Ongoing monitoring programs that provide molecular epidemiology surveillance will be necessary to detect emergence of new variants and make informed recommendations to develop reliable detection and vaccine methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Molecular and functional properties of P2X receptors--recent progress and persisting challenges.

PubMed

Kaczmarek-Hájek, Karina; Lörinczi, Eva; Hausmann, Ralf; Nicke, Annette

2012-09-01

ATP-gated P2X receptors are trimeric ion channels that assemble as homo- or heteromers from seven cloned subunits. Transcripts and/or proteins of P2X subunits have been found in most, if not all, mammalian tissues and are being discovered in an increasing number of non-vertebrates. Both the first crystal structure of a P2X receptor and the generation of knockout (KO) mice for five of the seven cloned subtypes greatly advanced our understanding of their molecular and physiological function and their validation as drug targets. This review summarizes the current understanding of the structure and function of P2X receptors and gives an update on recent developments in the search for P2X subtype-selective ligands. It also provides an overview about the current knowledge of the regulation and modulation of P2X receptors on the cellular level and finally on their physiological roles as inferred from studies on KO mice.

Lapatinib–induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors

PubMed Central

2013-01-01

Introduction Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Methods Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Results Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. Conclusions These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients. PMID:24216290

Lapatinib-induced NF-kappaB activation sensitizes triple-negative breast cancer cells to proteasome inhibitors.

PubMed

Chen, Yun-Ju; Yeh, Ming-Hsin; Yu, Meng-Chieh; Wei, Ya-Ling; Chen, Wen-Shu; Chen, Jhen-Yu; Shih, Chih-Yu; Tu, Chih-Yen; Chen, Chia-Hung; Hsia, Te-Chun; Chien, Pei-Hsuan; Liu, Shu-Hui; Yu, Yung-Luen; Huang, Wei-Chien

2013-11-12

Triple-negative breast cancer (TNBC), a subtype of breast cancer with negative expressions of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), is frequently diagnosed in younger women and has poor prognosis for disease-free and overall survival. Due to the lack of known oncogenic drivers for TNBC proliferation, clinical benefit from currently available targeted therapies is limited, and new therapeutic strategies are urgently needed. Triple-negative breast cancer cell lines were treated with proteasome inhibitors in combination with lapatinib (a dual epidermal growth factor receptor (EGFR)/HER2 tyrosine kinase inhibitor). Their in vitro and in vivo viability was examined by MTT assay, clonogenic analysis, and orthotopic xenograft mice model. Luciferase reporter gene, immunoblot, and RT-qPCR, immunoprecipitation assays were used to investigate the molecular mechanisms of action. Our data showed that nuclear factor (NF)-κB activation was elicited by lapatinib, independent of EGFR/HER2 inhibition, in TNBCs. Lapatinib-induced constitutive activation of NF-κB involved Src family kinase (SFK)-dependent p65 and IκBα phosphorylations, and rendered these cells more vulnerable to NF-κB inhibition by p65 small hairpin RNA. Lapatinib but not other EGFR inhibitors synergized the anti-tumor activity of proteasome inhibitors both in vitro and in vivo. Our results suggest that treatment of TNBCs with lapatinib may enhance their oncogene addiction to NF-κB, and thus augment the anti-tumor activity of proteasome inhibitors. These findings suggest that combination therapy of a proteasome inhibitor with lapatinib may benefit TNBC patients.

Epidemiological risk factors associated with inflammatory breast cancer subtypes.

PubMed

Atkinson, Rachel L; El-Zein, Randa; Valero, Vicente; Lucci, Anthony; Bevers, Therese B; Fouad, Tamer; Liao, Weiqin; Ueno, Naoto T; Woodward, Wendy A; Brewster, Abenaa M

2016-03-01

In this single-institution case-control study, we identified risk factors associated with inflammatory breast cancer (IBC) subtypes based on staining of estrogen receptor (ER), progesterone receptor (PR) and expression of human epidermal growth factor 2 (HER2neu) to determine distinct etiologic pathways. We identified 224 women with IBC and 396 cancer-free women seen at the MD Anderson Cancer Center. Multinomial logistic regression was used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for associations between breast cancer risk factors and the IBC tumor subtypes: luminal (ER+ and/or PR+/HER2neu-), HER2neu+ (any ER and PR, HER2neu+), and triple-negative (ER-/PR-/HER2neu-). In multivariable analysis, compared with women age ≥26 at first pregnancy, women age <26 had a higher risk of triple-negative IBC (OR 3.32, 95% CI 1.37-8.05). Women with a history of breast-feeding had a lower risk of triple-negative (OR 0.30; 95% CI 0.15-0.62) and luminal IBC (OR 0.35, 95% CI 0.18-0.68). A history of smoking was associated with an increased risk of luminal IBC (OR 2.37; 95% CI 1.24-4.52). Compared with normal-weight women, those who were overweight or obese (body mass index ≥25 kg/m(2)) had a higher risk of all three tumor subtypes (p < 0.01 for all subtypes). Overweight or obese status is important modifiable risk factor for IBC of any subtype. Modifiable risk factors, age at first pregnancy (≥26), breast-feeding, and smoking may be associated with specific IBC subtypes. These results highlight the importance of evaluating epidemiologic risk factors for IBC for the identification of subtype-specific prevention strategies.

Endothelin-1 and its receptors on haemorrhoidal tissue: a potential site for therapeutic intervention.

PubMed

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

2017-04-01

Haemorrhoids is a common anorectal condition affecting millions worldwide. We have studied the effect of endothelin-1 (ET-1) and the role of endothelin ET A and ET B receptors in haemorrhoid tissue. Protein expression of ET-1, ET A and ET B receptors were compared between haemorrhoids and normal rectal submucosa using Western blot analysis, with the localization of proteins determined by autoradiography and immunohistochemistry. Effects of ET-1 and sarafotoxin 6a on human colonic and rectal arteries and veins was assessed by wire myography and the involvement of receptor subtypes established by selective antagonists. Dense binding of [ 125 I]-ET-1 to haemorrhoidal sections was reduced by selective receptor antagonists. A higher density of ET B than ET A receptors was found in haemorrhoidal, than in control rectal tissue and confirmed by Western blot analysis. ET A and ET B receptors were localized to smooth muscle of haemorrhoidal arteries and veins, with ET B receptors on the endothelium. Human colonic and rectal arteries and veins were similarly sensitive to ET-1 and affected by the ET A selective antagonist, but sarafotoxin S6a-induced contractions were more pronounced in veins and antagonized by a selective ET B receptor antagonist. ET A and ET B receptors are present in human haemorrhoids with ET B receptors predominating. ET A receptors are activated by ET-1 to mediate a contraction in arteries and veins, but the latter are selectively activated by sarafotoxin S6a - a response that involves ET B receptors at low concentrations. Selective ET B agonists may have therapeutic potential to reduce congestion of the haemorrhoidal venous sinusoids. © 2017 The British Pharmacological Society.

Brain Serotonin Receptors and Transporters: Initiation vs. Termination of Escalated Aggression

PubMed Central

Takahashi, Aki; Quadros, Isabel M.; de Almeida, Rosa M. M.; Miczek, Klaus A.

2013-01-01

Rationale Recent findings have shown a complexly regulated 5-HT system as it is linked to different kinds of aggression. Objective We focus on (1) phasic and tonic changes of 5-HT and (2) state and trait of aggression, and emphasize the different receptor subtypes, their role in specific brain regions, feed-back regulation and modulation by other amines, acids and peptides. Results New pharmacological tools differentiate the first three 5-HT receptor families and their modulation by GABA, glutamate and CRF. Activation of 5-HT1A, 5-HT1B and 5-HT2A/2C receptors in mesocorticolimbic areas, reduce species-typical and other aggressive behaviors. In contrast, agonists at 5-HT1A and 5-HT1B receptors in the medial prefrontal cortex or septal area can increase aggressive behavior under specific conditions. Activation of serotonin transporters reduce mainly pathological aggression. Genetic analyses of aggressive individuals have identified several molecules that affect the 5-HT system directly (e.g., Tph2, 5-HT1B, 5-HT transporter, Pet1, MAOA) or indirectly (e.g., Neuropeptide Y, αCaMKII, NOS, BDNF). Dysfunction in genes for MAOA escalates pathological aggression in rodents and humans, particularly in interaction with specific experiences. Conclusions Feedback to autoreceptors of the 5-HT1 family and modulation via heteroreceptors are important in the expression of aggressive behavior. Tonic increase of the 5-HT2 family expression may cause escalated aggression, whereas the phasic increase of 5-HT2 receptors inhibits aggressive behaviors. Polymorphisms in the genes of 5-HT transporters or rate-limiting synthetic and metabolic enzymes of 5-HT modulate aggression, often requiring interaction with the rearing environment. PMID:20938650

Structure and Dynamics of the M3 Muscarinic Acetylcholine Receptor

PubMed Central

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.; Arlow, Daniel H.; Rosenbaum, Daniel M.; Rosemond, Erica; Green, Hillary F.; Liu, Tong; Chae, Pil Seok; Dror, Ron O.; Shaw, David E.; Weis, William I.; Wess, Jurgen; Kobilka, Brian

2012-01-01

Acetylcholine (ACh), the first neurotransmitter to be identified1, exerts many of its physiological actions via activation of a family of G protein-coupled receptors (GPCRs) known as muscarinic ACh receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G protein coupling preference and the physiological responses they mediate.2–4 Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences.5–6 We describe here the structure of the Gq/11-coupled M3 mAChR bound to the bronchodilator drug tiotropium and identify the binding mode for this clinically important drug. This structure, together with that of the Gi/o-coupled M2 receptor, offers new possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows the first structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and raise additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer new insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors. PMID:22358844

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists

PubMed Central

Hiranita, Takato; Kopajtic, Theresa A.; Rice, Kenner C.; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H.; McCurdy, Christopher R.

2016-01-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. PMID:27189970

Adrenoceptors in Brain: Cellular Gene Expression and Effects on Astrocytic Metabolism and [Ca2+]i

PubMed Central

Hertz, Leif; Lovatt, Ditte; Goldman, Steven A.; Nedergaard, Maiken

2010-01-01

Recent in vivo studies have established astrocytes as a major target for locus coeruleus activation (Bekar et al., Cereb. Cortex 18, 2789–2795), renewing interest in cell culture studies on noradrenergic effects on astrocytes in primary cultures and calling for additional information about the expression of adrenoceptor subtypes on different types of brain cells. In the present communication, mRNA expression of α1-, α2- and β-adrenergic receptors and their subtypes was determined in freshly-isolated, cell marker-defined populations of astrocytes, NG2-positive cells, microglia, endothelial cells, and Thy1-positive neurons (mainly glutamatergic projection neurons) in murine cerebral cortex. Immediately after dissection of frontal, parietal and occipital cortex of 10–12-week-old transgenic mice, which combined each cell-type marker with a specific fluorescent signal, the tissue was digested, triturated and centrifuged, yielding a solution of dissociated cells of all types, which were separated by fluorescence-activated cell sorting (FACS). mRNA expression in each cell fraction was determined by microarray analysis. α1A-Receptors were unequivocally expressed in astrocytes and NG2-positive cells, but absent in other cell types, and α1B-receptors were not expressed in any cell population. Among α2-receptors only α2A-receptors were expressed, unequivocally in astrocytes and NG-positive cells, tentatively in microglia and questionably in Thy1-positive neurons and endothelial cells. β1-Receptors were unequivocally expressed in astrocytes, tentatively in microglia, and questionably in neurons and endothelial cells, whereas β2-adrenergic receptors showed tentative expression in neurons and astrocytes and unequivocal expression in other cell types. This distribution was supported by immunochemical data and its relevance established by previous studies in well-differentiated primary cultures of mouse astrocytes, showing that stimulation of α2-adrenoceptors increases glycogen formation and oxidative metabolism, the latter by a mechanism depending on intramitochondrial Ca2+, whereas α1-adrenoceptor stimulation enhances glutamate uptake, and β-adrenoceptor activation causes glycogenolysis and increased Na+,K+-ATPase activity. The Ca2+- and cAMP-mediated association between energy-consuming and energy-yielding processes is emphasized. PMID:20380860

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

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

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

Structure and Receptor Binding of the Hemagglutinin from a Human H6N1 Influenza Virus

DOE PAGES

Tzarum, Netanel; de Vries, Robert P.; Zhu, Xueyong; ...

2015-03-11

Avian influenza viruses that cause infection and are transmissible in humans involve changes in the receptor binding site (RBS) of the viral hemagglutinin (HA) that alter receptor preference from α2-3-linked (avian-like) to α2-6-linked (human-like) sialosides. A human case of avian-origin H6N1 influenza virus was recently reported, but the molecular mechanisms contributing to it crossing the species barrier are unknown. We find that, although the H6 HA RBS contains D190V and G228S substitutions that potentially promote human receptor binding, recombinant H6 HA preferentially binds α2-3-linked sialosides, indicating no adaptation to human receptors. Crystal structures of H6 HA with avian and humanmore » receptor analogs reveal that H6 HA preferentially interacts with avian receptor analogs. Lastly, this binding mechanism differs from other HA subtypes due to a unique combination of RBS residues, highlighting additional variation in HA-receptor interactions and the challenges in predicting which influenza strains and subtypes can infect humans and cause pandemics.« less

GABAA receptor subtype involvement in addictive behaviour.

PubMed

Stephens, D N; King, S L; Lambert, J J; Belelli, D; Duka, T

2017-01-01

GABA A receptors form the major class of inhibitory neurotransmitter receptors in the mammalian brain. This review sets out to summarize the evidence that variations in genes encoding GABA A receptor isoforms are associated with aspects of addictive behaviour in humans, while animal models of addictive behaviour also implicate certain subtypes of GABA A receptor. In addition to outlining the evidence for the involvement of specific subtypes in addiction, we summarize the particular contributions of these isoforms in control over the functioning of brain circuits, especially the mesolimbic system, and make a first attempt to bring together evidence from several fields to understanding potential involvement of GABA A receptor subtypes in addictive behaviour. While the weight of the published literature is on alcohol dependency, the underlying principles outlined are relevant across a number of different aspects of addictive behaviour. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross-linkers

PubMed Central

Tian, Meilin; Ye, Shixin

2016-01-01

Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology. PMID:27713495

Adjuvant Trastuzumab in HER2-Positive Early Breast Cancer by Age and Hormone Receptor Status: A Cost-Utility Analysis.

PubMed

Leung, William; Kvizhinadze, Giorgi; Nair, Nisha; Blakely, Tony

2016-08-01

The anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab improves outcomes in patients with node-positive HER2+ early breast cancer. Given trastuzumab's high cost, we aimed to estimate its cost-effectiveness by heterogeneity in age and estrogen receptor (ER) and progesterone receptor (PR) status, which has previously been unexplored, to assist prioritisation. A cost-utility analysis was performed using a Markov macro-simulation model, with a lifetime horizon, comparing a 12-mo regimen of trastuzumab with chemotherapy alone using the latest (2014) effectiveness measures from landmark randomised trials. A New Zealand (NZ) health system perspective was adopted, employing high-quality national administrative data. Incremental quality-adjusted life-years for trastuzumab versus chemotherapy alone are two times higher (2.33 times for the age group 50-54 y; 95% CI 2.29-2.37) for the worst prognosis (ER-/PR-) subtype compared to the best prognosis (ER+/PR+) subtype, causing incremental cost-effectiveness ratios (ICERs) for the former to be less than half those of the latter for the age groups from 25-29 to 90-94 y (0.44 times for the age group 50-54 y; 95% CI 0.43-0.45). If we were to strictly apply an arbitrary cost-effectiveness threshold equal to the NZ gross domestic product per capita (2011 purchasing power parity [PPP]-adjusted: US$30,300; €23,700; £21,200), our study suggests that trastuzumab (2011 PPP-adjusted US$45,400/€35,900/£21,900 for 1 y at formulary prices) may not be cost-effective for ER+ (which are 61% of all) node-positive HER2+ early breast cancer patients but cost-effective for ER-/PR- subtypes (37% of all cases) to age 69 y. Market entry of trastuzumab biosimilars will likely reduce the ICER to below this threshold for premenopausal ER+/PR- cancer but not for ER+/PR+ cancer. Sensitivity analysis using the best-case effectiveness measure for ER+ cancer had the same result. A key limitation was a lack of treatment-effect data by hormone receptor subtype. Heterogeneity was restricted to age and hormone receptor status; tumour size/grade heterogeneity could be explored in future work. This study highlights how cost-effectiveness can vary greatly by heterogeneity in age and hormone receptor subtype. Resource allocation and licensing of subsidised therapies such as trastuzumab should consider demographic and clinical heterogeneity; there is currently a profound disconnect between how funding decisions are made (largely agnostic to heterogeneity) and the principles of personalised medicine.

Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies

PubMed Central

Lehmann, Brian D.; Bauer, Joshua A.; Chen, Xi; Sanders, Melinda E.; Chakravarthy, A. Bapsi; Shyr, Yu; Pietenpol, Jennifer A.

2011-01-01

Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem–like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted “driver” signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies. PMID:21633166

Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

PubMed

Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

2002-08-09

Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

Gene expression profiling assigns CHEK2 1100delC breast cancers to the luminal intrinsic subtypes.

PubMed

Nagel, Jord H A; Peeters, Justine K; Smid, Marcel; Sieuwerts, Anieta M; Wasielewski, Marijke; de Weerd, Vanja; Trapman-Jansen, Anita M A C; van den Ouweland, Ans; Brüggenwirth, Hennie; van I Jcken, Wilfred F J; Klijn, Jan G M; van der Spek, Peter J; Foekens, John A; Martens, John W M; Schutte, Mieke; Meijers-Heijboer, Hanne

2012-04-01

CHEK2 1100delC is a moderate-risk cancer susceptibility allele that confers a high breast cancer risk in a polygenic setting. Gene expression profiling of CHEK2 1100delC breast cancers may reveal clues to the nature of the polygenic CHEK2 model and its genes involved. Here, we report global gene expression profiles of a cohort of 155 familial breast cancers, including 26 CHEK2 1100delC mutant tumors. In line with previous work, all CHEK2 1100delC mutant tumors clustered among the hormone receptor-positive breast cancers. In the hormone receptor-positive subset, a 40-gene CHEK2 signature was subsequently defined that significantly associated with CHEK2 1100delC breast cancers. The identification of a CHEK2 gene signature implies an unexpected biological homogeneity among the CHEK2 1100delC breast cancers. In addition, all 26 CHEK2 1100delC tumors classified as luminal intrinsic subtype breast cancers, with 8 luminal A and 18 luminal B tumors. This biological make-up of CHEK2 1100delC breast cancers suggests that a relatively limited number of additional susceptibility alleles are involved in the polygenic CHEK2 model. Identification of these as-yet-unknown susceptibility alleles should be aided by clues from the 40-gene CHEK2 signature.

[(3)H]8-Ethyl-4-methyl-2-phenyl-(8R)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]-purin-5-one ([(3)H]PSB-11), a novel high-affinity antagonist radioligand for human A(3) adenosine receptors.

PubMed

Müller, Christa E; Diekmann, Martina; Thorand, Mark; Ozola, Vita

2002-02-11

This study describes the preparation and binding properties of [(3)H]PSB-11, a novel, potent, and selective antagonist radioligand for human A(3) adenosine receptors (ARs). [(3)H]PSB-11 binding to membranes of Chinese hamster ovary (CHO) cells expressing the human A(3) AR was saturable and reversible. Saturation experiments showed that [(3)H]PSB-11 labeled a single class of binding sites with high affinity (K(D)=4.9 nM) and limited capacity (B(max)=3500 fmol/mg of protein). PSB-11 is highly selective versus the other adenosine receptor subtypes. The new radioligand shows an extraordinarily low degree of non-specific binding rendering it a very useful tool for studying the (patho)physiological roles of A(3 )ARs.

Muscarinic receptor immunoreactivity in the superior salivatory nucleus neurons innervating the salivary glands of the rat.

PubMed

Ueda, Hirotaka; Mitoh, Yoshihiro; Fujita, Masako; Kobashi, Motoi; Yamashiro, Takashi; Sugimoto, Tomosada; Ichikawa, Hiroyuki; Matsuo, Ryuji

2011-07-15

The superior salivatory nucleus (SSN) contains preganglionic parasympathetic neurons to the submandibular and sublingual salivary glands. Cevimeline, a muscarinic acetylcholine receptor agonist, stimulates the salivary glands and is presently used as sialogogue in the treatment of dry mouth. Since cevimeline passes through the blood-brain barrier, it is also able to act on muscarinic acetylcholine receptors in the central nervous system. Our preliminary experiment using the whole-cell patch-clamp technique has shown that cevimeline excites SSN neurons in rat brain slices, suggesting that SSN neurons have muscarinic acetylcholine receptors; however, it is unclear which subtypes of muscarinic acetylcholine receptors exist in SSN neurons. In the present study, we investigated immunohistochemically muscarinic acetylcholine receptor subtypes, M1 receptor (M1R), M2R, M3R, M4R, and M5R in SSN neurons. SSN neurons innervating the salivary glands, retrogradely labeled with a fluorescent tracer from the chorda-lingual nerve, mostly expressed M3R immunoreactivity (-ir) (92.3%) but not M1R-ir. About half of such SSN neurons also showed M2R- (40.1%), M4R- (54.0%) and M5R-ir (46.0%); therefore, it is probable that SSN neurons co-express M3R-ir with at least two of the other muscarinic receptor subtypes. This is the first report to show that SSN neurons contain muscarinic acetylcholine receptors. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Hormone-Related Pathways and Risk of Breast Cancer Subtypes in African American Women

PubMed Central

Haddad, Stephen A.; Lunetta, Kathryn L.; Ruiz-Narváez, Edward A.; Bensen, Jeannette T.; Hong, Chi-Chen; Sucheston-Campbell, Lara E.; Yao, Song; Bandera, Elisa V.; Rosenberg, Lynn; Haiman, Christopher A.; Troester, Melissa A.; Ambrosone, Christine B.; Palmer, Julie R.

2016-01-01

Purpose We sought to investigate genetic variation in hormone pathways in relation to risk of overall and subtype-specific breast cancer in women of African ancestry (AA). Methods Genotyping and imputation yielded data on 143,934 SNPs in 308 hormone-related genes for 3663 breast cancer cases (1098 ER-, 1983 ER+, 582 ER unknown) and 4687 controls from the African American Breast Cancer Epidemiology and Risk (AMBER) Consortium. AMBER includes data from four large studies of AA women: the Carolina Breast Cancer Study, the Women's Circle of Health Study, the Black Women's Health Study, and the Multiethnic Cohort Study. Pathway- and gene-based analyses were conducted, and single SNP tests were run for the top genes. Results There were no strong associations at the pathway level. The most significantly associated genes were GHRH, CALM2, CETP, and AKR1C1 for overall breast cancer (gene-based nominal p ≤0.01); NR0B1, IGF2R, CALM2, CYP1B1, and GRB2 for ER+ breast cancer (p ≤0.02); and PGR, MAPK3, MAP3K1, and LHCGR for ER- disease (p ≤0.02). Single-SNP tests for SNPs with pairwise linkage disequilibrium r2 <0.8 in the top genes identified 12 common SNPs (in CALM2, CETP, NR0B1, IGF2R, CYP1B1, PGR, MAPK3, and MAP3K1) associated with overall or subtype-specific breast cancer after gene-level correction for multiple testing. Rs11571215 in PGR (progesterone receptor) was the SNP most strongly associated with ER- disease. Conclusion We identified eight genes in hormone pathways that contain common variants associated with breast cancer in AA women after gene-level correction for multiple testing. PMID:26458823

An immunocapture/scintillation proximity analysis of G alpha q/11 activation by native serotonin (5-HT)2A receptors in rat cortex: blockade by clozapine and mirtazapine.

PubMed

Mannoury La Cour, C; Chaput, C; Touzard, M; Millan, M J

2009-02-01

Though transduction mechanisms recruited by heterologously expressed 5-HT(2A) receptors have been extensively studied, their interaction with specific subtypes of G-protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5-HT, the prototypical 5-HT(2A) agonist, DOI, and Ro60,0175 all enhanced [(35)S]GTPgammaS binding to G alpha q/11 in rat cortex with pEC(50) values of 6.22, 7.24 and 6.35, respectively. No activation of G o or G s/olf was seen at equivalent concentrations of DOI. Stimulation of G alpha q/11 by 5-HT (30 microM) and DOI (30 microM) was abolished by the selective 5-HT(2A) vs. 5-HT(2C)/5-HT(2B) antagonists, ketanserin (pK(B) values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5-HT-induced [(35)S]GTPgammaS binding to G alpha q/11 was only weakly inhibited by the preferential 5-HT(2C) receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5-HT(2B) receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5-HT(2A) receptors, blocked the recruitment of G alpha q/11 by 5-HT and DOI with pK(B) values of 8.54 and 8.14, respectively. Its actions were mimicked by the "atypical" antidepressant and 5-HT(2A) receptor antagonist, mirtazapine, which likewise blocked 5-HT and DOI-induced G alpha q/11 protein activation with pK(B) values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5-HT(2A) receptors in rat frontal cortex specifically recruit G alpha q/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5-HT(2A) receptor-mediated G alpha q/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent. 2008 Wiley-Liss, Inc.

Temperature induced variation in gene expression of thyroid hormone receptors and deiodinases of European eel (Anguilla anguilla) larvae.

PubMed

Politis, S N; Servili, A; Mazurais, D; Zambonino-Infante, J-L; Miest, J J; Tomkiewicz, J; Butts, I A E

2018-04-01

Thyroid hormones (THs) are key regulators of growth, development, and metabolism in vertebrates and influence early life development of fish. TH is produced in the thyroid gland (or thyroid follicles) mainly as T4 (thyroxine), which is metabolized to T3 (3,5,3'-triiodothyronine) and T2 (3,5-diiodothyronine) by deiodinase (DIO) enzymes in peripheral tissues. The action of these hormones is mostly exerted by binding to a specific nuclear thyroid hormone receptor (THR). In this study, we i) cloned and characterized thr sequences, ii) investigated the expression pattern of the different subtypes of thrs and dios, and iii) studied how temperature affects the expression of those genes in artificially produced early life history stages of European eel (Anguilla anguilla), reared in different thermal regimes (16, 18, 20 and 22 °C) from hatch until first-feeding. We identified 2 subtypes of thr (thrα and thrβ) with 2 isoforms each (thrαA, thrαB, thrβA, thrβB) and 3 subtypes of deiodinases (dio1, dio2, dio3). All thr genes identified showed high similarity to the closely related Japanese eel (Anguilla japonica). We found that all genes investigated in this study were affected by larval age (in real time or at specific developmental stages), temperature, and/or their interaction. More specifically, the warmer the temperature the earlier the expression response of a specific target gene. In real time, the expression profiles appeared very similar and only shifted with temperature. In developmental time, gene expression of all genes differed across selected developmental stages, such as at hatch, during teeth formation or at first-feeding. Thus, we demonstrate that thrs and dios show sensitivity to temperature and are involved in and during early life development of European eel. Copyright © 2017 Elsevier Inc. All rights reserved.

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

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

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

The adenosine A2A receptor (A2AR) has long been implicated in cardiovascular disorders. As more selective A2AR ligands are being identified, its roles in other disorders, such as Parkinson’s disease, are starting to emerge, and A2AR antagonists are important drug candidates for nondopaminergic anti-Parkinson treatment. Here we report the crystal structure of A2A receptor bound to compound 1 (Cmpd-1), a novel A2AR/N-methyl D-aspartate receptor subtype 2B (NR2B) dual antagonist and potential anti-Parkinson candidate compound, at 3.5 Å resolution. The A2A receptor with a cytochrome b562-RIL (BRIL) fusion (A2AR–BRIL) in the intracellular loop 3 (ICL3) was crystallized in detergent micelles using vapor-phasemore » diffusion. Whereas A2AR–BRIL bound to the antagonist ZM241385 has previously been crystallized in lipidic cubic phase (LCP), structural differences in the Cmpd-1–bound A2AR–BRIL prevented formation of the lattice observed with the ZM241385–bound receptor. The crystals grew with a type II crystal lattice in contrast to the typical type I packing seen from membrane protein structures crystallized in LCP. Cmpd-1 binds in a position that overlaps with the native ligand adenosine, but its methoxyphenyl group extends to an exosite not previously observed in other A2AR structures. Structural analysis revealed that Cmpd-1 binding results in the unique conformations of two tyrosine residues, Tyr91.35 and Tyr2717.36, which are critical for the formation of the exosite. The structure reveals insights into antagonist binding that are not observed in other A2AR structures, highlighting flexibility in the binding pocket that may facilitate the development of A2AR-selective compounds for the treatment of Parkinson’s disease.« less

Dopamine D3 and D2 Receptor Mechanisms in the Abuse-Related Behavioral Effects of Cocaine: Studies with Preferential Antagonists in Squirrel Monkeys

PubMed Central

Grundt, Peter; Cao, Jianjing; Platt, Donna M.; Newman, Amy Hauck; Spealman, Roger D.

2010-01-01

Dopamine (DA) D3 and D2 receptor mechanisms are implicated in cocaine's abuse-related behavioral effects, but the relative contribution of the two receptor subtypes is only partially characterized. This study investigated the role of D3 and D2 subtype mechanisms by determining the degree to which the D3-preferring antagonist PG01037 [N-{4-[4-(2,3-dichlorophenyl)-piperazin- 1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl] and the D2-preferring antagonist L-741626 [3-[4-(4-chlorophenyl)-4- hydroxypiperidin-1-yl]methyl-1H-indole] attenuated several behavioral effects of cocaine in squirrel monkeys. Quantitative observational studies established doses of each antagonist that did not produce untoward effects, which were used in subsequent comparisons. In addition, the ability of the D3-preferring agonist PD128907 [(R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol)] and the D2-preferring agonist sumanirole [(R)-5,6-dihydro-5-(methylamino)-4H- imidazo[4,5,1-ij]quinolin-2(1H)-one(Z)-2-butenedioate] to reproduce cocaine's discriminative stimulus (DS) and priming effects were compared. In monkeys trained to discriminate cocaine from vehicle, both DA antagonists attenuated and both DA agonists partially reproduced cocaine's DS effects. PG01037 also selectively attenuated the cocaine-like DS effects of PD128907, whereas L-741626 attenuated the cocaine-like DS effects of both agonists. In self-administration studies, L-741626 nonselectively reduced cocaine- and food-maintained responding, whereas PG01037 was ineffective against either reinforcer. In studies involving reinstatement of extinguished cocaine seeking, both antagonists attenuated cocaine-induced reinstatement of responding, and both agonists induced at least partial reinstatement of cocaine seeking. L-741626 also attenuated sumanirole-induced, but not PD128907-induced, reinstatement of responding, whereas PG01037 was ineffective against either DA agonist. The results are consistent with a role for D3 and D2 receptor mechanisms in cocaine's DS effects and cocaine-induced reinstatement of drug seeking, but provide no evidence for a major role of D3 receptors in the direct reinforcing effects of cocaine. PMID:20494958

Expression Analysis of Dopamine Receptor Subtypes in Normal Human Pituitaries, Nonfunctioning Pituitary Adenomas and Somatotropinomas, and the Association between Dopamine and Somatostatin Receptors with Clinical Response to Octreotide-LAR in Acromegaly

PubMed Central

Neto, Leonardo Vieira; Machado, Evelyn de O.; Luque, Raul M.; Taboada, Giselle F.; Marcondes, Jorge B.; Chimelli, Leila M. C.; Quintella, Leonardo Pereira; Niemeyer, Paulo; de Carvalho, Denise P.; Kineman, Rhonda D.; Gadelha, Mônica R.

2009-01-01

Context: Dopamine receptor (DR) and somatostatin receptor subtype expression in pituitary adenomas may predict the response to postsurgical therapies. Objectives: Our objectives were to assess and compare the mRNA levels of DR1-5 and somatostatin receptors 1–5 in normal pituitaries (NPs), nonfunctioning pituitary adenomas (NFPAs), and somatotropinomas. In addition, we determined whether the level of DR expression correlates with the in vivo response to octreotide-LAR in acromegalic patients. Design and Patients: Eight NPs, 30 NFPAs, and 39 somatotropinomas were analyzed for receptor mRNA levels by real-time RT-PCR. The DR2 short variant was estimated as the DR2 long/DR2 total (DR2T). The relationship between DR expression and the postsurgical response to octreotide-LAR was assessed in 19 of the acromegalic patients. Results: DR3 was not detected. The relationship between expression levels of DR subtypes in NPs and somatotropinomas was DR2T⋙DR4≫DR5>DR1, whereas in NFPAs, DR2T⋙DR4≫DR1>DR5. The DR2 short variant was the predominant DR2 variant in the majority of samples. In acromegalics treated with octreotide-LAR, DR1 was negatively correlated with percent GH reduction (3 months: r = −0.67, P = 0.002; and 6 months: r = −0.58, P = 0.009), and DR5 was positively correlated with percent IGF-I reduction (3 months: r = 0.55, P = 0.01; and 6 months: r = 0.47, P = 0.04). Conclusions: DR2 is the predominant DR subtype in NPs, NFPAs, and somatotropinomas. The fact that DR1, DR4, and DR5 are also expressed in many adenomas tested suggests that these receptors might also play a role in the therapeutic impact of postsurgical medical therapies in patients with NFPA and acromegaly. This was supported by the finding that the in vivo response to octreotide-LAR was negatively associated with DR1 and positively associated with DR5. PMID:19293270

Selective mode of action of guanidine-containing non-peptides at human NPFF receptors.

PubMed

Findeisen, Maria; Würker, Cäcilia; Rathmann, Daniel; Meier, René; Meiler, Jens; Olsson, Roger; Beck-Sickinger, Annette G

2012-07-12

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By use of four nonpeptidic compounds and the peptide mimetics RF9 and BIBP3226, agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated. The binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF(1) but not in the NPFF(2) receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system.

Selective mode of action of guanidine-containing non-peptides at human NPFF receptors

PubMed Central

Findeisen, Maria; Würker, Cäcilia; Rathmann, Daniel; Meier, René; Meiler, Jens; Olsson, Roger; Beck-Sickinger, Annette G.

2012-01-01

The binding pocket of both NPFF receptors was investigated, focusing on subtype-selective behavior. By using four non-peptidic compounds and the peptide mimetics RF9 and BIBP3226 agonistic and antagonistic properties were characterized. A set of Ala receptor mutants was generated, the binding pocket was narrowed down to the upper part of transmembrane helices V, VI, VII, and the extracellular loop 2. Positions 5.27 and 6.59 have been shown to have a strong impact on receptor activation and were suggested to form an acidic, negatively charged binding pocket in both NPFF receptor subtypes. Additionally, position 7.35 was identified to play an important role in functional selectivity. According to docking experiments, the aryl group of AC-216 interacts with position 7.35 in the NPFF1 but not in the NPFF2 receptor. These results provide distinct insights into the receptor specific binding pockets, which is necessary for the development of drugs to address the NPFF system. PMID:22708927

Naftopidil inhibits 5-hydroxytryptamine-induced bladder contraction in rats.

PubMed

Sakai, Takumi; Kasahara, Ken-ichi; Tomita, Ken-ichi; Ikegaki, Ichiro; Kuriyama, Hiroshi

2013-01-30

Naftopidil is an α(1D) and α(1A) subtype-selective α(1)-adrenoceptor antagonist that has been used to treat lower urinary tract symptoms of benign prostatic hyperplasia. In this study, we investigated the effects of naftopidil on 5-hydroxytryptamine (5-HT)-induced rat bladder contraction (10(-8)-10(-4) M). Naftopidil (0.3, 1, and 3 μM) inhibited 5-HT-induced bladder contraction in a concentration-dependent manner. On the other hand, other α(1)-adrenoceptor antagonists, tamsulosin, silodosin or prazosin, did not inhibit 5-HT-induced bladder contraction. The 5-HT-induced bladder contraction was inhibited by both ketanserin and 4-(4-fluoronaphthalen-1-yl)-6-propan-2-ylpyrimidin-2-amine (RS127445), serotonin 5-HT(2A) and 5-HT(2B) receptor antagonists, respectively. In addition, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and α-methyl-5-HT, 5-HT(2A) and 5-HT(2) receptor agonists, respectively, induced bladder contraction. The 5-HT-induced bladder contraction was not inhibited by N-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]-N-pyridin-2-yl-cyclohexanecarboxamide (WAY-100635), [1-[2[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl-1-methyl-1H-indole-3-carboxylate (GR113808) or (R)-3-[2-[2-(4-methylpiperidin-1-yl)ethyl]pyrrolidine-1-sulphonyl]phenol (SB269970), 5-HT(1A), 5-HT(4) and 5-HT(7) receptor antagonists, respectively. Naftopidil inhibited both the 5-HT(2A) and 5-HT(2) receptor agonists-induced bladder contractions. Naftopidil binds to the human 5-HT(2A) and 5-HT(2B) receptors with pKi values of 6.55 and 7.82, respectively. These results suggest that naftopidil inhibits 5-HT-induced bladder contraction via blockade of the 5-HT(2A) and 5-HT(2B) receptors in rats. Furthermore, 5-HT-induced bladder contraction was enhanced in bladder strips obtained from bladder outlet obstructed rats, with this contraction inhibited by naftopidil. The beneficial effects of naftopidil on storage symptoms such as urinary frequency and nocturia in patients with benign prostatic hyperplasia may be due, in part, to the blockade of the 5-HT(2A) and 5-HT(2B) receptors in the bladder. Copyright © 2012 Elsevier B.V. All rights reserved.

CRF19_cpx is an Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba

PubMed Central

Kouri, Vivian; Khouri, Ricardo; Alemán‬, Yoan; Abrahantes, Yeissel; Vercauteren, Jurgen; Pineda-Peña, Andrea-Clemencia; Theys, Kristof; Megens, Sarah; Moutschen, Michel; Pfeifer, Nico; Van Weyenbergh, Johan; Pérez, Ana B.; Pérez, Jorge; Pérez, Lissette; Van Laethem, Kristel; Vandamme, Anne-Mieke

2015-01-01

Background Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba. Methods Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining. Findings Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis. Interpretation CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba. PMID:26137563

CRF19_cpx is an Evolutionary fit HIV-1 Variant Strongly Associated With Rapid Progression to AIDS in Cuba.

PubMed

Kouri, Vivian; Khouri, Ricardo; Alemán, Yoan; Abrahantes, Yeissel; Vercauteren, Jurgen; Pineda-Peña, Andrea-Clemencia; Theys, Kristof; Megens, Sarah; Moutschen, Michel; Pfeifer, Nico; Van Weyenbergh, Johan; Pérez, Ana B; Pérez, Jorge; Pérez, Lissette; Van Laethem, Kristel; Vandamme, Anne-Mieke

2015-03-01

Clinicians reported an increasing trend of rapid progression (RP) (AIDS within 3 years of infection) in Cuba. Recently infected patients were prospectively sampled, 52 RP at AIDS diagnosis (AIDS-RP) and 21 without AIDS in the same time frame (non-AIDS). 22 patients were sampled at AIDS diagnosis (chronic-AIDS) retrospectively assessed as > 3 years infected. Clinical, demographic, virological, epidemiological and immunological data were collected. Pol and env sequences were used for subtyping, transmission cluster analysis, and prediction of resistance, co-receptor use and evolutionary fitness. Host, immunological and viral predictors of RP were explored through data mining. Subtyping revealed 26 subtype B strains, 6 C, 6 CRF18_cpx, 9 CRF19_cpx, 29 BG-recombinants and other subtypes/URFs. All patients infected with CRF19 belonged to the AIDS-RP group. Data mining identified CRF19, oral candidiasis and RANTES levels as the strongest predictors of AIDS-RP. CRF19 was more frequently predicted to use the CXCR4 co-receptor, had higher fitness scores in the protease region, and patients had higher viral load at diagnosis. CRF19 is a recombinant of subtype D (C-part of Gag, PR, RT and nef), subtype A (N-part of Gag, Integrase, Env) and subtype G (Vif, Vpr, Vpu and C-part of Env). Since subtypes D and A have been associated with respectively faster and slower disease progression, our findings might indicate a fit PR driving high viral load, which in combination with co-infections may boost RANTES levels and thus CXCR4 use, potentially explaining the fast progression. We propose that CRF19 is evolutionary very fit and causing rapid progression to AIDS in many newly infected patients in Cuba.

Low gene expression levels of activating receptors of natural killer cells (NKG2E and CD94) in patients with fulminant type 1 diabetes.

PubMed

Nakata, Shinsuke; Imagawa, Akihisa; Miyata, Yugo; Yoshikawa, Atsushi; Kozawa, Junji; Okita, Kohei; Funahashi, Tohru; Nakamura, Seiji; Matsubara, Kenichi; Iwahashi, Hiromi; Shimomura, Iichiro

2013-01-01

Fulminant type 1 diabetes is an independent subtype of type 1 diabetes characterized by extremely rapid onset and absence of islet-related autoantibodies. However, detailed pathophysiology of this subtype is poorly understood. In this study, a comprehensive approach was applied to understand the pathogenesis of fulminant type 1 diabetes. We determined the genes that were differentially expressed in fulminant type 1 diabetes compared with type 1A diabetes and healthy control, using gene expression microarray in peripheral blood cells. Using volcano plot analysis, we found reduced expression of killer cell lectin-like receptor subfamily C, member 3 (KLRC3) which encodes NKG2E, a natural killer (NK) cell activating receptor, in fulminant type 1 diabetes, compared with healthy controls. This difference was confirmed by real-time RT-PCR among NK-enriched cells. The expression of KLRD1 (CD94), which forms heterodimer with NKG2E (KLRC3), was also reduced in NK-enriched cells in fulminant type 1 diabetes. Furthermore, flow cytometry showed significantly lower proportion of NK cells among peripheral blood mononuclear cells (PBMCs) in fulminant type 1 diabetes than in healthy controls. In patients with fulminant type 1 diabetes, the relative proportion of NK cells correlated significantly with the time period between onset of fever to the appearance of hyperglycemic-related symptoms. We conclude the presence of reduced NK activating receptor gene expression and low proportion of NK cells in fulminant type 1 diabetes. Copyright © 2013 Elsevier B.V. All rights reserved.

Collection and Testing of Respiratory Samples

ClinicalTrials.gov

2017-04-03

QIAGEN ResPlex II Advanced Panel; Influenza A; Respiratory Syncytial Virus Infections; Infection Due to Human Parainfluenza Virus 1; Parainfluenza Type 2; Parainfluenza Type 3; Parainfluenza Type 4; Human Metapneumovirus A/B; Rhinovirus; Coxsackie Virus/Echovirus; Adenovirus Types B/C/E; Coronavirus Subtypes 229E; Coronavirus Subtype NL63; Coronavirus Subtype OC43; Coronavirus Subtype HKU1; Human Bocavirus; Artus Influenza A/B RT-PCR Test; Influenza B

The N-terminal domain of GluR6-subtype glutamate receptor ion channels

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

Kumar, Janesh; Schuck, Peter; Jin, Rongsheng

2009-09-25

The amino-terminal domain (ATD) of glutamate receptor ion channels, which controls their selective assembly into AMPA, kainate and NMDA receptor subtypes, is also the site of action of NMDA receptor allosteric modulators. Here we report the crystal structure of the ATD from the kainate receptor GluR6. The ATD forms dimers in solution at micromolar protein concentrations and crystallizes as a dimer. Unexpectedly, each subunit adopts an intermediate extent of domain closure compared to the apo and ligand-bound complexes of LIVBP and G protein-coupled glutamate receptors (mGluRs), and the dimer assembly has a markedly different conformation from that found in mGluRs.more » This conformation is stabilized by contacts between large hydrophobic patches in the R2 domain that are absent in NMDA receptors, suggesting that the ATDs of individual glutamate receptor ion channels have evolved into functionally distinct families.« less

Notch signaling is a potent inducer of growth arrest and apoptosis in a wide range of B-cell malignancies

PubMed Central

Zweidler-McKay, Patrick A.; He, Yiping; Xu, Lanwei; Rodriguez, Carlos G.; Karnell, Fredrick G.; Carpenter, Andrea C.; Aster, Jon C.; Allman, David; Pear, Warren S.

2005-01-01

Although Notch receptor expression on malignant B cells is widespread, the effect of Notch signaling in these cells is poorly understood. To investigate Notch signaling in B-cell malignancy, we assayed the effect of Notch activation in multiple murine and human B-cell tumors, representing both immature and mature subtypes. Expression of constitutively active, truncated forms of the 4 mammalian Notch receptors (ICN1-4) inhibited growth and induced apoptosis in both murine and human B-cell lines but not T-cell lines. Similar results were obtained in human precursor B-cell acute lymphoblastic leukemia lines when Notch activation was achieved by coculture with fibroblasts expressing the Notch ligands Jagged1 or Jagged2. All 4 truncated Notch receptors, as well as the Jagged ligands, induced Hes1 transcription. Retroviral expression of Hairy/Enhancer of Split-1 (Hes1) recapitulated the Notch effects, suggesting that Hes1 is an important mediator of Notch-induced growth arrest and apoptosis in B cells. Among the B-cell malignancies that were susceptible to Notch-mediated growth inhibition/apoptosis were mature B-cell and therapy-resistant B-cell malignancies, including Hodgkin, myeloma, and mixed-lineage leukemia (MLL)–translocated cell lines. These results suggest that therapies capable of activating Notch/Hes1 signaling may have therapeutic potential in a wide range of human B-cell malignancies. PMID:16118316

Lysophosphatidic Acid Signals through Multiple Receptors in Osteoclasts to Elevate Cytosolic Calcium Concentration, Evoke Retraction, and Promote Cell Survival*

PubMed Central

Lapierre, Danielle M.; Tanabe, Natsuko; Pereverzev, Alexey; Spencer, Martha; Shugg, Ryan P. P.; Dixon, S. Jeffrey; Sims, Stephen M.

2010-01-01

Lysophosphatidic acid (LPA) is a bioactive phospholipid whose functions are mediated by multiple G protein-coupled receptors. We have shown that osteoblasts produce LPA, raising the possibility that it mediates intercellular signaling among osteoblasts and osteoclasts. Here we investigated the expression, signaling and function of LPA receptors in osteoclasts. Focal application of LPA elicited transient increases in cytosolic calcium concentration ([Ca2+]i), with 50% of osteoclasts responding at ∼400 nm LPA. LPA-induced elevation of [Ca2+]i was blocked by pertussis toxin or the LPA1/3 receptor antagonist VPC-32183. LPA caused sustained retraction of osteoclast lamellipodia and disrupted peripheral actin belts. Retraction was insensitive to VPC-32183 or pertussis toxin, indicating involvement of a distinct signaling pathway. In this regard, inhibition of Rho-associated kinase stimulated respreading after LPA-induced retraction. Real-time reverse transcription-PCR revealed transcripts encoding LPA1 and to a lesser extent LPA2, LPA4, and LPA5 receptor subtypes. LPA induced nuclear translocation of NFATc1 and enhanced osteoclast survival, effects that were blocked by VPC-32183 or by a specific peptide inhibitor of NFAT activation. LPA slightly reduced the resorptive activity of osteoclasts in vitro. Thus, LPA binds to at least two receptor subtypes on osteoclasts: LPA1, which couples through Gi/o to elevate [Ca2+]i, activate NFATc1, and promote survival, and a second receptor that likely couples through G12/13 and Rho to evoke and maintain retraction through reorganization of the actin cytoskeleton. These findings reveal a signaling axis in bone through which LPA, produced by osteoblasts, acts on multiple receptor subtypes to induce pleiotropic effects on osteoclast activity and function. PMID:20551326

Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes

PubMed Central

Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R.

2016-01-01

In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947–1961], 43 from France [1986–2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2′) A5(B3′), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements. PMID:27189984

The role of α1-adrenergic receptors in regulating metabolism: increased glucose tolerance, leptin secretion and lipid oxidation.

PubMed

Shi, Ting; Papay, Robert S; Perez, Dianne M

2017-04-01

The role of α 1 -adrenergic receptors (α 1 -ARs) and their subtypes in metabolism is not well known. Most previous studies were performed before the advent of transgenic mouse models and utilized transformed cell lines and poorly selective antagonists. We have now studied the metabolic regulation of the α 1A - and α 1B -AR subtypes in vivo using knock-out (KO) and transgenic mice that express a constitutively active mutant (CAM) form of the receptor, assessing subtype-selective functions. CAM mice increased glucose tolerance while KO mice display impaired glucose tolerance. CAM mice increased while KO decreased glucose uptake into white fat tissue and skeletal muscle with the CAM α 1A -AR showing selective glucose uptake into the heart. Using indirect calorimetry, both CAM mice demonstrated increased whole body fatty acid oxidation, while KO mice preferentially oxidized carbohydrate. CAM α 1A -AR mice displayed significantly decreased fasting plasma triglycerides and glucose levels while α 1A -AR KO displayed increased levels of triglycerides and glucose. Both CAM mice displayed increased plasma levels of leptin while KO mice decreased leptin levels. Most metabolic effects were more efficacious with the α 1A -AR subtype. Our results suggest that stimulation of α 1 -ARs results in a favorable metabolic profile of increased glucose tolerance, cardiac glucose uptake, leptin secretion and increased whole body lipid metabolism that may contribute to its previously recognized cardioprotective and neuroprotective benefits.

Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid.

PubMed

Einsiedel, Jürgen; Weber, Klaus; Thomas, Christoph; Lehmann, Thomas; Hübner, Harald; Gmeiner, Peter

2003-10-06

Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.

Inhibition of Nuclear Transcription Factor-κB and Activation of Peroxisome Proliferator-Activated Receptors in HepG2 Cells by Cucurbitane-Type Triterpene Glycosides from Momordica charantia

PubMed Central

Nhiem, Nguyen Xuan; Yen, Pham Hai; Ngan, Nguyen Thi Thanh; Quang, Tran Hong; Kiem, Phan Van; Minh, Chau Van; Tai, Bui Huu; Cuong, Nguyen Xuan; Song, Seok Bean

2012-01-01

Abstract Momordica charantia: is used to treat various diseases, including inflammatory conditions. Previous reports indicated that the extract of this plant inhibits activation of nuclear transcription factor-κB (NF-κB) but activates peroxisome proliferator-activated receptor (PPAR). Additionally, cucurbitane-type triterpene glycosides are the main bioactive components of the fruit of M. charantia. Therefore, we investigated the anti-inflammatory activity of 17 cucurbitane-type triterpene glycosides (1–17) isolated from this plant. Their inhibition of NF-κB and activation of PPAR activities in HepG2 cells were measured using luciferase reporter and PPAR subtype transactivation assays. Compounds 6 and 8 were found to inhibit NF-κB activation stimulated by tumor necrosis factor-α (TNFα) in a dose-dependent manner. With 50% inhibition concentration (IC50) values of 0.4 μM, compounds 6 and 8 were more potent inhibitors than the positive control, sulfasalazine (IC50=0.9 μM). Compounds 4, 6, and 8 also inhibited TNFα-induced expressions of inducible nitric oxide synthase and cyclooxygenase-2 mRNA. However, only compound 13 significantly increased PPARγ transactivation. PMID:22248180

Detection of hepatitis C virus subtypes 6a, 6n, 6w and mixed infections using a modified multiplex real-time polymerase chain reaction protocol.

PubMed

Lee, Yuan-Ming; Chen, Yen-Ju; Lee, Cheng-Ming; Kuo, Lou-Hui; Wong, Wing-Wai; Chen, Yi-Ming Arthur

2011-12-01

In the past few years, many new subtypes in hepatitis C virus (HCV) genotype 6 have been identified. The aim of this study was to modify the multiplex real-time polymerase chain reaction (RT-PCR) protocol and use it to determine the HCV subtypes of a group of Taiwanese injection drug users (IDUs). We used 76 serum specimens collected in northern Taiwan in 2008. Multiplex RT-PCR was used for HCV subtyping among those serum samples having anti-HCV antibodies. Twenty cases were randomly selected for comparison with subtyping results from Inno-LiPa II tests and phylogenetic tree analysis using NS5B sequences. Multiplex RT-PCR assays showed that 60.5% (46/76) of IDUs had single HCV infection. Three out of 76 (3.9%) had double HCV infection (1b/6a, 2a/2b and 2b/6a). Besides this, 27.6% (21/76) had no HCV signal. One IDU had subtype 6n and two had subtype 6w infection. Inno-LiPa II tests misclassified all 6n and 6w cases as 1b subtype. Our modified multiplex RT-PCR protocol can be used to support molecular epidemiological studies and laboratory diagnoses of different HCV subtypes including genotype 6. Copyright © 2011. Published by Elsevier B.V.

Differential manipulation of arrestin-3 binding to basal and agonist-activated G protein-coupled receptors.

PubMed

Prokop, Susanne; Perry, Nicole A; Vishnivetskiy, Sergey A; Toth, Andras D; Inoue, Asuka; Milligan, Graeme; Iverson, Tina M; Hunyady, Laszlo; Gurevich, Vsevolod V

2017-08-01

Non-visual arrestins interact with hundreds of different G protein-coupled receptors (GPCRs). Here we show that by introducing mutations into elements that directly bind receptors, the specificity of arrestin-3 can be altered. Several mutations in the two parts of the central "crest" of the arrestin molecule, middle-loop and C-loop, enhanced or reduced arrestin-3 interactions with several GPCRs in receptor subtype and functional state-specific manner. For example, the Lys139Ile substitution in the middle-loop dramatically enhanced the binding to inactive M 2 muscarinic receptor, so that agonist activation of the M 2 did not further increase arrestin-3 binding. Thus, the Lys139Ile mutation made arrestin-3 essentially an activation-independent binding partner of M 2 , whereas its interactions with other receptors, including the β 2 -adrenergic receptor and the D 1 and D 2 dopamine receptors, retained normal activation dependence. In contrast, the Ala248Val mutation enhanced agonist-induced arrestin-3 binding to the β 2 -adrenergic and D 2 dopamine receptors, while reducing its interaction with the D 1 dopamine receptor. These mutations represent the first example of altering arrestin specificity via enhancement of the arrestin-receptor interactions rather than selective reduction of the binding to certain subtypes. Copyright © 2017. Published by Elsevier Inc.

Tachykinin receptors mediating airway marcomolecular secretion

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

Gentry, S.E.

1991-01-01

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

Potencies of agonists acting at tachykinin receptors in the oestrogen-primed rat uterus: effects of peptidase inhibitors.

PubMed

Fisher, L; Pennefather, J N

1997-09-24

The uterotonic potencies of the naturally occurring mammalian tachykinins and the synthetic subtype-selective agonist analogues of these agents [Lys5,MeLeu9,Nlel0]neurokinin A-(4-10) and [Nle10]neurokinin A-(4-10) (tachykinin NK2 receptor-selective), [Sar9,Met(O2)11]substance P (tachykinin NK1 receptor-selective) and senktide (tachykinin NK3 receptor-selective) were determined using preparations from oestradiol-treated rats. The endopeptidase 24.11 inhibitor, N-[N-[1-(S)-carboxyl-3-phenylpropyl]-(S)-phenyl-alanyl-(S)-isoserine+ ++ (SCH 39370), potentiated responses to neurokinin A, neurokinin B and substance P, but not to [Lys5,MeLeu9,Nle10)]neurokinin A-(4-10) or senktide. [Nle10]neurokinin A-(4-10) effects were potentiated by SCH 39370 with amastatin and those to [Sar9,Met(O2)11]substance P were potentiated by SCH 39370 and captopril in combination. In the presence of optimal concentrations of peptidase inhibitors the relative order of agonist potency was: neurokinin A > substance P > neurokinin B for the naturally occurring mammalian tachykinins and [Lys5,MeLeu9,Nle10]neurokinin A-(4-10) > [Nle10]neurokinin A-(4-10) > [Sar9,Met(O2)11]substance P > senktide for the synthetic tachykinin analogues. Thus, while a tachykinin NK2 receptor predominates in the oestrogen-primed uterus, a tachykinin NK1 receptor may also be present. The non-peptide tachykinin NK3 receptor antagonist, SR 142801, did not antagonise the effects of senktide suggesting that tachykinin NK3 receptors do not mediate its relatively minor effect on the uterus of the oestrogen-primed rat.

Modulation of 3H-noradrenaline release by presynaptic opioid, cannabinoid and bradykinin receptors and β-adrenoceptors in mouse tissues

PubMed Central

Trendelenburg, A U; Cox, S L; Schelb, V; Klebroff, W; Khairallah, L; Starke, K

2000-01-01

Release-modulating opioid and cannabinoid (CB) receptors, β-adrenoceptors and bradykinin receptors at noradrenergic axons were studied in mouse tissues (occipito-parietal cortex, heart atria, vas deferens and spleen) preincubated with 3H-noradrenaline. Experiments using the OP1 receptor-selective agonists DPDPE and DSLET, the OP2-selective agonists U50488H and U69593, the OP3-selective agonist DAMGO, the ORL1 receptor-selective agonist nociceptin, and a number of selective antagonists showed that the noradrenergic axons innervating the occipito-parietal cortex possess release-inhibiting OP3 and ORL1 receptors, those innervating atria OP1, ORL1 and possibly OP3 receptors, and those innervating the vas deferens all four opioid receptor types. Experiments using the non-selective CB agonists WIN 55,212-2 and CP 55,940 and the CB1-selective antagonist SR 141716A indicated that the noradrenergic axons of the vas deferens possess release-inhibiting CB1 receptors. Presynaptic CB receptors were not found in the occipito-parietal cortex, in atria or in the spleen. Experiments using the non-selective β-adrenoceptor agonist isoprenaline and the β2-selective agonist salbutamol, as well as subtype-selective antagonists, demonstrated the occurrence of release-enhancing β2-adrenoceptors at the sympathetic axons of atria and the spleen, but demonstrated their absence in the occipito-parietal cortex and the vas deferens. Experiments with bradykinin and the B2-selective antagonist Hoe 140 showed the operation of release-enhancing B2 receptors at the sympathetic axons of atria, the vas deferens and the spleen, but showed their absence in the occipito-parietal cortex. The experiments document a number of new presynaptic receptor locations. They confirm and extend the existence of marked tissue and species differences in presynaptic receptors at noradrenergic neurons. PMID:10807669

Bombyx neuropeptide G protein-coupled receptor A7 is the third cognate receptor for short neuropeptide F from silkworm.

PubMed

Ma, Qiang; Cao, Zheng; Yu, Yena; Yan, Lili; Zhang, Wenjuan; Shi, Ying; Zhou, Naiming; Huang, Haishan

2017-12-15

The short neuropeptide F (sNPF) neuropeptides, closely related to vertebrate neuropeptide Y (NPY), have been suggested to exert pleiotropic effects on many physiological processes in insects. In the silkworm ( Bombyx mori ) two orphan G protein-coupled receptors, Bombyx neuropeptide G protein-coupled receptor (BNGR) A10 and A11, have been identified as cognate receptors for sNPFs, but other sNPF receptors and their signaling mechanisms in B. mori remain unknown. Here, we cloned the full-length cDNA of the orphan receptor BNGR-A7 from the brain of B. mori larvae and identified it as a receptor for Bombyx sNPFs. Further characterization of signaling and internalization indicated that BNGR-A7, -A10, and -A11 are activated by direct interaction with synthetic Bombyx sNPF-1 and -3 peptides. This activation inhibited forskolin or adipokinetic hormone-induced adenylyl cyclase activity and intracellular Ca 2+ mobilization via a G i/o -dependent pathway. Upon activation by sNPFs, BNGR-A7, -A10, and -A11 evoked ERK1/2 phosphorylation and underwent internalization. On the basis of these findings, we designated the receptors BNGR-A7, -A10, and -A11 as Bommo -sNPFR-1, -2, and -3, respectively. Moreover, the results obtained with quantitative RT-PCR analysis revealed that the three Bombyx sNPF receptor subtypes exhibit differential spatial and temporal expression patterns, suggesting possible roles of sNPF signaling in the regulation of a wide range of biological processes. Our findings provide the first in-depth information on sNPF signaling for further elucidation of the roles of the Bombyx sNPF/sNPFR system in the regulation of physiological activities. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Alpha1- and alpha2-containing GABAA receptor modulation is not necessary for benzodiazepine-induced hyperphagia.

PubMed

Morris, H V; Nilsson, S; Dixon, C I; Stephens, D N; Clifton, P G

2009-06-01

Benzodiazepines increase food intake, an effect attributed to their ability to enhance palatability. We investigated which GABA(A) receptor subtypes may be involved in mediating benzodiazepine-induced hyperphagia. The role of the alpha2 subtype was investigated by observing the effects of midazolam, on the behavioural satiety sequence in mice with targeted deletion of the alpha2 gene (alpha2 knockout). Midazolam (0.125, 0.25 and 0.5mg/kg) increased food intake and the amount of time spent feeding in alpha2 knockout mice, suggesting that BZ-induced hyperphagia does not involve alpha2-containing GABA(A) receptors. We further investigated the roles of alpha1- and alpha3-containing GABA(A) receptors in mediating BZ-induced hyperphagia. We treated alpha2(H101R) mice, in which alpha2-containing receptors are rendered benzodiazepine insensitive, with L-838417, a compound which acts as a partial agonist at alpha2-, alpha3- and alpha5-receptors but is inactive at alpha1-containing receptors. L-838417 (10 and 30 mg/kg) increased food intake and the time spent feeding in both wildtype and alpha2(H101R) mice, demonstrating that benzodiazepine-induced hyperphagia does not require alpha1- and alpha2-containing GABA(A) receptors. These observations, together with evidence against the involvement of alpha5-containing GABA(A) receptors, suggest that alpha3-containing receptors mediate BZ-induced hyperphagia in the mouse.

Dopamine Receptor-Specific Contributions to the Computation of Value.

PubMed

Burke, Christopher J; Soutschek, Alexander; Weber, Susanna; Raja Beharelle, Anjali; Fehr, Ernst; Haker, Helene; Tobler, Philippe N

2018-05-01

Dopamine is thought to play a crucial role in value-based decision making. However, the specific contributions of different dopamine receptor subtypes to the computation of subjective value remain unknown. Here we demonstrate how the balance between D1 and D2 dopamine receptor subtypes shapes subjective value computation during risky decision making. We administered the D2 receptor antagonist amisulpride or placebo before participants made choices between risky options. Compared with placebo, D2 receptor blockade resulted in more frequent choice of higher risk and higher expected value options. Using a novel model fitting procedure, we concurrently estimated the three parameters that define individual risk attitude according to an influential theoretical account of risky decision making (prospect theory). This analysis revealed that the observed reduction in risk aversion under amisulpride was driven by increased sensitivity to reward magnitude and decreased distortion of outcome probability, resulting in more linear value coding. Our data suggest that different components that govern individual risk attitude are under dopaminergic control, such that D2 receptor blockade facilitates risk taking and expected value processing.

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

PubMed

Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

2017-10-16

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

P2X purinergic receptor ligands: recently patented compounds.

PubMed

Gunosewoyo, Hendra; Kassiou, Michael

2010-05-01

P2X channels are ionotropic purinergic receptors that are currently under scrutiny as attractive targets for novel therapeutics in areas including chronic inflammation, pain and depression. Their wide expression in the CNS, recent advances in the biochemical and pharmacological properties as well as increasing numbers of patents published in this research domain demand a review in this field. The patent literature covering novel drug-like antagonists for each P2X receptor subtype (P2X1R to P2X7R) up to December 2009 is described in this review article together with their recent highlights in pharmacology. Readers will gain an up-to-date overview of patents covering drug-like antagonists for seven P2X receptor subtypes within the last 4 years. P2X7R antagonists and other P2X inhibitors will probably be on the market for combating rheumatoid arthritis and other diseases. Some P2X7R antagonists are already in Phase I and II clinical trials.

Allosteric Modulation of Metabotropic Glutamate Receptors

PubMed Central

Sheffler, Douglas J.; Gregory, Karen J.; Rook, Jerri M.; Conn, P. Jeffrey

2013-01-01

The development of receptor subtype-selective ligands by targeting allosteric sites of G protein-coupled receptors (GPCRs) has proven highly successful in recent years. One GPCR family that has greatly benefited from this approach is the metabotropic glutamate receptors (mGlus). These family C GPCRs participate in the neuromodulatory actions of glutamate throughout the CNS, where they play a number of key roles in regulating synaptic transmission and neuronal excitability. A large number of mGlu subtype-selective allosteric modulators have been identified, the majority of which are thought to bind within the transmembrane regions of the receptor. These modulators can either enhance or inhibit mGlu functional responses and, together with mGlu knockout mice, have furthered the establishment of the physiologic roles of many mGlu subtypes. Numerous pharmacological and receptor mutagenesis studies have been aimed at providing a greater mechanistic understanding of the interaction of mGlu allosteric modulators with the receptor, which have revealed evidence for common allosteric binding sites across multiple mGlu subtypes and the presence for multiple allosteric sites within a single mGlu subtype. Recent data have also revealed that mGlu allosteric modulators can display functional selectivity toward particular signal transduction cascades downstream of an individual mGlu subtype. Studies continue to validate the therapeutic utility of mGlu allosteric modulators as a potential therapeutic approach for a number of disorders including anxiety, schizophrenia, Parkinson’s disease, and Fragile X syndrome. PMID:21907906

Body water balance and body temperature in vasopressin V1b receptor knockout mice.

PubMed

Daikoku, R; Kunitake, T; Kato, K; Tanoue, A; Tsujimoto, G; Kannan, H

2007-10-30

In an attempt to determine whether there is a specific vasopressin receptor (V(1b)) subtype involved in the regulation of body water balance and temperature, vasopressin V(1b) receptor knockout mice were used. Daily drinking behavior and renal excretory function were examined in V(1b)-deficient (V(1b)(-/-)) and control (V(1b)(+/+)) mice under the basal and stress-induced condition. In addition, body temperature and locomotor activity were measured with a biotelemetry system. The baseline daily water intake and urine volume were larger in V(1b)(-/-) mice than in V(1b)(+/+) mice. V(1b)(-/-) mice (V(1b)(-/-)) had significantly higher locomotor activity than wild-type, whereas the body temperature and oxygen consumption were lower in V(1b)(-/-) than in the V(1b)(+/+) mice. Next, the V(1b)(-/-) and V(1b)(+/+) mice were subjected to water deprivation for 48 hr. Under this condition, their body temperature decreased with the time course, which was significantly larger for V(1b)(-/-) than for V(1b)(+/+) mice. Central vasopressin has been reported to elicit drinking behavior and antipyretic action, and the V(1b) receptor has been reported to be located in the kidney. Thus, the findings suggest that the V(1b) receptor may be, at least in part, involved in body water balance and body temperature regulation.

Regular and low-dose aspirin, other non-steroidal anti-inflammatory medications and prospective risk of HER2-defined breast cancer: the California Teachers Study.

PubMed

Clarke, Christina A; Canchola, Alison J; Moy, Lisa M; Neuhausen, Susan L; Chung, Nadia T; Lacey, James V; Bernstein, Leslie

2017-05-01

Regular users of aspirin may have reduced risk of breast cancer. Few studies have addressed whether risk reduction pertains to specific breast cancer subtypes defined jointly by hormone receptor (estrogen and progesterone receptor) and human epidermal growth factor receptor 2 (HER2) expression. This study assessed the prospective risk of breast cancer (overall and by subtype) according to use of aspirin and other non-steroidal anti-inflammatory medications (NSAIDs) in a cohort of female public school professionals in California. In 1995 - 1996, participants in the California Teachers Study completed a baseline questionnaire on family history of cancer and other conditions, use of NSAIDs, menstrual and reproductive history, self-reported weight and height, living environment, diet, alcohol use, and physical activity. In 2005-2006, 57,164 participants provided some updated information, including use of NSAIDs and 1457 of these participants developed invasive breast cancer before January 2013. Multivariable Cox proportional hazards regression models provided hazard rate ratios (HRR) for the association between NSAID use and risk of invasive breast cancer as well as hormone receptor- and HER2-defined subtypes. Developing breast cancer was associated inversely with taking three or more tablets of low-dose aspirin per week (23% of participants). Among women reporting this exposure, the HRR was 0.84 (95% confidence interval (CI) 0.72-0.98) compared to those not taking NSAIDs and this was particularly evident in women with the hormone receptor-positive/HER2-negative subtype (HRR = 0.80, 95% CI 0.66-0.96). Use of three or more tablets of "other" NSAIDs was marginally associated with lower risk of breast cancer (HRR = 0.79, 95% CI 0.62-1.00). Other associations with NSAIDs were generally null. Our observation of reduced risk of breast cancer, among participants who took three or more tablets of low-dose aspirin weekly, is consistent with other reports looking at aspirin without differentiation by dose. This is the first report to suggest that the reduction in risk occurs for low-dose aspirin and not for regular-dose aspirin and only among women with the hormone receptor-positive/HER2-negative subtype. This preliminary study builds on previous knowledge and further supports the need for formal cancer chemoprevention studies of low-dose aspirin.

Differential Modulation of Ethanol-Induced Sedation and Hypnosis by Metabotropic Glutamate Receptor Antagonists in C57BL/6J Mice

PubMed Central

Sharko, Amanda C.; Hodge, Clyde W.

2008-01-01

Background Emerging evidence implicates metabotropic glutamate receptor (mGluR) function in the neurobiological effects of ethanol. The recent development of subtype specific mGluR antagonists has made it possible to examine the roles of specific mGluRs in biochemical and behavioral responses to ethanol. The purpose of the present study was to determine if mGluRs modulate the acute sedative-hypnotic properties of ethanol in mice. Methods C57BL / 6J mice were tested for locomotor activity (sedation) and duration of loss of the righting reflex (hypnosis) following acute systemic administration of ethanol alone or in combination with the mGluR5-selective antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP), the mGluR1-selective antagonist, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), or the mGluR2 / 3-selective antagonist (2S)-2-Amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495)). Results MPEP (10 and 30 mg / kg) significantly enhanced both the sedative and hypnotic effects of ethanol, while LY341495 (10 and 30 mg / kg) significantly reduced the sedative-hypnotic effects of ethanol. CPCCOEt had no effect at any concentration tested. Further loss of righting reflex experiments revealed that LY341495 (30 mg / kg) significantly reduced hypnosis induced by the gamma-aminobutyric acid type A (GABAA) positive modulators, pentobarbital (50 mg / kg) and midazolam (60 mg / kg), and the N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine (150 mg / kg), while MPEP (30 mg / kg) only significantly enhanced the hypnotic properties of ketamine (150 mg / kg). Conclusions These findings suggest that specific subtypes of the metabotropic glutamate receptor differentially modulate the sedative-hypnotic properties of ethanol through separate mechanisms of action, potentially involving GABAA and NMDA receptors. PMID:18070246

Effect of HIV type 1 subtype on virological and immunological response to combination antiretroviral therapy: evidence for a more rapid viral suppression for subtype A than subtype B-infected Greek individuals.

PubMed

Paraskevis, Dimirios; Touloumi, Giota; Bakoyannis, Giorgos; Paparizos, Vassilios; Lazanas, Marios; Gargalianos, Panagiotis; Chryssos, Georgios; Antoniadou, Anastasia; Psichogiou, Mina; Panos, Georgios; Katsarou, Olga; Sambatakou, Helen; Kordossis, Theodoros; Hatzakis, Angelos

2013-03-01

Whether response to combination antiretroviral therapy (cART) differs between those infected with HIV-1 subtype A or B remains unclear. We compared virological and immunological response to cART in individuals infected with subtype A or B in an ethnically homogeneous population. Data derived from the Athens Multicenter AIDS Cohort Study (AMACS) and analysis were restricted to those of Greek origin. Time to virological response (confirmed HIV-RNA <500 copies/ml) and time to failure (>500 copies/ml at any time or no response by month 6) were analyzed using survival models and CD4 changes after cART initiation using piecewise linear mixed effects models. Of the 571 subjects included in the analysis, 412 (72.2%) were infected with subtype B and 159 (27.8%) with subtype A. After adjusting for various prognostic factors, the rate of virological response was higher for those infected with subtype A versus B (adjusted HR: 1.35; 95% CI: 1.08-1.68; p=0.009). Subtype A was also marginally associated with a lower hazard of virological failure compared to subtype B (HR=0.73; 95% CI: 0.53-1.02; p=0.062). Further adjustment for treatment adherence did not substantially changed the main results. No significant differences were observed in the rates of CD4 increases by subtype. The overall median (95% CI) CD4 increase at 2 years of cART was 193 (175, 212) cells/μl. Our study, based on one of the largest homogeneous groups of subtype A and B infections in Europe, showed that individuals infected with subtype A had an improved virological but similar immunological response to cART compared to those infected with subtype B.

HIV subtype, epidemiological and mutational correlations in patients from Paraná, Brazil.

PubMed

Silva, Monica Maria Gomes da; Telles, Flavio Queiroz; da Cunha, Clovis Arns; Rhame, Frank S

2010-01-01

Analyze patients with HIV infection from Curitiba, Paraná, their epidemiological characteristics and HIV RAM. Patients regularly followed in an ID Clinic had their medical data evaluated and cases of virological failure were analyzed with genotypic report. Patients with complete medical charts were selected (n = 191). Demographic and clinical characteristics were compared. One hundred thirty two patients presented with subtype B infection (69.1%), 41 subtype C (21.5%), 10 subtype F (5.2%), 7 BF (3.7%) and 1 CF (0.5%). Patients with subtype B infection had been diagnosed earlier than patients with subtype non-B. Also, subtype B infection was more frequent in men who have sex with men, while non-B subtypes occurred more frequently in heterosexuals and women. Patients with previous history of three classes of ARVs (n = 161) intake were selected to evaluate resistance. For RT inhibitors, 41L and 210W were more frequently observed in subtype B than in non-B strains. No differences between subtypes and mutations were observed to NNTRIs. Mutations at 10, 32 and 63 position of protease were more observed in subtype B viruses than non-B, while positions 20 and 36 of showed more amino acid substitutions in subtype non-B viruses. Patients with history of NFV intake were evaluated to resistance pathway. The 90M pathway was more frequent in subtypes B and non-B. Mutations previously reported as common in non-B viruses, such as 65R and 106M, were uncommon in our study. Mutations 63P and 36I, previously reported as common in HIV-1 subtypes B and C from Brazil, respectively, were common. There is a significant frequency of HIV-1 non-B infections in Paraná state, with isolates classified as subtypes C, F, BF and BC. Patients with subtype C infection were more frequently female, heterosexual and had a longer average time of HIV diagnosis.

Establishment of estrogen receptor 1 (ESR1)-knockout medaka: ESR1 is dispensable for sexual development and reproduction in medaka, Oryzias latipes.

PubMed

Tohyama, Saki; Ogino, Yukiko; Lange, Anke; Myosho, Taijun; Kobayashi, Tohru; Hirano, Yu; Yamada, Gen; Sato, Tomomi; Tatarazako, Norihisa; Tyler, Charles R; Iguchi, Taisen; Miyagawa, Shinichi

2017-08-01

Estrogens play fundamental roles in regulating reproductive activities and they act through estrogen receptor (ESR) in all vertebrates. Most vertebrates have two ESR subtypes (ESR1 and ESR2), whereas teleost fish have at least three (Esr1, Esr2a and Esr2b). Intricate functionalization has been suggested among the Esr subtypes, but to date, distinct roles of Esr have been characterized in only a limited number of species. Study of loss-of-function in animal models is a powerful tool for application to understanding vertebrate reproductive biology. In the current study, we established esr1 knockout (KO) medaka using a TALEN approach and examined the effects of Esr1 ablation. Unexpectedly, esr1 KO medaka did not show any significant defects in their gonadal development or in their sexual characteristics. Neither male or female esr1 KO medaka exhibited any significant changes in sexual differentiation or reproductive activity compared with wild type controls. Interestingly, however, estrogen-induced vitellogenin gene expression, an estrogen-responsive biomarker in fish, was limited in the liver of esr1 KO males. Our findings, in contrast to mammals, indicate that Esr1 is dispensable for normal development and reproduction in medaka. We thus provide an evidence for estrogen receptor functionalization between mammals and fish. Our findings will also benefit interpretation of studies into the toxicological effects of estrogenic chemicals in fish. © 2017 Japanese Society of Developmental Biologists.

Structure and dynamics of the M3 muscarinic acetylcholine receptor

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

Kruse, Andrew C.; Hu, Jianxin; Pan, Albert C.

2012-03-01

Acetylcholine, the first neurotransmitter to be identified, exerts many of its physiological actions via activation of a family of G-protein-coupled receptors (GPCRs) known as muscarinic acetylcholine receptors (mAChRs). Although the five mAChR subtypes (M1-M5) share a high degree of sequence homology, they show pronounced differences in G-protein coupling preference and the physiological responses they mediate. Unfortunately, despite decades of effort, no therapeutic agents endowed with clear mAChR subtype selectivity have been developed to exploit these differences. We describe here the structure of the G{sub q/11}-coupled M3 mAChR ('M3 receptor', from rat) bound to the bronchodilator drug tiotropium and identify themore » binding mode for this clinically important drug. This structure, together with that of the G{sub i/o}-coupled M2 receptor, offers possibilities for the design of mAChR subtype-selective ligands. Importantly, the M3 receptor structure allows a structural comparison between two members of a mammalian GPCR subfamily displaying different G-protein coupling selectivities. Furthermore, molecular dynamics simulations suggest that tiotropium binds transiently to an allosteric site en route to the binding pocket of both receptors. These simulations offer a structural view of an allosteric binding mode for an orthosteric GPCR ligand and provide additional opportunities for the design of ligands with different affinities or binding kinetics for different mAChR subtypes. Our findings not only offer insights into the structure and function of one of the most important GPCR families, but may also facilitate the design of improved therapeutics targeting these critical receptors.« less

Cloning and expression of melatonin receptors in the mudskipper Boleophthalmus pectinirostris: their role in synchronizing its semilunar spawning rhythm.

PubMed

Hong, Lu Yan; Hong, Wan Shu; Zhu, Wen Bo; Shi, Qiong; You, Xin Xin; Chen, Shi Xi

2014-01-01

The mudskipper Boleophthalmus pectinirostris, a burrow-dwelling fish inhabiting intertidal mudflats, spawns only once during the spawning season around either the first or last lunar quarters. To understand the molecular mechanisms regulating this semilunar spawning rhythm, we cloned all melatonin receptor subtypes (mtnr1a1.4, mtnr1a1.7, mtnr1b, and mtnr1c). Expression of three melatonin receptor subtypes (except mtnr1c) was found in the ovaries. In contrast, the expression of all receptor subtypes was found in the diencephalon and the pituitary. In the fully-grown follicles, only mtnr1a1.7 mRNA was detected in both the isolated follicle layers and denuded oocytes. Interestingly, the transcript levels of both mtnr1a1.4 in the diencephalon and mtnr1a1.7 in the ovary displayed two cycles within one lunar month, and peaked around the first and last lunar quarters. We used 17α,20β-dihydroxy-4-pregnen-3-one (DHP), a maturation-inducing hormone, as a biomarker to examine the involvement of melatonin receptors in the control of the spawning cycle. Melatonin significantly increased the plasma DHP level 1h post intraperitoneal injection. Melatonin also directly stimulated ovarian fragments in vitro to produce a significantly higher amount of DHP. Taken together, these results provided the first evidence that melatonin receptors were involved in the synchronization of the semilunar spawning rhythm in the female mudskipper by acting through the HPG axis and/or directly on ovarian tissues to stimulate the production of DHP. Copyright © 2013 Elsevier Inc. All rights reserved.

Function and distribution of 5-HT2 receptors in the honeybee (Apis mellifera).

PubMed

Thamm, Markus; Rolke, Daniel; Jordan, Nadine; Balfanz, Sabine; Schiffer, Christian; Baumann, Arnd; Blenau, Wolfgang

2013-01-01

Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca(2+) imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca(2+) concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors.

Function and Distribution of 5-HT2 Receptors in the Honeybee (Apis mellifera)

PubMed Central

Thamm, Markus; Rolke, Daniel; Jordan, Nadine; Balfanz, Sabine; Schiffer, Christian; Baumann, Arnd; Blenau, Wolfgang

2013-01-01

Background Serotonin plays a pivotal role in regulating and modulating physiological and behavioral processes in both vertebrates and invertebrates. In the honeybee (Apis mellifera), serotonin has been implicated in division of labor, visual processing, and learning processes. Here, we present the cloning, heterologous expression, and detailed functional and pharmacological characterization of two honeybee 5-HT2 receptors. Methods Honeybee 5-HT2 receptor cDNAs were amplified from brain cDNA. Recombinant cell lines were established constitutively expressing receptor variants. Pharmacological properties of the receptors were investigated by Ca2+ imaging experiments. Quantitative PCR was applied to explore the expression patterns of receptor mRNAs. Results The honeybee 5-HT2 receptor class consists of two subtypes, Am5-HT2α and Am5-HT2β. Each receptor gene also gives rise to alternatively spliced mRNAs that possibly code for truncated receptors. Only activation of the full-length receptors with serotonin caused an increase in the intracellular Ca2+ concentration. The effect was mimicked by the agonists 5-methoxytryptamine and 8-OH-DPAT at low micromolar concentrations. Receptor activities were blocked by established 5-HT receptor antagonists such as clozapine, methiothepin, or mianserin. High transcript numbers were detected in exocrine glands suggesting that 5-HT2 receptors participate in secretory processes in the honeybee. Conclusions This study marks the first molecular and pharmacological characterization of two 5-HT2 receptor subtypes in the same insect species. The results presented should facilitate further attempts to unravel central and peripheral effects of serotonin mediated by these receptors. PMID:24324783

P2X and P2Y nucleotide receptors as targets in cardiovascular disease.

PubMed

Kennedy, Charles; Chootip, Krongkarn; Mitchell, Callum; Syed, Nawazish-i-Husain; Tengah, Asrin

2013-03-01

Endogenous nucleotides have widespread actions in the cardiovascular system, but it is only recently that the P2X and P2Y receptor subtypes, at which they act, have been identified and subtype-selective agonists and antagonists developed. These advances have greatly increased our understanding of the physiological and pathophysiological functions of P2X and P2Y receptors, but investigation of the clinical usefulness of selective ligands is at an early stage. Nonetheless, the evidence considered in this review demonstrates clearly that various cardiovascular disorders, including vasospasm, hypertension, congestive heart failure and cardiac damage during ischemic episodes, may be viable targets. With further development of novel, selective agonists and antagonists, our understanding will continue to improve and further therapeutic applications are likely to be discovered.

The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

PubMed Central

Wang, Rui; Mellem, Jerry E.; Jensen, Michael; Brockie, Penelope J.; Walker, Craig S.; Hoerndli, Frédéric J.; Madsen, David M.; Maricq, Andres V.

2012-01-01

Summary The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. PMID:22958824

The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

PubMed

Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

2012-09-06

The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. Copyright © 2012 Elsevier Inc. All rights reserved.

Dysregulation of ErbB Receptor Trafficking and Signaling in Demyelinating Charcot-Marie-Tooth Disease

PubMed Central

Lee, Samuel M.; Chin, Lih-Shen; Li, Lian

2016-01-01

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with the majority of cases involving demyelination of peripheral nerves. The pathogenic mechanisms of demyelinating CMT remain unclear, and no effective therapy currently exists for this disease. The discovery that mutations in different genes can cause a similar phenotype of demyelinating peripheral neuropathy raises the possibility that there may be convergent mechanisms leading to demyelinating CMT pathogenesis. Increasing evidence indicates that ErbB receptor-mediated signaling plays a major role in the control of Schwann cell-axon communication and myelination in the peripheral nervous system. Recent studies reveal that several demyelinating CMT-linked proteins are novel regulators of endocytic trafficking and/or phosphoinositide metabolism that may affect ErbB receptor signaling. Emerging data have begun to suggest that dysregulation of ErbB receptor trafficking and signaling in Schwann cells may represent a common pathogenic mechanism in multiple subtypes of demyelinating CMT. In this review, we focus on the roles of ErbB receptor trafficking and signaling in regulation of peripheral nerve myelination and discuss the emerging evidence supporting the potential involvement of altered ErbB receptor trafficking and signaling in demyelinating CMT pathogenesis and the possibility of modulating these trafficking and signaling processes for treating demyelinating peripheral neuropathy. PMID:26732592

Dysregulation of ErbB Receptor Trafficking and Signaling in Demyelinating Charcot-Marie-Tooth Disease.

PubMed

Lee, Samuel M; Chin, Lih-Shen; Li, Lian

2017-01-01

Charcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy with the majority of cases involving demyelination of peripheral nerves. The pathogenic mechanisms of demyelinating CMT remain unclear, and no effective therapy currently exists for this disease. The discovery that mutations in different genes can cause a similar phenotype of demyelinating peripheral neuropathy raises the possibility that there may be convergent mechanisms leading to demyelinating CMT pathogenesis. Increasing evidence indicates that ErbB receptor-mediated signaling plays a major role in the control of Schwann cell-axon communication and myelination in the peripheral nervous system. Recent studies reveal that several demyelinating CMT-linked proteins are novel regulators of endocytic trafficking and/or phosphoinositide metabolism that may affect ErbB receptor signaling. Emerging data have begun to suggest that dysregulation of ErbB receptor trafficking and signaling in Schwann cells may represent a common pathogenic mechanism in multiple subtypes of demyelinating CMT. In this review, we focus on the roles of ErbB receptor trafficking and signaling in regulation of peripheral nerve myelination and discuss the emerging evidence supporting the potential involvement of altered ErbB receptor trafficking and signaling in demyelinating CMT pathogenesis and the possibility of modulating these trafficking and signaling processes for treating demyelinating peripheral neuropathy.

Genetic alterations activating kinase and cytokine receptor signaling in high-risk acute lymphoblastic leukemia

PubMed Central

Roberts, Kathryn G.; Morin, Ryan D.; Zhang, Jinghui; Hirst, Martin; Zhao, Yongjun; Su, Xiaoping; Chen, Shann-Ching; Payne-Turner, Debbie; Churchman, Michelle; Harvey, Richard C.; Chen, Xiang; Kasap, Corynn; Yan, Chunhua; Becksfort, Jared; Finney, Richard P.; Teachey, David T.; Maude, Shannon L.; Tse, Kane; Moore, Richard; Jones, Steven; Mungall, Karen; Birol, Inanc; Edmonson, Michael N.; Hu, Ying; Buetow, Kenneth E.; Chen, I-Ming; Carroll, William L.; Wei, Lei; Ma, Jing; Kleppe, Maria; Levine, Ross L.; Garcia-Manero, Guillermo; Larsen, Eric; Shah, Neil P.; Devidas, Meenakshi; Reaman, Gregory; Smith, Malcolm; Paugh, Steven W.; Evans, William E.; Grupp, Stephan A.; Jeha, Sima; Pui, Ching-Hon; Gerhard, Daniela S.; Downing, James R.; Willman, Cheryl L.; Loh, Mignon; Hunger, Stephen P.; Marra, Marco; Mullighan, Charles G.

2012-01-01

SUMMARY Genomic profiling has identified a subtype of high-risk B-progenitor acute lymphoblastic leukemia (B-ALL) with alteration of IKZF1, a gene expression profile similar to BCR-ABL1-positive ALL and poor outcome (Ph-like ALL). The genetic alterations that activate kinase signaling in Ph-like ALL are poorly understood. We performed transcriptome and whole genome sequencing on 15 cases of Ph-like ALL, and identified rearrangements involving ABL1, JAK2, PDGFRB, CRLF2 and EPOR, activating mutations of IL7R and FLT3, and deletion of SH2B3, which encodes the JAK2 negative regulator LNK. Importantly, several of these alterations induce transformation that is attenuated with tyrosine kinase inhibitors, suggesting the treatment outcome of these patients may be improved with targeted therapy. PMID:22897847

S2RSLDB: a comprehensive manually curated, internet-accessible database of the sigma-2 receptor selective ligands.

PubMed

Nastasi, Giovanni; Miceli, Carla; Pittalà, Valeria; Modica, Maria N; Prezzavento, Orazio; Romeo, Giuseppe; Rescifina, Antonio; Marrazzo, Agostino; Amata, Emanuele

2017-01-01

Sigma (σ) receptors are accepted as a particular receptor class consisting of two subtypes: sigma-1 (σ 1 ) and sigma-2 (σ 2 ). The two receptor subtypes have specific drug actions, pharmacological profiles and molecular characteristics. The σ 2 receptor is overexpressed in several tumor cell lines, and its ligands are currently under investigation for their role in tumor diagnosis and treatment. The σ 2 receptor structure has not been disclosed, and researchers rely on σ 2 receptor radioligand binding assay to understand the receptor's pharmacological behavior and design new lead compounds. Here we present the sigma-2 Receptor Selective Ligands Database (S2RSLDB) a manually curated database of the σ 2 receptor selective ligands containing more than 650 compounds. The database is built with chemical structure information, radioligand binding affinity data, computed physicochemical properties, and experimental radioligand binding procedures. The S2RSLDB is freely available online without account login and having a powerful search engine the user may build complex queries, sort tabulated results, generate color coded 2D and 3D graphs and download the data for additional screening. The collection here reported is extremely useful for the development of new ligands endowed of σ 2 receptor affinity, selectivity, and appropriate physicochemical properties. The database will be updated yearly and in the near future, an online submission form will be available to help with keeping the database widely spread in the research community and continually updated. The database is available at http://www.researchdsf.unict.it/S2RSLDB.

Solution conformation of a neuronal nicotinic acetylcholine receptor antagonist {alpha}-conotoxin OmIA that discriminates {alpha}3 vs. {alpha}6 nAChR subtypes

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

Chi, Seung-Wook; Kim, Do-Hyoung; Olivera, Baldomero M.

2006-06-23

{alpha}-Conotoxin OmIA from Conus omaria is the only {alpha}-conotoxin that shows a {approx}20-fold higher affinity to the {alpha}3{beta}2 over the {alpha}6{beta}2 subtype of nicotinic acetylcholine receptor. We have determined a three-dimensional structure of {alpha}-conotoxin OmIA by nuclear magnetic resonance spectroscopy. {alpha}-Conotoxin OmIA has an '{omega}-shaped' overall topology with His{sup 5}-Asn{sup 12} forming an {alpha}-helix. Structural features of {alpha}-conotoxin OmIA responsible for its selectivity are suggested by comparing its surface characteristics with other functionally related {alpha}4/7 subfamily conotoxins. Reduced size of the hydrophilic area in {alpha}-conotoxin OmIA seems to be associated with the reduced affinity towards the {alpha}6{beta}2 nAChR subtype.

Genotype diversity of hepatitis C virus (HCV) in HCV-associated liver disease patients in Indonesia.

PubMed

Utama, Andi; Tania, Navessa Padma; Dhenni, Rama; Gani, Rino Alvani; Hasan, Irsan; Sanityoso, Andri; Lelosutan, Syafruddin A R; Martamala, Ruswhandi; Lesmana, Laurentius Adrianus; Sulaiman, Ali; Tai, Susan

2010-09-01

Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

Medicinal chemistry of P2X receptors: allosteric modulators.

PubMed

Müller, Christa E

2015-01-01

P2X receptors are trimeric ligand-gated ion channels whose potential as novel drug targets for a number of diseases has been recognized. They are mainly involved in inflammatory processes, including neuroinflammation, and pain sensation. The orthosteric binding site is lined by basic amino acid residues that bind the negatively charged agonist ATP. Therefore it is not easy to develop orthosteric ligands that possess drug-like properties for such a highly polar binding site. However, ligand-gated ion channels offer multiple additional binding sites for allosteric ligands, positive or negative allosteric modulators enhancing or blocking receptor function. So far, the P2X3 (and P2X2/3), as well as the P2X7 receptor subtype have been the main focus of drug development efforts. A number of potent and selective allosteric antagonists have been developed to block these receptors. We start to see the development of novel allosteric ligands also for the other P2X receptor subtypes, P2X1, P2X2 and especially P2X4. The times when only poor, non-selective, non-drug-like tools for studying P2X receptor function were available have been overcome. The first clinical studies with allosteric P2X3 and P2X7 antagonists suggest that P2X therapeutics may soon become a reality.

Identification of Copy Number Aberrations in Breast Cancer Subtypes Using Persistence Topology

PubMed Central

Arsuaga, Javier; Borrman, Tyler; Cavalcante, Raymond; Gonzalez, Georgina; Park, Catherine

2015-01-01

DNA copy number aberrations (CNAs) are of biological and medical interest because they help identify regulatory mechanisms underlying tumor initiation and evolution. Identification of tumor-driving CNAs (driver CNAs) however remains a challenging task, because they are frequently hidden by CNAs that are the product of random events that take place during tumor evolution. Experimental detection of CNAs is commonly accomplished through array comparative genomic hybridization (aCGH) assays followed by supervised and/or unsupervised statistical methods that combine the segmented profiles of all patients to identify driver CNAs. Here, we extend a previously-presented supervised algorithm for the identification of CNAs that is based on a topological representation of the data. Our method associates a two-dimensional (2D) point cloud with each aCGH profile and generates a sequence of simplicial complexes, mathematical objects that generalize the concept of a graph. This representation of the data permits segmenting the data at different resolutions and identifying CNAs by interrogating the topological properties of these simplicial complexes. We tested our approach on a published dataset with the goal of identifying specific breast cancer CNAs associated with specific molecular subtypes. Identification of CNAs associated with each subtype was performed by analyzing each subtype separately from the others and by taking the rest of the subtypes as the control. Our results found a new amplification in 11q at the location of the progesterone receptor in the Luminal A subtype. Aberrations in the Luminal B subtype were found only upon removal of the basal-like subtype from the control set. Under those conditions, all regions found in the original publication, except for 17q, were confirmed; all aberrations, except those in chromosome arms 8q and 12q were confirmed in the basal-like subtype. These two chromosome arms, however, were detected only upon removal of three patients with exceedingly large copy number values. More importantly, we detected 10 and 21 additional regions in the Luminal B and basal-like subtypes, respectively. Most of the additional regions were either validated on an independent dataset and/or using GISTIC. Furthermore, we found three new CNAs in the basal-like subtype: a combination of gains and losses in 1p, a gain in 2p and a loss in 14q. Based on these results, we suggest that topological approaches that incorporate multiresolution analyses and that interrogate topological properties of the data can help in the identification of copy number changes in cancer. PMID:27600228

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.

Asparagine, valine, and threonine in the third extracellular loop of muscarinic receptor have essential roles in the positive cooperativity of strychnine-like allosteric modulators.

PubMed

Jakubík, J; Krejcí, A; Dolezal, V

2005-05-01

We have investigated allosteric interactions of four closely related strychnine-like substances: Wieland-Gumlich aldehyde (WGA), propargyl Wieland-Gumlich aldehyde, strychnine, and brucine with N-methylscopolamine (NMS) on M(3) subtype of muscarinic receptor genetically modified in the second or the third extracellular loop to corresponding loops of M(2) subtype (M(3)o2 and M(3)o3 chimera). The M(3)o2 chimeric receptor The exhibited no change in either affinity of strychnine, brucine, and WGA or in cooperativity of brucine or WGA, whereas both parameters for propargyl-WGA changed. In contrast, there was a change in affinity of all tested modulators (except for brucine) and in their cooperativity in the M(3)o3 chimera. Directions of affinity changes in both chimeras were always toward values of the donor M(2) subtype, but changes in cooperativity were variable. Compared with the native M(3) receptor, strychnine displayed a slight increase in positive cooperativity and propargyl-WGA a robust decrease in negative cooperativity at M(3)o2 chimera. Similar changes were found in the M(3)o3 chimera. Interestingly, cooperativity of brucine and WGA at the M(3)o3 chimera changed from negative to positive. This is the first evidence of constitution of positive cooperativity of WGA by switching sequences of two parental receptors, both exhibiting negative cooperativity. Gradual replacement of individual amino acids revealed that only three residues (NVT of the o3 loop of the M(2) receptor) are involved in this effect. Data suggest that these amino acids are essential for propagation of a conformation change resulting in positive cooperativity induced by these modulators.

Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for determination of HIV-1 viral loads in a cohort of Canadian patients with diverse HIV subtype infections.

PubMed

Church, Deirdre; Gregson, Daniel; Lloyd, Tracie; Klein, Marina; Beckthold, Brenda; Laupland, Kevin; Gill, M John

2011-01-01

HIV clinics in Canada provide care to an increasing number of patients born outside of Canada with HIV-1 non-B subtype infections. Because the Easy Q HIV-1 v1.2 assay (EQ; bioMérieux) failed to detect some non-B subtype infections, a multiassay HIV-1 viral load (VL) study was conducted with patients with diverse HIV subtype infections. Patients were enrolled from the Southern Alberta HIV Clinic (SAC), Calgary, Alberta, Canada (n = 349) and the McGill HIV Clinic (MHC), Montreal, Quebec, Canada (n = 20) and had four or five tubes of blood drawn for testing by EQ and three other commercial HIV VL assays: (i) the Versant 3.0 HIV-1 test, with the Versant 440 instrument (branched DNA [bDNA]; Siemens), (ii) the RealTime HIV-1 test, with the m2000rt instrument (m2000rt; Abbott Molecular Diagnostics), and (iii) the COBAS AmpliPrep TaqMan HIV-1 48 test (CAP-CTM; Roche Molecular Diagnostics). Blood was processed according to the individual manufacturer's requirements and stored frozen at -86°C. The HIV subtype was known for patients who had undergone HIV genotypic resistance testing (Virco, Belgium). Data analyses were done using standard statistical methods within Stata 9.0 (StataCorp, College Station, TX). A total of 371 samples were tested on 369 patients, of whom 291 (81%) had a Virco genotype result of B (195; 53%) or non-B (96; 26%) subtypes A to D and F to K, as well as circulating recombinant forms (CRFs) (i.e., CRF01_AE and CRF02_AG). Most (58/78; 74%) patients of unknown subtype were recent African emigrants who likely have non-subtype B infection. Overall bias was small in pairwise Bland-Altman plots, but the limits of agreement between assays were wide. Discordant viral load results occurred for 98 samples and were due to missing values, false negatives, and significant underquantification that varied by HIV subtype. Results were obtained for all 371 samples with m2000rt, but for only 357 (97%) with CAP-CTM, 338 (92%) with EQ, and 276 (75%) with bDNA due to errors/equipment failures. False-negative results (nondetection of viral RNA versus other assay results) occurred for all platforms, as follows: for m2000rt, 8 (2%) [B(4) and non-B(4) subtypes], CAP-CTM, 9 (2.5%) [B(6) and non-B(3) subtypes]; EQ, 20 (6%) [B(7) and non-B(13) subtypes]; bDNA, 5 (2%) [B(1) and C(4)]. EQ and bDNA had the highest rates of underquantification by ≥ 1.0 log(10) copies/ml, mainly for HIV non-B subtypes. Performance significantly varied between HIV VL platforms according to subtype. HIV viral diversity in the population being tested must be considered in selection of the viral load platform.

Adjuvant Trastuzumab in HER2-Positive Early Breast Cancer by Age and Hormone Receptor Status: A Cost-Utility Analysis

PubMed Central

Leung, William; Kvizhinadze, Giorgi; Nair, Nisha; Blakely, Tony

2016-01-01

Background The anti–human epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab improves outcomes in patients with node-positive HER2+ early breast cancer. Given trastuzumab’s high cost, we aimed to estimate its cost-effectiveness by heterogeneity in age and estrogen receptor (ER) and progesterone receptor (PR) status, which has previously been unexplored, to assist prioritisation. Methods and Findings A cost-utility analysis was performed using a Markov macro-simulation model, with a lifetime horizon, comparing a 12-mo regimen of trastuzumab with chemotherapy alone using the latest (2014) effectiveness measures from landmark randomised trials. A New Zealand (NZ) health system perspective was adopted, employing high-quality national administrative data. Incremental quality-adjusted life-years for trastuzumab versus chemotherapy alone are two times higher (2.33 times for the age group 50–54 y; 95% CI 2.29–2.37) for the worst prognosis (ER−/PR−) subtype compared to the best prognosis (ER+/PR+) subtype, causing incremental cost-effectiveness ratios (ICERs) for the former to be less than half those of the latter for the age groups from 25–29 to 90–94 y (0.44 times for the age group 50–54 y; 95% CI 0.43–0.45). If we were to strictly apply an arbitrary cost-effectiveness threshold equal to the NZ gross domestic product per capita (2011 purchasing power parity [PPP]–adjusted: US$30,300; €23,700; £21,200), our study suggests that trastuzumab (2011 PPP-adjusted US$45,400/€35,900/£21,900 for 1 y at formulary prices) may not be cost-effective for ER+ (which are 61% of all) node-positive HER2+ early breast cancer patients but cost-effective for ER−/PR− subtypes (37% of all cases) to age 69 y. Market entry of trastuzumab biosimilars will likely reduce the ICER to below this threshold for premenopausal ER+/PR− cancer but not for ER+/PR+ cancer. Sensitivity analysis using the best-case effectiveness measure for ER+ cancer had the same result. A key limitation was a lack of treatment-effect data by hormone receptor subtype. Heterogeneity was restricted to age and hormone receptor status; tumour size/grade heterogeneity could be explored in future work. Conclusions This study highlights how cost-effectiveness can vary greatly by heterogeneity in age and hormone receptor subtype. Resource allocation and licensing of subsidised therapies such as trastuzumab should consider demographic and clinical heterogeneity; there is currently a profound disconnect between how funding decisions are made (largely agnostic to heterogeneity) and the principles of personalised medicine. PMID:27504960

The Role of Adenosine A2A Receptor, CYP450s, and PPARs in the Regulation of Vascular Tone

PubMed Central

Khayat, Maan T.

2017-01-01

Adenosine is an endogenous mediator involved in a myriad of physiologic functions, including vascular tone regulation. It is also implicated in some pathologic conditions. Four distinct receptor subtypes mediate the effects of adenosine, such as its role in the regulation of the vascular tone. Vascular tone regulation is a complex and continuous process which involves many mechanisms and mediators that are not fully disclosed. The vascular endothelium plays a pivotal role in regulating blood flow to and from all body organs. Also, the vascular endothelium is not merely a physical barrier; it is a complex tissue with numerous functions. Among adenosine receptors, A2A receptor subtype (A2AAR) stands out as the primary receptor responsible for the vasodilatory effects of adenosine. This review focuses on important effectors of the vascular endothelium, including adenosine, adenosine receptors, EETs (epoxyeicosatrienoic acids), HETEs (hydroxyeicosatetraenoic acids), PPARs (peroxisome proliferator-activated receptors), and KATP channels. Given the impact of vascular tone regulation in cardiovascular physiology and pathophysiology, better understanding of the mechanisms affecting it could have a significant potential for developing therapeutic agents for cardiovascular diseases. PMID:28884118

Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

PubMed

Viswanathan, Karthik; Koh, Xiaoying; Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M; Sasisekharan, Ram

2010-10-29

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.

Determinants of Glycan Receptor Specificity of H2N2 Influenza A Virus Hemagglutinin

PubMed Central

Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M.; Sasisekharan, Ram

2010-01-01

The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA. PMID:21060797

Ventricular, but not atrial, M2-muscarinic receptors increase in the canine pacing-overdrive model of heart failure.

PubMed

Wilkinson, M; Giles, A; Armour, J A; Cardinal, R

1996-01-01

To investigate the effects of heart failure induced by chronic rapid ventricular pacing (six weeks) on canine atrial and ventricular muscarinic receptors. Dogs (n = 4) were fitted with a bipolar pacing electrode connected to a Medtronic pacemaker set at 240 stimuli/min. Pacing was maintained for six weeks. Tissue samples obtained from the left atrium and ventral wall of the left ventricle were frozen at -70 degrees C. Control tissue was obtained from normal dogs (n = 6) following anesthesia and thoracotomy. M2-muscarinic receptors were characterized and quantified in tissue micropunches using the hydrophilic ligand [3H] N-methyl-scopolamine (NMS). Cardiac tissue bound [3H] NMS with the specificity of an M2 subtype. Tachycardia-induced heart failure did not affect atrial muscarinic receptors but signify left ventricular myocytes (control 160.0 +/- 10.0 fmol/mg protein versus heart failure 245.0 +/- 25.0 fmol/mg protein; P < 0.01). Canine ventricular muscarinic receptors display a specificity for the M2 subtype. In contrast to previous work, tachycardia-induced heart failure was accompanied by an increase (+ 53%) in ventricular, but not atrial, M2 receptors compared with normal dogs.

The Role of mGlu Receptors in Hippocampal Plasticity Deficits in Neurological and Psychiatric Disorders: Implications for Allosteric Modulators as Novel Therapeutic Strategies

PubMed Central

Senter, Rebecca K.; Ghoshal, Ayan; Walker, Adam G.; Xiang, Zixiu; Niswender, Colleen M.; Conn, P. Jeffrey

2016-01-01

Long-term potentiation (LTP) and long-term depression (LTD) are two distinct forms of synaptic plasticity that have been extensively characterized at the Schaffer collateral-CA1 (SC-CA1) synapse and the mossy fiber (MF)-CA3 synapse within the hippocampus, and are postulated to be the molecular underpinning for several cognitive functions. Deficits in LTP and LTD have been implicated in the pathophysiology of several neurological and psychiatric disorders. Therefore, there has been a large effort focused on developing an understanding of the mechanisms underlying these forms of plasticity and novel therapeutic strategies that improve or rescue these plasticity deficits. Among many other targets, the metabotropic glutamate (mGlu) receptors show promise as novel therapeutic candidates for the treatment of these disorders. Among the eight distinct mGlu receptor subtypes (mGlu1-8), the mGlu1,2,3,5,7 subtypes are expressed throughout the hippocampus and have been shown to play important roles in the regulation of synaptic plasticity in this brain area. However, development of therapeutic agents that target these mGlu receptors has been hampered by a lack of subtype-selective compounds. Recently, discovery of allosteric modulators of mGlu receptors has provided novel ligands that are highly selective for individual mGlu receptor subtypes. The mGlu receptors modulate the multiple forms of synaptic plasticity at both SC-CA1 and MF synapses and allosteric modulators of mGlu receptors have emerged as potential therapeutic agents that may rescue plasticity deficits and improve cognitive function in patients suffering from multiple neurological and psychiatric disorders. PMID:27296640

Structural characterization of B and non-B subtypes of HIV-protease: insights into the natural susceptibility to drug resistance development.

PubMed

Sanches, Mario; Krauchenco, Sandra; Martins, Nadia H; Gustchina, Alla; Wlodawer, Alexander; Polikarpov, Igor

2007-06-15

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.

Surface expression of hippocampal NMDA GluN2B receptors regulated by fear conditioning determines its contribution to memory consolidation in adult rats.

PubMed

Sun, Yan-Yan; Cai, Wei; Yu, Jie; Liu, Shu-Su; Zhuo, Min; Li, Bao-Ming; Zhang, Xue-Han

2016-08-04

The number and subtype composition of N-methyl-d-aspartate receptor (NMDAR) at synapses determines their functional properties and role in learning and memory. Genetically increased or decreased amount of GluN2B affects hippocampus-dependent memory in the adult brain. But in some experimental conditions (e.g., memory elicited by a single conditioning trial (1 CS-US)), GluN2B is not a necessary factor, which indicates that the precise role of GluN2B in memory formation requires further exploration. Here, we examined the role of GluN2B in the consolidation of fear memory using two training paradigms. We found that GluN2B was only required for the consolidation of memory elicited by five conditioning trials (5 CS-US), not by 1 CS-US. Strikingly, the expression of membrane GluN2B in CA1was training-strength-dependently increased after conditioning, and that the amount of membrane GluN2B determined its involvement in memory consolidation. Additionally, we demonstrated the increases in the activities of cAMP, ERK, and CREB in the CA1 after conditioning, as well as the enhanced intrinsic excitability and synaptic efficacy in CA1 neurons. Up-regulation of membrane GluN2B contributed to these enhancements. These studies uncover a novel mechanism for the involvement of GluN2B in memory consolidation by its accumulation at the cell surface in response to behavioral training.

One-pot synthesis and sigma receptor binding studies of novel spirocyclic-2,6-diketopiperazine derivatives.

PubMed

Ghandi, Mehdi; Sherafat, Fatemeh; Sadeghzadeh, Masoud; Alirezapour, Behrouz

2016-06-01

New spirocyclic-2,6-diketopiperazine derivatives containing benzylpiperidine and cycloalkane moieties were synthesized by a one-pot two-step sequential Ugi/intramolecular N-amidation process in moderate to good yields. The in vitro ligand-binding profile studies performed on the sigma-1 and sigma-2 receptors revealed that the σ1 affinities and subtype selectivities of three spirocyclic piperidine derivatives are generally comparable to those of spirocycloalkane analogues. Compared to the low σ1 affinities obtained for cycloalkyl-substituted spirocyclic-2,6-diketopiperazines with n=2, those with n=1 proved to have optimal fitting with σ2 subtype by exhibiting higher affinities. Moreover, the best binding affinity and subtype selectivity was identified for compound 3c with Kiσ1=5.9±0.5nM and Kiσ2=563±21nM as well as 95-fold σ1/σ2 selectivity ratio, respectively. Copyright © 2016. Published by Elsevier Ltd.

Expression and sub-cellular localization of an epigenetic regulator, co-activator arginine methyltransferase 1 (CARM1), is associated with specific breast cancer subtypes and ethnicity

PubMed Central

2013-01-01

Background Co-Activator Arginine Methyltransferase 1(CARM1) is an Estrogen Receptor (ER) cofactor that remodels chromatin for gene regulation via methylation of Histone3. We investigated CARM1 levels and localization across breast cancer tumors in a cohort of patients of either European or African ancestry. Methods We analyzed CARM1 levels using tissue microarrays with over 800 histological samples from 549 female cancer patients from the US and Nigeria, Africa. We assessed associations between CARM1 expression localized to the nucleus and cytoplasm for 11 distinct variables, including; ER status, Progesterone Receptor status, molecular subtypes, ethnicity, HER2+ status, other clinical variables and survival. Results We found that levels of cytoplasmic CARM1 are distinct among tumor sub-types and increased levels are associated with ER-negative (ER-) status. Higher nuclear CARM1 levels are associated with HER2 receptor status. EGFR expression also correlates with localization of CARM1 into the cytoplasm. This suggests there are distinct functions of CARM1 among molecular tumor types. Our data reveals a basal-like subtype association with CARM1, possibly due to expression of Epidermal Growth Factor Receptor (EGFR). Lastly, increased cytoplasmic CARM1, relative to nuclear levels, appear to be associated with self-identified African ethnicity and this result is being further investigated using quantified genetic ancestry measures. Conclusions Although it is known to be an ER cofactor in breast cancer, CARM1 expression levels are independent of ER. CARM1 has distinct functions among molecular subtypes, as is indicative of its sub-cellular localization and it may function in subtype etiology. These sub-cellular localization patterns, indicate a novel role beyond its ER cofactor function in breast cancer. Differential localization among ethnic groups may be due to ancestry-specific polymorphisms which alter the gene product. PMID:23663560

The sleep-modulating peptide orexin-B protects midbrain dopamine neurons from degeneration, alone or in cooperation with nicotine.

PubMed

Guerreiro, Serge; Florence, Clélia; Rousseau, Erwann; Hamadat, Sabah; Hirsch, Etienne C; Michel, Patrick P

2015-01-01

To determine whether orexinergic hypothalamic peptides can influence the survival of brainstem dopamine (DA) neurons, we used a model system of rat midbrain cultures in which DA neurons degenerate spontaneously and progressively as they mature. We established that orexin (OX)-B provides partial but significant protection to spontaneously dying DA neurons, whereas the homologous peptide OXA has only marginal effects. Importantly, DA neurons rescued by OXB accumulated DA efficiently by active transport, suggesting that they were functional. G-protein-coupled OX1 and OX2 receptors were both present on DA neurons, but the protective effect of OXB was attributable solely to OX2 receptors; a selective inhibitor of this receptor subtype, N-ethyl-2-[(6-methoxy-3-pyridinyl)[(2-methylphenyl)sulfonyl]amino]-N-(3-pyridinylmethyl)-acetamide (EMPA), suppressed this effect, whereas a selective agonist, [Ala(11), d-Leu(15)]OXB, reproduced it. Survival promotion by OXB required intracellular calcium mobilization via inositol-1,4,5-triphosphate and ryanodine receptors. Nicotine, a well known neuroprotective molecule for DA neurons, improved OXB-mediated rescue through the activation of α-bungarotoxin-sensitive (presumably α7) nicotinic receptors, although nicotine had no effect on its own. Altogether, our data suggest that the loss of hypothalamic orexinergic neurons that occurs in Parkinson's disease might confer an increased vulnerability to midbrain DA neurons in this disorder. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Interactions Between Vitamin D and Breast Cancer

DTIC Science & Technology

2010-07-01

BCL2 VDR C CYP24A1 CYP27B1 HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF D CDKN1A IGFBP3 SPP1 AR PTHLH AMH FABP5 PPARG GSTM1 GDC RTC PPC E...HR SNAI2 MYC PTGS2 HPGD PTGER4 DUSP10 IL6 TGFB1 TNF H CDKN1A IGFBP3 SPP1 AR PTHLH AMH FABP5 PPARG GSTM1 GDC RTC PPC GDC: Genomic DNA...cyclooxygenase) C07 Hs.655491 NM_000860 HPGD Hydroxyprostaglandin dehydrogenase 15-(NAD) C08 Hs.199248 NM_000958 PTGER4 Prostaglandin E receptor 4 (subtype

SB-205384 Is a Positive Allosteric Modulator of Recombinant GABAA Receptors Containing Rat α3, α5, or α6 Subunit Subtypes Coexpressed with β3 and γ2 Subunits

PubMed Central

Heidelberg, Laura S.; Warren, James W.

2013-01-01

Many drugs used to treat anxiety are positive modulators of GABAA receptors, which mediate fast inhibitory neurotransmission. The GABAA receptors can be assembled from a combination of at least 16 different subunits. The receptor’s subunit composition determines its pharmacologic and functional properties, and subunit expression varies throughout the brain. A primary goal for new treatments targeting GABAA receptors is the production of subunit-selective modulators acting upon a discrete population of receptors. The anxiolytic 4-amino-7-hydroxy-2-methyl-5,6,7,8,-tetrahydrobenzo[b]thieno[2,3-b]pyridine-3-carboxylic acid, but-2-ynyl ester (SB-205384) is widely considered to be selective for α3-containing GABAA receptors. However, it has been tested only on α1-, α2-, and α3-containing receptors. We examined the activity of SB-205384 at recombinant receptors containing the six different α subunits and found that receptors containing the α3, α5, and α6 subunits were potentiated by SB-205384, with the α6 subunit conferring the greatest responsiveness. Properties associated with chimeric α1/α6 subunits suggested that multiple structural domains influence sensitivity to SB-205384. Point mutations of residues within the extracellular N-terminal domain identified a leucine residue located in loop E of the agonist binding site as an important determinant of high sensitivity to modulation. In the α6 subunit the identity of this residue is species-dependent, with the leucine found in rat subunits but not in human. Our results indicate that SB-205384 is not an α3-selective modulator, and instead acts at several GABAA receptor isoforms. These findings have implications for the side-effect profile of this anxiolytic as well as for its use in neuronal and animal studies as a marker for contribution from α3-containing receptors. PMID:23902941

Differential Regulation of Cell Proliferation and Apoptosis by Melatonin Receptor Subtype-Signaling in the Adult Murine Brain.

PubMed

Fredrich, Michaela; Christ, Elmar; Korf, Horst-Werner

2018-06-27

Background/Aims: Zeitgeber time (ZT)-dependent changes in cell proliferation and apoptosis are regulated by melatonin receptor (MT)-mediated signaling in the adult hippocampus and hypothalamic-hypophyseal system. There are two G-protein-coupled MT-subtypes, MT1 and MT2. Therefore, the present study examined which MT-subtype is required for regulation of ZT-dependent changes in cell proliferation and/or apoptosis in the adult murine brain and pituitary. Adult melatonin-proficient (C3H) mice with targeted deletion of MT1 (MT1 KO) or MT2 (MT2 KO) were adapted to a 12-hour light, 12-hour dark photoperiod and sacrificed at ZT00, ZT06, ZT12, and ZT18. Immunohistochemistry for Ki67 or activated caspase-3 served to quantify proliferating and apoptotic cells in the hippocampal subgranular zone (SGZ) and granule cell layer, the hypothalamic median eminence (ME), and the hypophyseal pars tuberalis. ZT-dependent changes in cell proliferation were found exclusively in the SGZ and ME of MT1 KO mice, while apoptosis showed no ZT-dependent changes in the regions analyzed, neither in MT1 nor in MT2 KO mice. Comparison with our previous studies in C3H mice with functional MTs and MT1/2 KO mice revealed that MT2-mediated signaling is required and sufficient for ZT-dependent changes in cell proliferation in the SGZ and ME, while ZT-dependent changes in apoptosis require signaling from both MT-subtypes. Our results indicate that generation and timing of ZT-dependent changes in cell proliferation and apoptosis by melatonin require different MT-subtype-constellations and emphasize the importance to shed light on the specific function of each receptor-subtype in different tissues and physiological conditions.
. ©2018S. Karger AG, Basel.

Adrenoceptor function and expression in bladder urothelium and lamina propria.

PubMed

Moro, Christian; Tajouri, Lotti; Chess-Williams, Russ

2013-01-01

To investigate the role of adrenoceptor subtypes in regulating the spontaneous contractile activity of the inner lining of the urinary bladder (urothelium/lamina propria). The responses of isolated strips of porcine urothelium/lamina propria to noradrenaline, phenylephrine, and isoprenaline were obtained in the absence and presence of receptor subtype-selective antagonists. Quantitative reverse-transcriptase polymerase chain reaction was undertaken to assess the expression of adrenoceptor genes. The tissues expressed all α1- and β-adrenoceptor subtypes, with the α1A-, α1B-, and β2-adrenoceptors the predominant receptors at the messenger RNA level. In the functional experiments, the rate of phasic contractions and the basal tension were increased by the α1-adrenoceptor agonists phenylephrine (100 μM) and A61603 (10 μM). The rate and tension responses to phenylephrine were reduced by low concentrations of tamsulosin (3 nM) and RS100329 (10 nM) but were unaffected by BMY7378 (100 nM), prazosin (10 nM), and RS17053 (1 μM). In contrast, isoprenaline and salbutamol (both 1 μM) induced a relaxation of tissues and slowing of phasic contractions. The rate and tension responses to isoprenaline were inhibited by propranolol (100 nM) or a combination of CGP20712A (30 nM) and ICI118551 (70 nM). The rate responses were also significantly inhibited by ICI118551 alone (70 nM). Although all α1- and β-adrenoceptor subtypes were expressed in the pig urothelium/lamina propria, the α1A/L-adrenoceptor appeared to mediate increases in the contractile rate and tension. The β-adrenoceptor induced inhibition of spontaneous contractile activity appears to be predominately mediated by β2-adrenoceptors, with β1- and β2-adrenoceptors possibly involved in the tension responses. Copyright © 2013 Elsevier Inc. All rights reserved.

Muscarinic Stimulation Facilitates Sarcoplasmic Reticulum Ca Release by Modulating Ryanodine Receptor 2 Phosphorylation Through Protein Kinase G and Ca/Calmodulin-Dependent Protein Kinase II.

PubMed

Ho, Hsiang-Ting; Belevych, Andriy E; Liu, Bin; Bonilla, Ingrid M; Radwański, Przemysław B; Kubasov, Igor V; Valdivia, Héctor H; Schober, Karsten; Carnes, Cynthia A; Györke, Sándor

2016-11-01

Although the effects and the underlying mechanism of sympathetic stimulation on cardiac Ca handling are relatively well established both in health and disease, the modes of action and mechanisms of parasympathetic modulation are poorly defined. Here, we demonstrate that parasympathetic stimulation initiates a novel mode of excitation-contraction coupling that enhances the efficiency of cardiac sarcoplasmic reticulum Ca store utilization. This efficient mode of excitation-contraction coupling involves reciprocal changes in the phosphorylation of ryanodine receptor 2 at Ser-2808 and Ser-2814. Specifically, Ser-2808 phosphorylation was mediated by muscarinic receptor subtype 2 and activation of PKG (protein kinase G), whereas dephosphorylation of Ser-2814 involved activation of muscarinic receptor subtype 3 and decreased reactive oxygen species-dependent activation of CaMKII (Ca/calmodulin-dependent protein kinase II). The overall effect of these changes in phosphorylation of ryanodine receptor 2 is an increase in systolic Ca release at the low sarcoplasmic reticulum Ca content and a paradoxical reduction in aberrant Ca leak. Accordingly, cholinergic stimulation of cardiomyocytes isolated from failing hearts improved Ca cycling efficiency by restoring altered ryanodine receptor 2 phosphorylation balance. © 2016 American Heart Association, Inc.

Spreading of HIV-1 subtype G and envB/gagG recombinant strains among injecting drug users in Lisbon, Portugal.

PubMed

Esteves, Aida; Parreira, Ricardo; Piedade, João; Venenno, Teresa; Franco, Margarida; Germano de Sousa, José; Patrício, Luis; Brum, Paula; Costa, António; Canas-Ferreira, Wanda F

2003-06-01

We have evaluated the genetic diversity of HIV-1 strains infecting injecting drug users (IDUs) in Lisbon, Portugal. Heteroduplex mobility assay and/or phylogenetic analysis revealed that env (C2V3C3 or gp41) subtype B is present in 63.7% of the 135 viral samples studied, followed by subtypes G (23.7%), A (6.7%), F (5.2%), and D (0.7%). Similar analysis of gag (p24/p7) performed on 91 of the specimens demonstrated that 49.5% of the infections were caused by subtype G viruses; other gag subtypes identified were B (39.5%), F (3.3%), A and D (1.1.% each), and the recombinant circulating form CRF02_AG (5.5%). Discordant env/gag sub-types were detected in 34.1% of the strains and may reflect the presence of dual infections and/or recombinant viruses. The presumptive B/G recombinant form was highly predominant (21 of 31). The genetic pattern of HIV-1 subtype B and G strains is suggestive of multiple introductions and recombination episodes and of a longstanding presence of both subtypes in the country. C2V3C3 amino acid sequences from IDU-derived subtype G viruses presented highly significant signatures, which distinguish the variants from this transmission group. The unusually high prevalence of subtype G sequences (34.1%), independent of the geographic origin of the infected individuals, makes this IDU HIV-1 epidemic unique.

Trace metals in the brain: allosteric modulators of ligand-gated receptor channels, the case of ATP-gated P2X receptors.

PubMed

Huidobro-Toro, J Pablo; Lorca, Ramón A; Coddou, Claudio

2008-03-01

Zinc and copper are indispensable trace metals for life with a recognized role as catalysts in enzyme actions. We now review evidence supporting the role of trace metals as novel allosteric modulators of ionotropic receptors: a new and fundamental physiological role for zinc and copper in neuronal and brain excitability. The review is focussed on ionotropic receptor channels including nucleotide receptors, in particular the P2X receptor family. Since zinc and copper are stored within synaptic vesicles in selected brain regions, and released to the synaptic cleft upon electrical nerve ending depolarization, it is plausible that zinc and copper reach concentrations in the synapse that profoundly affect ligand-gated ionic channels, including the ATP-gated currents of P2X receptors. The identification of key P2X receptor amino acids that act as ligands for trace metal coordination, carves the structural determinants underlying the allosteric nature of the trace metal modulation. The recognition that the identified key residues such as histidines, aspartic and glutamic acids or cysteines in the extracellular domain are different for each P2X receptor subtype and may be different for each metal, highlights the notion that each P2X receptor subtype evolved independent strategies for metal coordination, which form upon the proper three-dimensional folding of the receptor channels. The understanding of the molecular mechanism of allosteric modulation of ligand-operated ionic channels by trace metals is a new contribution to metallo-neurobiology.

Molecular modeling of interactions of the non-peptide antagonist YM087 with the human vasopressin V1a, V2 receptors and with oxytocin receptors.

NASA Astrophysics Data System (ADS)

Giełdoń, Artur; Kaźmierkiewicz, Rajmund; Ślusarz, Rafał; Ciarkowski, Jerzy

2001-12-01

The nonapeptide hormones arginine vasopressin (CYFQNCPRG-NH2, AVP) and oxytocin (CYIQNCPLG-NH2, OT), control many essential functions in mammals. Their main activities include the urine concentration (via stimulation of AVP V2 receptors, V2R, in the kidneys), blood pressure regulation (via stimulation of vascular V1a AVP receptors, V1aR), ACTH control (via stimulation of V1b receptors, V1bR, in the pituitary) and labor and lactation control (via stimulation of OT receptors, OTR, in the uterus and nipples, respectively). All four receptor subtypes belong to the GTP-binding (G) protein-coupled receptor (GPCR) family. This work consists of docking of YM087, a potent non-peptide V1aR and V2R - but not OTR - antagonist, into the receptor models based on relatively new theoretical templates of rhodopsin (RD) and opiate receptors, proposed by Mosberg et al. (Univ. of Michigan, Ann Arbor, USA). It is simultaneously demonstrated that this RD template satisfactorily compares with the first historical GPCR structure of bovine rhodopsin (Palczewski et al., 2000) and that homology-modeling of V2R, V1aR and OTR using opiate receptors as templates is rational, based on relatively high (20-60%) sequence homology among the set of 4 neurophyseal and 4 opiate receptors. YM087 was computer-docked to V1aR, V2R and OTR using the AutoDock (Olson et al., Scripps Research Institute, La Jolla, USA) and subsequently relaxed using restrained simulated annealing and molecular dynamics, as implemented in AMBER program (Kollman et al., University of California, San Francisco, USA). From about 80 diverse configurations, sampled for each of the three ligand/receptor systems, 3 best energy-relaxed complexes were selected for mutual comparisons. Similar docking modes were found for the YM087/V1aR and YM087/V2R complexes, diverse from those of the YM087/OTR complexes, in agreement with the molecular affinity data.

Effect of MR Imaging Contrast Thresholds on Prediction of Neoadjuvant Chemotherapy Response in Breast Cancer Subtypes: A Subgroup Analysis of the ACRIN 6657/I-SPY 1 TRIAL

PubMed Central

Li, Wen; Arasu, Vignesh; Newitt, David C.; Jones, Ella F.; Wilmes, Lisa; Gibbs, Jessica; Kornak, John; Joe, Bonnie N.; Esserman, Laura J.; Hylton, Nola M.

2016-01-01

Functional tumor volume (FTV) measurements by dynamic contrast-enhanced magnetic resonance imaging can predict treatment outcomes for women receiving neoadjuvant chemotherapy for breast cancer. Here, we explore whether the contrast thresholds used to define FTV could be adjusted by breast cancer subtype to improve predictive performance. Absolute FTV and percent change in FTV (ΔFTV) at sequential time-points during treatment were calculated and investigated as predictors of pathologic complete response at surgery. Early percent enhancement threshold (PEt) and signal enhancement ratio threshold (SERt) were varied. The predictive performance of resulting FTV predictors was evaluated using the area under the receiver operating characteristic curve. A total number of 116 patients were studied both as a full cohort and in the following groups defined by hormone receptor (HR) and HER2 receptor subtype: 45 HR+/HER2−, 39 HER2+, and 30 triple negatives. High AUCs were found at different ranges of PEt and SERt levels in different subtypes. Findings from this study suggest that the predictive performance to treatment response by MRI varies by contrast thresholds, and that pathologic complete response prediction may be improved through subtype-specific contrast enhancement thresholds. A validation study is underway with a larger patient population. PMID:28066808

Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes.

PubMed

Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R

2016-06-13

In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Blockade of Cocaine or σ Receptor Agonist Self Administration by Subtype-Selective σ Receptor Antagonists.

PubMed

Katz, Jonathan L; Hiranita, Takato; Kopajtic, Theresa A; Rice, Kenner C; Mesangeau, Christophe; Narayanan, Sanju; Abdelazeem, Ahmed H; McCurdy, Christopher R

2016-07-01

The identification of sigma receptor (σR) subtypes has been based on radioligand binding and, despite progress with σ1R cellular function, less is known about σR subtype functions in vivo. Recent findings that cocaine self administration experience will trigger σR agonist self administration was used in this study to assess the in vivo receptor subtype specificity of the agonists (+)-pentazocine, PRE-084 [2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate hydrochloride], and 1,3-di-o-tolylguanidine (DTG) and several novel putative σR antagonists. Radioligand binding studies determined in vitro σR selectivity of the novel compounds, which were subsequently studied for self administration and antagonism of cocaine, (+)-pentazocine, PRE-084, or DTG self administration. Across the dose ranges studied, none of the novel compounds were self administered, nor did they alter cocaine self administration. All compounds blocked DTG self administration, with a subset also blocking (+)-pentazocine and PRE-084 self administration. The most selective of the compounds in binding σ1Rs blocked cocaine self administration when combined with a dopamine transport inhibitor, either methylphenidate or nomifensine. These drug combinations did not decrease rates of responding maintained by food reinforcement. In contrast, the most selective of the compounds in binding σ2Rs had no effect on cocaine self administration in combination with either dopamine transport inhibitor. Thus, these results identify subtype-specific in vivo antagonists, and the utility of σR agonist substitution for cocaine self administration as an assay capable of distinguishing σR subtype selectivity in vivo. These results further suggest that effectiveness of dual σR antagonism and dopamine transport inhibition in blocking cocaine self administration is specific for σ1Rs and further support this dual targeting approach to development of cocaine antagonists. U.S. Government work not protected by U.S. copyright.

Imaging features of breast cancers on digital breast tomosynthesis according to molecular subtype: association with breast cancer detection.

PubMed

Lee, Su Hyun; Chang, Jung Min; Shin, Sung Ui; Chu, A Jung; Yi, Ann; Cho, Nariya; Moon, Woo Kyung

2017-12-01

To evaluate imaging features of breast cancers on digital breast tomosynthesis (DBT) according to molecular subtype and to determine whether the molecular subtype affects breast cancer detection on DBT. This was an institutional review board--approved study with a waiver of informed consent. DBT findings of 288 invasive breast cancers were reviewed according to Breast Imaging Reporting and Data System lexicon. Detectability of breast cancer was quantified by the number of readers (0-3) who correctly detected the cancer in an independent blinded review. DBT features and the cancer detectability score according to molecular subtype were compared using Fisher's exact test and analysis of variance. Of 288 invasive cancers, 194 were hormone receptor (HR)-positive, 48 were human epidermal growth factor receptor 2 (HER2) positive and 46 were triple negative breast cancers. The most common DBT findings were irregular spiculated masses for HR-positive cancer, fine pleomorphic or linear branching calcifications for HER2 positive cancer and irregular masses with circumscribed margins for triple negative breast cancers (p < 0.001). Cancer detectability on DBT was not significantly different according to molecular subtype (p = 0.213) but rather affected by tumour size, breast density and presence of mass or calcifications. Breast cancers showed different imaging features according to molecular subtype; however, it did not affect the cancer detectability on DBT. Advances in knowledge: DBT showed characteristic imaging features of breast cancers according to molecular subtype. However, cancer detectability on DBT was not affected by molecular subtype of breast cancers.

HIV-1 pol mutation frequency by subtype and treatment experience: extension of the HIVseq program to seven non-B subtypes.

PubMed

Rhee, Soo-Yon; Kantor, Rami; Katzenstein, David A; Camacho, Ricardo; Morris, Lynn; Sirivichayakul, Sunee; Jorgensen, Louise; Brigido, Luis F; Schapiro, Jonathan M; Shafer, Robert W

2006-03-21

HIVseq was developed in 2000 to make published data on the frequency of HIV-1 group M protease and reverse transcriptase (RT) mutations available in real time to laboratories and researchers sequencing these genes. Because most published protease and RT sequences belonged to subtype B, the initial version of HIVseq was based on this subtype. As additional non-B sequences from persons with well-characterized antiretroviral treatment histories have become available, the program has been extended to subtypes A, C, D, F, G, CRF01, and CRF02. The latest frequency of each protease and RT mutation according to subtype and drug-class exposure was calculated using published sequences in the Stanford HIV RT and Protease Sequence Database. Each mutation was hyperlinked to published reports of viruses containing the mutation. As of September 2005, the mean number of protease sequences per non-B subtype was 534 from protease inhibitor-naive persons and 133 from protease inhibitor-treated persons, representing 13.2% and 2.3%, respectively, of the data available for subtype B. The mean number of RT sequences per non-B subtype was 373 from RT inhibitor-naive persons and 288 from RT inhibitor-treated persons, representing 17.9% and 3.8%, respectively, of the data available for subtype B. HIVseq allows users to examine protease and RT mutations within the context of previously published sequences of these genes. The publication of additional non-B protease and RT sequences from persons with well-characterized treatment histories, however, will be required to perform the same types of analysis possible with the much larger number of subtype B sequences.

GPER-1 and estrogen receptor-β ligands modulate aldosterone synthesis.

PubMed

Caroccia, Brasilina; Seccia, Teresa M; Campos, Abril Gonzalez; Gioco, Francesca; Kuppusamy, Maniselvan; Ceolotto, Giulio; Guerzoni, Eugenia; Simonato, Francesca; Mareso, Sara; Lenzini, Livia; Fassina, Ambrogio; Rossi, Gian Paolo

2014-11-01

Fertile women have lower blood pressure and cardiovascular risk than age-matched men, which suggests that estrogens exert cardiovascular protective effects. However, whether 17 β-estradiol (E2) blunts aldosterone secretion, and thereby affects the gender dimorphism of blood pressure, is unknown. We therefore sought for the estrogen receptor (ER) subtypes in human adrenocortical tissues ex vivo by performing gene and protein expression studies. We also investigated the effect of E2 on aldosterone synthesis and the involved receptors through in vitro functional experiments in the adrenocortical cells HAC15. We found that in the human adrenal cortex and aldosterone-producing adenoma cells, the most expressed ERs were the ERβ and the G protein-coupled receptor-1 (GPER-1), respectively. After selective ERβ blockade, E2 (10 nmol/L) markedly increased both the expression of aldosterone synthase and the production of aldosterone (+5- to 7-fold vs baseline, P < .001). Under the same condition, the GPER-1 receptor agonist 1-[4-(6-bromo-benzo (1, 3)dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c] quinolin-8-yl]-ethanone (G-1) (10 nmol/L) mimicked this effect, which was abrogated by cotreatment with either the GPER-1 receptor antagonist (3aS*,4R*,9bR*)-4-(6-Bro-mo-1,3-benzodioxol-5-yl)-3a,4,5,9b-3H-cyclopenta[c]quinoline (G-15), or a selective protein kinase A inhibitor 8-Bromo-2-monobutyryladenosine-3,5-cyclic mono-phosphorothioate, Rp-isomer. Silencing of the ERβ significantly raised aldosterone synthase expression and aldosterone production. Conversely, silencing of the GPER-1 lowered aldosterone synthase gene and protein expression. Moreover, it blunted the stimulatory effect of E2 on aldosterone synthase that was seen during ERβ blockade. These results support the conclusion that in humans, E2 inhibits aldosterone synthesis by acting via ERβ. Pharmacologic disinhibition of ERβ unmasks a potent secretagogue effect of E2 that involves GPER-1 and protein kinase A signaling.

Targeting Non-proteolytic Protein Ubiquitination for the Treatment of Diffuse Large B Cell Lymphoma.

PubMed

Yang, Yibin; Kelly, Priscilla; Shaffer, Arthur L; Schmitz, Roland; Yoo, Hee Min; Liu, Xinyue; Huang, Da Wei; Webster, Daniel; Young, Ryan M; Nakagawa, Masao; Ceribelli, Michele; Wright, George W; Yang, Yandan; Zhao, Hong; Yu, Xin; Xu, Weihong; Chan, Wing C; Jaffe, Elaine S; Gascoyne, Randy D; Campo, Elias; Rosenwald, Andreas; Ott, German; Delabie, Jan; Rimsza, Lisa; Staudt, Louis M

2016-04-11

Chronic active B cell receptor (BCR) signaling, a hallmark of the activated B cell-like (ABC) subtype of diffuse large B cell lymphoma (DLBCL), engages the CARD11-MALT1-BCL10 (CBM) adapter complex to activate IκB kinase (IKK) and the classical NF-κB pathway. Here we show that the CBM complex includes the E3 ubiquitin ligases cIAP1 and cIAP2, which are essential mediators of BCR-dependent NF-κB activity in ABC DLBCL. cIAP1/2 attach K63-linked polyubiquitin chains on themselves and on BCL10, resulting in the recruitment of IKK and the linear ubiquitin chain ligase LUBAC, which is essential for IKK activation. SMAC mimetics target cIAP1/2 for destruction, and consequently suppress NF-κB and selectively kill BCR-dependent ABC DLBCL lines, supporting their clinical evaluation in patients with ABC DLBCL. Copyright © 2016 Elsevier Inc. All rights reserved.

Signaling via the transcriptionally regulated activin receptor 2B is a novel mediator of neuronal cell death during chicken ciliary ganglion development.

PubMed

Koszinowski, S; Buss, K; Kaehlcke, K; Krieglstein, K

2015-04-01

The TGF-β ligand superfamily members activin A and BMP control important aspects of embryonic neuronal development and differentiation. Both are known to bind to activin receptor subtypes IIA (ActRIIA) and IIB, while in the avian ciliary ganglion (CG), so far only ActRIIA-expression has been described. We show that the expression of ACVR2B, coding for the ActRIIB, is tightly regulated during CG development and the knockdown of ACVR2B expression leads to a deregulation in the execution of neuronal apoptosis and therefore affects ontogenetic programmed cell death in vivo. While the differentiation of choroid neurons was impeded in the knockdown, pointing toward a reduction in activin A-mediated neural differentiation signaling, naturally occurring neuronal cell death in the CG was not prevented by follistatin treatment. Systemic injections of the BMP antagonist noggin, on the other hand, reduced the number of apoptotic neurons to a similar extent as ACVR2B knockdown. We therefore propose a novel pathway in the regulation of CG neuron ontogenetic programmed cell death, which could be mediated by BMP and signals via the ActRIIB. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

Somatostatin receptor subtype-4 agonist NNC 26-9100 decreases extracellular and intracellular Aβ₁₋₄₂ trimers.

PubMed

Sandoval, Karin E; Farr, Susan A; Banks, William A; Crider, Albert M; Morley, John E; Witt, Ken A

2012-05-15

Soluble amyloid β-protein (Aβ) oligomers are primary mediators of synaptic dysfunction associated with the progression of Alzheimer's disease. Such Aβ oligomers exist dependent on their rates of aggregation and metabolism. Use of selective somatostatin receptor-subtype agonists have been identified as a potential means to mitigate Aβ accumulation in the brain, via regulation of the enzyme neprilysin. Herein, we first evaluated the impact of the somatostatin receptor subtype-4 agonist 1-[3-[N-(5-Bromopyridin-2-yl)-N-(3,4-dichlorobenzyl)amino]propyl]-3-[3-(1H-imidazol-4-yl)propyl]thiourea (NNC 26-9100) on learning and memory in 12-month SAMP8 mice (i.c.v. injection). NNC 26-9100 (0.2 μg-dose) was shown to enhance both learning (T-maze) and memory (object recognition) compared to vehicle controls. Cortical and hippocampal tissues were evaluated subsequent to NNC 26-9100 (0.2 μg) or vehicle administration for changes in neprilysin activity, along with protein expression of amyloid-precursor protein (APP), neprilysin, and Aβ₁₋₄₂ oligomers within respective cellular fractions (extracellular, intracellular and membrane). NNC 26-9100 increased neprilysin activity in cortical tissue, with an associated protein expression increase in the extracellular fraction and decreased in the intracellular fraction. A decrease in intracellular APP expression was found with treatment in both cortical and hippocampal tissues. NNC 26-9100 also significantly decreased expression of Aβ₁₋₄₂ trimers within both the extracellular and intracellular cortical fractions. No expression changes were found in membrane fractions for any protein. These finding suggest the potential use of selective SSTR4 agonists to mitigate toxic oligomeric forms of Aβ₁₋₄₂ in critical regions of the brain identified with learning and memory decline. Copyright © 2012 Elsevier B.V. All rights reserved.

A new serotonin 5-HT6 receptor antagonist with procognitive activity - Importance of a halogen bond interaction to stabilize the binding

NASA Astrophysics Data System (ADS)

González-Vera, Juan A.; Medina, Rocío A.; Martín-Fontecha, Mar; Gonzalez, Angel; de La Fuente, Tania; Vázquez-Villa, Henar; García-Cárceles, Javier; Botta, Joaquín; McCormick, Peter J.; Benhamú, Bellinda; Pardo, Leonardo; López-Rodríguez, María L.

2017-01-01

Serotonin 5-HT6 receptor has been proposed as a promising therapeutic target for cognition enhancement though the development of new antagonists is still needed to validate these molecules as a drug class for the treatment of Alzheimer’s disease and other pathologies associated with memory deficiency. As part of our efforts to target the 5-HT6 receptor, new benzimidazole-based compounds have been designed and synthesized. Site-directed mutagenesis and homology models show the importance of a halogen bond interaction between a chlorine atom of the new class of 5-HT6 receptor antagonists identified herein and a backbone carbonyl group in transmembrane domain 4. In vitro pharmacological characterization of 5-HT6 receptor antagonist 7 indicates high affinity and selectivity over a panel of receptors including 5-HT2B subtype and hERG channel, which suggests no major cardiac issues. Compound 7 exhibited in vivo procognitive activity (1 mg/kg, ip) in the novel object recognition task as a model of memory deficit.

Classifier ensemble based on feature selection and diversity measures for predicting the affinity of A(2B) adenosine receptor antagonists.

PubMed

Bonet, Isis; Franco-Montero, Pedro; Rivero, Virginia; Teijeira, Marta; Borges, Fernanda; Uriarte, Eugenio; Morales Helguera, Aliuska

2013-12-23

A(2B) adenosine receptor antagonists may be beneficial in treating diseases like asthma, diabetes, diabetic retinopathy, and certain cancers. This has stimulated research for the development of potent ligands for this subtype, based on quantitative structure-affinity relationships. In this work, a new ensemble machine learning algorithm is proposed for classification and prediction of the ligand-binding affinity of A(2B) adenosine receptor antagonists. This algorithm is based on the training of different classifier models with multiple training sets (composed of the same compounds but represented by diverse features). The k-nearest neighbor, decision trees, neural networks, and support vector machines were used as single classifiers. To select the base classifiers for combining into the ensemble, several diversity measures were employed. The final multiclassifier prediction results were computed from the output obtained by using a combination of selected base classifiers output, by utilizing different mathematical functions including the following: majority vote, maximum and average probability. In this work, 10-fold cross- and external validation were used. The strategy led to the following results: i) the single classifiers, together with previous features selections, resulted in good overall accuracy, ii) a comparison between single classifiers, and their combinations in the multiclassifier model, showed that using our ensemble gave a better performance than the single classifier model, and iii) our multiclassifier model performed better than the most widely used multiclassifier models in the literature. The results and statistical analysis demonstrated the supremacy of our multiclassifier approach for predicting the affinity of A(2B) adenosine receptor antagonists, and it can be used to develop other QSAR models.

Definition of the Cellular Mechanisms which Distinguish Between Estrogen Receptor Agonists and Antagonists

DTIC Science & Technology

2001-07-01

hormones: 10-7 M 17p3- estradiol for ERa and ERI3, 10-7 M progesterone for PR-A and PR-B, 10-7 M dexamethasone for GR, 10-7 M 5ot-dihydrotestosterone...cyproterone acetate, d-Ald.: d-aldosterone, DHEA: dehydroepiandrosterone, DOC: 11-deoxycorticosterone, Dex: dexamethasone, MPA: medroxyprogesterone , OH-F...two receptors are not functionally equivalent and that tory activities by altering ER structure and indepen- each subtype plays a unique role in ER

An intracellular loop 2 amino acid residue determines differential binding of arrestin to the dopamine D2 and D3 receptors.

PubMed

Lan, Hongxiang; Teeter, Martha M; Gurevich, Vsevolod V; Neve, Kim A

2009-01-01

Dopamine D(2) and D(3) receptors are similar subtypes with distinct interactions with arrestins; the D(3) receptor mediates less agonist-induced translocation of arrestins than the D(2) receptor. The goals of this study were to compare nonphosphorylated arrestin-binding determinants in the second intracellular domain (IC2) of the D(2) and D(3) receptors to identify residues that contribute to the differential binding of arrestin to the subtypes. Arrestin 3 bound to glutathione transferase (GST) fusion proteins of the D(2) receptor IC2 more avidly than to the D(3) receptor IC2. Mutagenesis of the fusion proteins identified a residue at the C terminus of IC2, Lys149, that was important for the preferential binding of arrestin 3 to D(2)-IC2; arrestin binding to D(2)-IC2-K149C was greatly decreased compared with wild-type D(2)-IC2, whereas binding to the reciprocal mutant D(3)-IC2-C147K was enhanced compared with wild-type D(3)-IC2. Mutating this lysine in the full-length D(2) receptor to cysteine decreased the ability of the D(2) receptor to mediate agonist-induced arrestin 3 translocation to the membrane and decreased agonist-induced receptor internalization in human embryonic kidney 293 cells. The reciprocal mutation in the D(3) receptor increased receptor-mediated translocation of arrestin 3 without affecting agonist-induced receptor internalization. G protein-coupled receptor crystal structures suggest that Lys149, at the junction of IC2 and the fourth membrane-spanning helix, has intramolecular interactions that contribute to maintaining an inactive receptor state. It is suggested that the preferential agonist-induced binding of arrestin3 to the D(2) receptor over the D(3) receptor is due in part to Lys149, which could be exposed as a result of receptor activation.

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.

Emerging molecular therapeutic targets for cholangiocarcinoma.

PubMed

Rizvi, Sumera; Gores, Gregory J

2017-09-01

Cholangiocarcinomas (CCAs) are diverse epithelial tumors arising from the liver or large bile ducts with features of cholangiocyte differentiation. CCAs are classified anatomically into intrahepatic (iCCA), perihilar (pCCA), and distal CCA (dCCA). Each subtype has distinct risk factors, molecular pathogenesis, therapeutic options, and prognosis. CCA is an aggressive malignancy with a poor overall prognosis and median survival of less than 2years in patients with advanced disease. Potentially curative surgical treatment options are limited to the subset of patients with early-stage disease. Presently, the available systemic medical therapies for advanced or metastatic CCA have limited therapeutic efficacy. Molecular alterations define the differences in biological behavior of each CCA subtype. Recent comprehensive genetic analysis has better characterized the genomic and transcriptomic landscape of each CCA subtype. Promising candidates for targeted, personalized therapy have emerged, including potential driver fibroblast growth factor receptor (FGFR) gene fusions and somatic mutations in isocitrate dehydrogenase (IDH)1/2 in iCCA, protein kinase cAMP-activated catalytic subunit alpha (PRKACA) or beta (PRKACB) gene fusions in pCCA, and ELF3 mutations in dCCA/ampullary carcinoma. A precision genomic medicine approach is dependent on an enhanced understanding of driver mutations in each subtype and stratification of patients according to their genetic drivers. We review the current genomic landscape of CCA, the potentially actionable molecular aberrations in each CCA subtype, and the role of immunotherapy in CCA. Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Tracking Progesterone Receptor-Mediated Actions in Breast Cancer

PubMed Central

Knutson, Todd P.; Lange, Carol A.

2014-01-01

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

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential.

PubMed

Arakawa, Hiroshi; Kamioka, Hiroki; Jomura, Tomoko; Koyama, Satoshi; Idota, Yoko; Yano, Kentaro; Kojima, Hajime; Ogihara, Takuo

2017-01-01

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids). Hepatocyte spheroids could be used up to 21 d after seeding (pre-culture for 7 d and exposure to inducer for up to 14 d), based on preliminary evaluation of basal activities of CYP subtypes and mRNA expression of the corresponding transcription factor and xenobiotic receptors (aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR) and pregnane X receptor (PXR)). After 2 d exposure of hepatocyte spheroids to omeprazole, phenobarbital and rifampicin (typical inducers of CYP1A2, 2B6 and 3A4, respectively), CYP1A2, 2B6 and 3A4 mRNA expression levels were significantly increased. The mRNA induction of CYP2B6 remained reasonably stable between days 2 and 14 of exposure to inducers, while induction of both CYP1A2 and 3A4 continued to increase up to day 14. These enzyme activities were all significantly increased compared with the control until day 14. Our findings indicate that our 3D hepatocyte spheroids system would be especially suitable for long-term testing of enzyme activity induction by drugs, either to predict or to verify clinical events.

Muscarinic receptor subtypes involved in urothelium-derived relaxatory effects in the inflamed rat urinary bladder.

PubMed

Andersson, M; Aronsson, P; Doufish, D; Lampert, A; Tobin, G

2012-09-25

Functional studies have shown altered cholinergic mechanisms in the inflamed bladder, which partly depend on muscarinic receptor-induced release of nitric oxide (NO). The current study aimed to characterize which muscarinic receptor subtypes that are involved in the regulation of the nitrergic effects in the bladder cholinergic response during cystitis. For this purpose, in vitro examinations of carbachol-evoked contractions of inflamed and normal bladder preparations were performed. The effects of antagonists with different selectivity for the receptor subtypes were assessed on intact and urothelium-denuded bladder preparations. In preparations from cyclophosphamide (CYP; in order to induce cystitis) pre-treated rats, the response to carbachol was about 75% of that of normal preparations. Removal of the urothelium or administration of a nitric oxide synthase inhibitor re-established the responses in the inflamed preparations. Administration of 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) inhibited the carbachol-induced contractile responses of preparations from CYP pre-treated rats less potently than controls. Pirenzepine and p-fluoro-hexahydro-sila-diphenidol (pFHHSiD) affected the carbachol-induced contractile responses to similar extents in preparations of CYP pre-treated and control rats. However, the Schild slopes for the three antagonists were all significantly different from unity in the preparations from CYP pre-treated rats. Again, L-NNA or removal of the urothelium eliminated any difference compared to normal preparations. This study confirms that muscarinic receptor stimulation in the inflamed rat urinary bladder induces urothelial release of NO, which counteracts detrusor contraction. Copyright © 2012 Elsevier B.V. All rights reserved.

[The potential of group II metabotropic glutamate receptor antagonists as a novel antidepressant].

PubMed

Chaki, Shigeyuki

2012-08-01

Recently, abnormalities of glutamatergic transmission have been implicated in the pathophysiology of depression. Moreover, both ketamine, an NMDA receptor antagonist, and riluzole, a modulator of glutamatergic, transmission have been reported to be effective for the treatment of patients with treatment-refractory depression. Based on these findings, extensive studies to develop agents acting on glutamatergic transmission have been conducted. Glutamate receptors are divided into two main subtypes, ionotropic glutamate receptors and metabotropic glutamate (mGlu) receptors, both of which have subtypes. Of these, much attention has been paid to mGlu2/3 receptors. mGlu2/3 receptor antagonists such as MGS0039 and LY341495 have been reported to exert antidepressant effects in animal models of depression including the forced swim test, tail suspension test, learned helplessness paradigm, olfactory bulmectomy model and isolation rearing model, and to enhance serotonin release in the prefrontal cortex and dopamine release in the nucleus accumbens. Moreover, activation of AMPA receptor and mTOR signaling have been suggested to be involved in the antidepressant effects of mGlu2/3 receptor antagonists, as demonstrated in the actions of ketamine. Thus, mGlu2/3 receptor antagonists may share some neural networks with ketamine in exerting their antidepressant effects. In addition, the potential of other agents targeting glutamatergic transmission for novel antidepressants is being investigated.

Pharmacological characterization of nucleotide P2Y receptors on endothelial cells of the mouse aorta

PubMed Central

Guns, Pieter-Jan D F; Korda, András; Crauwels, Herta M; Van Assche, Tim; Robaye, Bernard; Boeynaems, Jean-Marie; Bult, Hidde

2005-01-01

Nucleotides regulate various effects including vascular tone. This study was aimed to characterize P2Y receptors on endothelial cells of the aorta of C57BL6 mice. Five adjacent segments (width 2 mm) of the thoracic aorta were mounted in organ baths to measure isometric force development. Nucleotides evoked complete (adenosine 5′ triphosphate (ATP), uridine 5′ triphosphate (UTP), uridine 5′ diphosphate (UDP); >90%) or partial (adenosine 5′ diphosphate (ADP)) relaxation of phenylephrine precontracted thoracic aortic rings of C57BL6 mice. Relaxation was abolished by removal of the endothelium and was strongly suppressed (>90%) by inhibitors of nitric oxide synthesis. The rank order of potency was: UDP∼UTP∼ADP>adenosine 5′-[γ-thio] triphosphate (ATPγS)>ATP, with respective pD2 values of 6.31, 6.24, 6.22, 5.82 and 5.40. These results are compatible with the presence of P2Y1 (ADP>ATP), P2Y2 or P2Y4 (ATP and UTP) and P2Y6 (UDP) receptors. P2Y4 receptors were not involved, since P2Y4-deficient mice displayed unaltered responses to ATP and UTP. The purinergic receptor antagonist suramin exerted surmountable antagonism for all agonists. Its apparent pKb for ATP (4.53±0.07) was compatible with literature, but the pKb for UTP (5.19±0.03) was significantly higher. This discrepancy suggests that UTP activates supplementary non-P2Y2 receptor subtype(s). Further, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) showed surmountable (UTP, UDP), nonsurmountable (ADP) or no antagonism (ATP). Finally, 2′-deoxy-N6-methyladenosine3′,5′-bisphosphate (MRS2179) inhibited ADP-evoked relaxation only. Taken together, these results point to the presence of functional P2Y1 (ADP), P2Y2 (ATP, UTP) and P2Y6 (UDP) receptors on murine aorta endothelial cells. The identity of the receptor(s) mediating the action of UTP is not fully clear and other P2Y subtypes might be involved in UTP-evoked vasodilatation. PMID:15997227

Ocular Purine Receptors as Drug Targets in the Eye.

PubMed

Jacobson, Kenneth A; Civan, Mortimer M

2016-10-01

Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A 1 , A 2A , and A 3 . An A 1 AR agonist is in clinical trials for glaucoma, A 2A AR reduces neuroinflammation, A 3 AR protects retinal ganglion cells from apoptosis, and both A 3 AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y 2 and P2Y 6 , lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases.

Ocular Purine Receptors as Drug Targets in the Eye

PubMed Central

Civan, Mortimer M.

2016-01-01

Abstract Agonists and antagonists of various subtypes of G protein coupled adenosine receptors (ARs), P2Y receptors (P2YRs), and ATP-gated P2X receptor ion channels (P2XRs) are under consideration as agents for the treatment of ocular diseases, including glaucoma and dry eye. Numerous nucleoside and nonnucleoside modulators of the receptors are available as research tools and potential therapeutic molecules. Three of the 4 subtypes of ARs have been exploited with clinical candidate molecules for treatment of the eye: A1, A2A, and A3. An A1AR agonist is in clinical trials for glaucoma, A2AAR reduces neuroinflammation, A3AR protects retinal ganglion cells from apoptosis, and both A3AR agonists and antagonists had been reported to lower intraocular pressure (IOP). Extracellular concentrations of endogenous nucleotides, including dinucleoside polyphosphates, are increased in pathological states, activating P2Y and P2XRs throughout the eye. P2YR agonists, including P2Y2 and P2Y6, lower IOP. Antagonists of the P2X7R prevent the ATP-induced neuronal apoptosis in the retina. Thus, modulators of the purinome in the eye might be a source of new therapies for ocular diseases. PMID:27574786

Benzodiazepines modulate the A2 adenosine binding sites on 108CC15 neuroblastoma X glioma hybrid cells.

PubMed Central

Snell, C. R.; Snell, P. H.

1984-01-01

We have demonstrated high affinity diazepam binding sites of the Ro5-4864 benzodiazepine receptor subtype on 108CC15 neuroblastoma X glioma hybrid cells. These cells were previously shown to have purinoceptors of the A2 adenosine subtype and we have now found that [3H]-adenosine can be displaced from this binding site by the benzodiazepines and related compounds that can also bind to the Ro5-4864 site. Diazepam was found to have no intrinsic activity at the A2-receptor as measured by the stimulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) production in this cell line. At concentrations sufficient to compete for the A2-receptor, diazepam was shown to facilitate, by approximately 2 fold, the stimulation of cyclic AMP by adenosine. These effects are not due to inhibition of adenosine uptake or phosphodiesterase activity, but are probably a consequence of modulation of the coupling of the A2-receptor to cyclic AMP production in this hybrid cell line. PMID:6150742

Neonatal seizures alter NMDA glutamate receptor GluN2A and 3A subunit expression and function in hippocampal CA1 neurons

PubMed Central

Zhou, Chengwen; Sun, Hongyu; Klein, Peter M.; Jensen, Frances E.

2015-01-01

Neonatal seizures are commonly caused by hypoxic and/or ischemic injury during birth and can lead to long-term epilepsy and cognitive deficits. In a rodent hypoxic seizure (HS) model, we have previously demonstrated a critical role for seizure-induced enhancement of the AMPA subtype of glutamate receptor (GluA) in epileptogenesis and cognitive consequences, in part due to GluA maturational upregulation of expression. Similarly, as the expression and function of the N-Methyl-D-aspartate (NMDA) subtype of glutamate receptor (GluN) is also developmentally controlled, we examined how early life seizures during the critical period of synaptogenesis could modify GluN development and function. In a postnatal day (P)10 rat model of neonatal seizures, we found that seizures could alter GluN2/3 subunit composition of GluNs and physiological function of synaptic GluNs. In hippocampal slices removed from rats within 48–96 h following seizures, the amplitudes of synaptic GluN-mediated evoked excitatory postsynaptic currents (eEPSCs) were elevated in CA1 pyramidal neurons. Moreover, GluN eEPSCs showed a decreased sensitivity to GluN2B selective antagonists and decreased Mg2+ sensitivity at negative holding potentials, indicating a higher proportion of GluN2A and GluN3A subunit function, respectively. These physiological findings were accompanied by a concurrent increase in GluN2A phosphorylation and GluN3A protein. These results suggest that altered GluN function and expression could potentially contribute to future epileptogenesis following neonatal seizures, and may represent potential therapeutic targets for the blockade of future epileptogenesis in the developing brain. PMID:26441533

Rectification properties and Ca2+ permeability of glutamate receptor channels in hippocampal cells.

PubMed

Lerma, J; Morales, M; Ibarz, J M; Somohano, F

1994-07-01

Excitatory amino acids exert a depolarizing action on central nervous system cells through an increase in cationic conductances. Non-NMDA receptors have been considered to be selectively permeable to Na+ and K+, while Ca2+ influx has been thought to occur through the NMDA receptor subtype. Recently, however, the expression of cloned non-NMDA receptor subunits has shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are permeable to Ca2+ whenever the receptor lacks a particular subunit (edited GluR-B). The behaviour of recombinant glutamate receptor channels predicts that Ca2+ would only permeate through receptors that show strong inward rectification and vice versa, i.e. AMPA receptors with linear current-voltage relationships would be impermeable to Ca2+. Using the whole-cell configuration of the patch-clamp technique, we have studied the Ca2+ permeability and the rectifying properties of AMPA receptors, when activated by kainate, in hippocampal neurons kept in culture or acutely dissociated from differentiated hippocampus. Cells were classified according to whether they showed outward rectifying (type I), inward rectifying (type II) or almost linear (type III) current-voltage relationships for kainate-activated responses. AMPA receptors of type I cells (52.2%) were mostly Ca(2+)-impermeable (PCa/PCs = 0.1), while type II cells (6.5%) expressed Ca(2+)-permeable receptors (PCa/PCs = 0.9). Type III cells (41.3%) showed responses with low but not negligible Ca2+ permeability (PCa/PCs = 0.18). The degree of Ca2+ permeability and inward rectification were well correlated in cultured cells, i.e. more inward rectification corresponded to higher Ca2+ permeability.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery and structural optimization of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-ones as RORc inverse agonists.

PubMed

Wu, Xi-Shan; Wang, Rui; Xing, Yan-Li; Xue, Xiao-Qian; Zhang, Yan; Lu, Yong-Zhi; Song, Yu; Luo, Xiao-Yu; Wu, Chun; Zhou, Yu-Lai; Jiang, Jian-Qin; Xu, Yong

2016-11-01

Retinoic acid receptor-related orphan nuclear receptors (RORs) are orphan nuclear receptors that show constitutive activity in the absence of ligands. Among 3 subtypes of RORs, RORc is a promising therapeutic target for the treatment of Th17-mediated autoimmune diseases. Here, we report novel RORc inverse agonists discovered through structure-based drug design. Based on the structure of compound 8, a previously described agonist of RORa, a series of 4-(4-(benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives were designed and synthesized. The interaction between the compounds and RORc was detected at molecular level using AlphaScreen assay. The compounds were further examined in 293T cells transfected with RORc and luciferase reporter gene. Thermal stability shift assay was used to evaluate the effects of the compounds on protein stability. A total of 27 derivatives were designed and synthesized. Among them, the compound 22b was identified as the most potent RORc inverse agonist. Its IC 50 values were 2.39 μmol/L in AlphaScreen assay, and 0.82 μmol/L in inhibition of the cell-based luciferase reporter activity. Furthermore, the compound 22b displayed a 120-fold selectivity for RORc over other nuclear receptors. Moreover, a molecular docking study showed that the structure-activity relationship was consistent with the binding mode of compound 22b in RORc. 4-(4-(Benzyloxy)phenyl)-3,4-dihydropyrimidin-2(1H)-one derivatives are promising candidates for the treatment of Th17-mediated autoimmune diseases, such as rheumatoid arthritis, psoriasis, and multiple sclerosis.

Molecular breast cancer subtypes prevalence in an indigenous Sub Saharan African population

PubMed Central

Galukande, Moses; Wabinga, Henry; Mirembe, Florence; Karamagi, Charles; Asea, Alexzander

2014-01-01

Introduction Sub-Saharan Africa is predicted to face an unprecedented growth of cancers including breast cancer. There are indications of a significant burden of aggressive and late stage breast disease among premenopausal women in sub-Saharan Africa; because hormonal status tests are not routinely done, many women are given anti-hormonal therapy empirically. There is paucity of data on breast cancer molecular subtypes and their characteristics among women in sub Saharan Africa. The objective is to determine the prevalence of breast cancer molecular phenotypes among Ugandan women. Methods This was a cross sectional descriptive study, conducted at a tertiary hospital in Africa. Eligible participants’ formalin fixed and paraffin embedded sections were evaluated. H & E stains and Immunochemistry (Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal growth factor Receptor (HER2)) were performed. Ethical approval was obtained. Results A total of 226 patient samples were evaluated. The mean age was 45 years (SD 14);the prevalence of Triple Negative Breast Cancer (TNBC) was 34% (77/226), Luminal A 38% (83/226), HER2 positive was 22% (49/226), and Luminal B was 5% (13/226). High-grade (III) tumors were 68%, stage III and IV constituted 75% of presentations. Histological type was mostly invasive ductal carcinoma. Most patients (55%) were from rural areas. Conclusion Ugandan women had an over representation of TNBC and high-grade breast tumors. Underlying reasons ought to be investigated. The empirical use of tamoxifen (anti-hormonal therapy) should be reexamined. PMID:25309649

Molecular breast cancer subtypes prevalence in an indigenous Sub Saharan African population.

PubMed

Galukande, Moses; Wabinga, Henry; Mirembe, Florence; Karamagi, Charles; Asea, Alexzander

2014-01-01

Sub-Saharan Africa is predicted to face an unprecedented growth of cancers including breast cancer. There are indications of a significant burden of aggressive and late stage breast disease among premenopausal women in sub-Saharan Africa; because hormonal status tests are not routinely done, many women are given anti-hormonal therapy empirically. There is paucity of data on breast cancer molecular subtypes and their characteristics among women in sub Saharan Africa. The objective is to determine the prevalence of breast cancer molecular phenotypes among Ugandan women. This was a cross sectional descriptive study, conducted at a tertiary hospital in Africa. Eligible participants' formalin fixed and paraffin embedded sections were evaluated. H & E stains and Immunochemistry (Estrogen Receptor (ER), Progesterone Receptor (PR), Human Epidermal growth factor Receptor (HER2)) were performed. Ethical approval was obtained. A total of 226 patient samples were evaluated. The mean age was 45 years (SD 14);the prevalence of Triple Negative Breast Cancer (TNBC) was 34% (77/226), Luminal A 38% (83/226), HER2 positive was 22% (49/226), and Luminal B was 5% (13/226). High-grade (III) tumors were 68%, stage III and IV constituted 75% of presentations. Histological type was mostly invasive ductal carcinoma. Most patients (55%) were from rural areas. Ugandan women had an over representation of TNBC and high-grade breast tumors. Underlying reasons ought to be investigated. The empirical use of tamoxifen (anti-hormonal therapy) should be reexamined.

Both retinoic-acid-receptor- and retinoid-X-receptor-dependent signalling pathways mediate the induction of the brown-adipose-tissue-uncoupling-protein-1 gene by retinoids.

PubMed Central

Alvarez, R; Checa, M; Brun, S; Viñas, O; Mampel, T; Iglesias, R; Giralt, M; Villarroya, F

2000-01-01

The intracellular pathways and receptors mediating the effects of retinoic acid (RA) on the brown-fat-uncoupling-protein-1 gene (ucp-1) have been analysed. RA activates transcription of ucp-1 and the RA receptor (RAR) is known to be involved in this effect. However, co-transfection of an expression vector for retinoid-X receptor (RXR) increases the action of 9-cis RA but not the effects of all-trans RA on the ucp-1 promoter in brown adipocytes. Either RAR-specific ¿p-[(E)-2-(5,6,7,8,-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid¿ or RXR-specific [isopropyl-(E,E)-(R,S)-11-methoxy-3,7, 11-trimethyldodeca-2,4-dienoate, or methoprene] synthetic compounds increase the expression of UCP-1 mRNA and the activity of chloramphenicol acetyltransferase expression vectors driven by the ucp-1 promoter. The RXR-mediated action of 9-cis RA requires the upstream enhancer region at -2469/-2318 in ucp-1. During brown-adipocyte differentiation RXRalpha and RXRgamma mRNA expression is induced in parallel with UCP-1 mRNA, whereas the mRNA for the three RAR subtypes, alpha, beta and gamma, decreases. Co-transfection of murine expression vectors for the different RAR and RXR subtypes indicates that RARalpha and RARbeta as well as RXRalpha are the major retinoid-receptor subtypes capable of mediating the responsiveness of ucp-1 to retinoids. It is concluded that the effects of retinoids on ucp-1 transcription involve both RAR- and RXR-dependent signalling pathways. The responsiveness of brown adipose tissue to retinoids in vivo relies on a complex combination of the capacity of RAR and RXR subtypes to mediate ucp-1 induction and their distinct expression in the differentiated brown adipocyte. PMID:10600643

Colonic inflammation increases the contribution of muscarinic M2 receptors to carbachol-induced contraction of the rat colon.

PubMed

Jragh, Dina M; Khan, Islam; Oriowo, Mabayoje A

2011-01-01

Carbachol-induced contraction of the rat colon is impaired in rats with trinitrobenzene sulfonic acid (TNBS)-induced colitis. The main objective of this study was to examine the effect of colitis on the expression and function of muscarinic (M) receptor subtypes in the rat colon. Rats (n = 80) were treated with TNBS and used 5 days later for measurement of contractility, myeloperoxidase activity, histology and expression of muscarinic receptor isoforms using Western blot analysis. Carbachol produced concentration-dependent contractions of colonic segments from control (n = 40) and TNBS-treated (n = 40) rats with no significant difference in potency. However, the maximum response to carbachol was significantly reduced in colon segments of TNBS-treated rats. The selective muscarinic receptor antagonists 4-diphenylacetoxy-N-methyl piperidine (4-DAMP, M(3)), pirenzepine (M(1)) and methoctramine (M(2)) antagonized carbachol-induced contraction in control (9.1 ± 0.1, 6.7 ± 0.3 and 6.0 ± 0.1, respectively) and TNBS-treated rats (9.2 ± 0.2, 6.9 ± 0.2, 6.7 ± 0.2). The -logK(B) values in control rats are consistent with an action of carbachol on muscarinic M(3) receptors. There was no significant difference in -logK(B) values for 4-DAMP and pirenzepine in control and TNBS-treated rats, but methoctramine was fivefold more potent in TNBS-treated rats, possibly indicating an increased contribution of muscarinic M(2) receptors to carbachol-induced contraction in the inflamed colon. The expression of M(2) receptors was also significantly increased in colon segments from TNBS-treated rats, confirming the increased role of muscarinic M(2) receptors in the inflamed colon. The data show that while only M(3) receptors appeared to mediate carbachol-induced contraction in control segments, expression of both M(2) and M(3) receptors was increased in the inflamed rat colon. Copyright © 2011 S. Karger AG, Basel.

Prostaglandin E2 modulates dendritic cell function via EP2 and EP4 receptor subtypes.

PubMed

Harizi, Hedi; Grosset, Christophe; Gualde, Norbert

2003-06-01

We have reported previously that PGE(2) inhibits dendritic cells (DC) functions. Because E prostanoid receptor (EPR) subtypes involved in this action are unknown, expression and functions of these receptors were examined in DC. Western blot and flow cytometry analyses showed that all EPRs were coexpressed in DC. In a dose-dependent manner, lipopolysaccharide (LPS) enhanced EP(2)R/EP(4)R but not EP(1)R/EP(3)R expressions. NS-398, a cyclooxygenase (COX)-2-selective inhibitor, suppressed LPS-enhanced EP(2)R/EP(4)R expression, suggesting that COX-2-issued prostaglandin E(2) (PGE(2)) modulates DC function through stimulation of specific EPR subtypes. Using selective agonists, we found that butaprost, an EP(2)R agonist, and PGE(1) alcohol, an EP(2)R and EP(2)R/EP(4)R agonist, inhibited major histocompatibility complex class II expression and enhanced interleukin-10 production from DC. However, no effect was observed with sulprostone and 17-phenyl-omega-trinor-PGE(2), selective agonists for EP(1)R and EP(1)R/EP(3)R, respectively. Treatment of DC with dibutyryl cyclic adenosine monophosphate (cAMP), an analog of cAMP, mimics PGE(2)-induced, inhibitory effects. Taken together, our data demonstrate that EP(2)R/EP(4)R are efficient for mediating PGE(2)-induced modulation of DC functions.

Quantification of mu and non-mu opiate receptors in temporal lobe epilepsy using positron emission tomography.

PubMed

Mayberg, H S; Sadzot, B; Meltzer, C C; Fisher, R S; Lesser, R P; Dannals, R F; Lever, J R; Wilson, A A; Ravert, H T; Wagner, H N

1991-07-01

Alterations in a variety of neurotransmitter systems have been identified in experimental models of epilepsy and in brain tissue from patients with intractable temporal lobe seizures. The availability of new high-affinity radioligands permits the study of some neuroreceptors in vivo with positron emission tomography (PET). We previously characterized the in vivo binding of 11C-carfentanil, a potent and selective mu opiate receptor agonist, and described increases in 11C-carfentanil binding in the temporal neocortex of patients with unilateral temporal lobe epilepsy. These studies have been extended to 11C-diprenorphine, which labels mu, kappa, and delta opiate receptor subtypes. Paired measurements of opiate receptor binding were performed with PET using 11C-carfentanil and 11C-diprenorphine in patients with unilateral temporal lobe seizures. Carfentanil binding, reflecting changes in mu opiate receptors, was increased in the temporal neocortex and decreased in the amygdala on the side of the epileptic focus. Diprenorphine binding, reflecting mu as well as non-mu opiate subtypes, was not significantly different among regions in the focus and nonfocus temporal lobes. Regional glucose metabolism, measured using 18F-2-fluoro-2-deoxyglucose, was decreased in the mesial and lateral aspects of the temporal lobe ipsilateral to the epileptogenic focus. The variation in pattern of carfentanil and diprenorphine binding supports a differential regulation of opiate subtypes in unilateral temporal lobe epilepsy.

Design and synthesis of alkoxyindolyl-3-acetic acid analogs as peroxisome proliferator-activated receptor-γ/δ agonists.

PubMed

Gim, Hyo Jin; Li, Hua; Lee, Eun; Ryu, Jae-Ha; Jeon, Raok

2013-01-15

A series of carbazole or phenoxazine containing alkoxyindole-3-acetic acid analogs were prepared as PPARγ/δ agonists and their transactivation activities for PPAR receptor subtypes (α, γ and δ) were investigated. Structure-activity relationship studies disclosed the effect of the lipophilic tail, attaching position of the alkoxy group and N-benzyl substitution at indole. Compound 1b was the most potent for PPARδ and 3b for PPARγ. Molecular modeling suggested two different binding modes of our alkoxyindole-3-acetic acid analogs providing the insight into their PPAR activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Clinical Overestimation of HER2 Positivity in Early Estrogen and Progesterone Receptor–Positive Breast Cancer and the Value of Molecular Subtyping Using BluePrint

PubMed Central

Langenhoven, Lizanne; Grant, Kathleen A.; van der Merwe, Lize; Kotze, Maritha J.

2017-01-01

Purpose Human epidermal growth factor receptor 2 (HER2) positivity is an important prognostic and predictive indicator in breast cancer. HER2 status is determined by immunohistochemistry and fluorescent in situ hybridization (FISH), which are potentially inaccurate techniques as a result of several technical factors, polysomy of chromosome 17, and amplification or overexpression of CEP17 (centromeric probe for chromosome 17) and/or HER2. In South Africa, HER2-positive tumors are excluded from a MammaPrint (MP; Agendia BV, Amsterdam, Netherlands) pretest algorithm. Clinical HER2 status has been reported to correlate poorly with molecular subtype. The aim of this study was to investigate the correlation of clinical HER2 status with BluePrint (BP) molecular subtyping. Methods Clinico-pathologic and genomic information was extracted from a prospectively collected central MP database containing records of 256 estrogen receptor–positive and/or progesterone receptor–positive tumors. Twenty-one tumors considered HER2 positive on immunohistochemistry or FISH were identified for this study. Results The median age of patients was 56 years (range, 34 to 77 years), with a median tumor size of 16 mm (3 to 27 mm). Four (19%) tumors were confirmed HER2-enriched subtype, six (29%) were luminal A, and 11 (52%) were luminal B. The positive predictive values of HER2/CEP17 ratio ≥ 2 and HER2 copy number ≥ 6 were only 29% and 40%, respectively. The differences in means for HER2/CEP17 ratio were significant between BP HER2-enriched versus luminal (P = .0249; 95% CI, 0.12 to 1.21) and MP high-risk versus low-risk tumors (P = .0002; 95% CI, 0.40 to 1.06). Conclusion Of the 21 tumors considered clinically HER2 positive, only four were HER2-enriched subtype with BP, indicating an overestimation of HER2 positivity. FISH testing has a poor positive predictive value. PMID:28831439

Structural and dynamic features of HLA-B27 subtypes.

PubMed

Uchanska-Ziegler, Barbara; Ziegler, Andreas; Schmieder, Peter

2013-07-01

The differential association of HLA-B27 subtypes with ankylosing spondylitis provides the rationale for a comparative investigation of these proteins. Results from the last 2 years of research on minimally distinct HLA-B27 subtypes, primarily using biochemical and biophysical techniques, are presented and discussed. We summarize evidence that micropolymorphisms within the molecules' peptide-binding groove influence wide-ranging biochemical, biophysical and antigenic properties of HLA-B27 molecules, and suggest that distinct, subtype and peptide-dependent dynamics of peptide - heavy chain - β(2)-microglobulin heterotrimers could be instrumental for an understanding of the initiation of disease processes that are connected with certain HLA-B27 subtypes. The results indicate that mAbs that bind only to structurally distinguishable subsets of HLA-B27 molecules as well as techniques that assess the flexibility of these antigens may hold the key to comprehend molecular events contributing to the initial stages of disease pathogenesis in spondyloarthropathies.

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

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

Haga, Kazuko; Kruse, Andrew C.; Asada, Hidetsugu

2012-03-15

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structuremore » of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.« less

P2 receptor signaling in neurons and glial cells of the central nervous system.

PubMed

Köles, Laszlo; Leichsenring, Anna; Rubini, Patrizia; Illes, Peter

2011-01-01

Purine and pyrimidine nucleotides are extracellular signaling molecules in the central nervous system (CNS) leaving the intracellular space of various CNS cell types via nonexocytotic mechanisms. In addition, ATP is a neuro-and gliotransmitter released by exocytosis from neurons and neuroglia. These nucleotides activate P2 receptors of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. In mammalians, seven P2X and eight P2Y receptor subunits occur; three P2X subtypes form homomeric or heteromeric P2X receptors. P2Y subtypes may also hetero-oligomerize with each other as well as with other G protein-coupled receptors. P2X receptors are able to physically associate with various types of ligand-gated ion channels and thereby to interact with them. The P2 receptor homomers or heteromers exhibit specific sensitivities against pharmacological ligands and have preferential functional roles. They may be situated at both presynaptic (nerve terminals) and postsynaptic (somatodendritic) sites of neurons, where they modulate either transmitter release or the postsynaptic sensitivity to neurotransmitters. P2 receptors exist at neuroglia (e.g., astrocytes, oligodendrocytes) and microglia in the CNS. The neuroglial P2 receptors subserve the neuron-glia cross talk especially via their end-feets projecting to neighboring synapses. In addition, glial networks are able to communicate through coordinated oscillations of their intracellular Ca(2+) over considerable distances. P2 receptors are involved in the physiological regulation of CNS functions as well as in its pathophysiological dysregulation. Normal (motivation, reward, embryonic and postnatal development, neuroregeneration) and abnormal regulatory mechanisms (pain, neuroinflammation, neurodegeneration, epilepsy) are important examples for the significance of P2 receptor-mediated/modulated processes. Copyright © 2011 Elsevier Inc. All rights reserved.

Is androgen receptor targeting an emerging treatment strategy for triple negative breast cancer?

PubMed

Anestis, Aristomenis; Karamouzis, Michalis V; Dalagiorgou, Georgia; Papavassiliou, Athanasios G

2015-06-01

Triple negative breast cancer (TNBC) is an aggressive breast cancer subtype. The absence of expression and/or amplification of estrogen and progesterone receptor as well as ERBB-2 prevent the use of currently available endocrine options and/or ERBB-2-directed drugs and indicates chemotherapy as the main current therapy. TNBC represents approximately 15% of breast cancer cases with high index of heterogeneity. Here, we review the role of androgen receptor in breast carcinogenesis and its association with alterations in the expression pattern and functional roles of regulatory molecules and signal transduction pathways in TNBC. Additionally, based on the so far preclinical and clinical published data, we evaluate the perspectives for using and/or developing androgen receptor targeting strategies for specific TNBC subtypes. Copyright © 2015 Elsevier Ltd. All rights reserved.

ROLE OF ESTROGEN RECEPTOR-α ON FOOD DEMAND ELASTICITY

PubMed Central

Minervini, Vanessa; Rowland, Neil E.; Robertson, Kimberly L.; Foster, Thomas C.

2016-01-01

Estrogens have been shown to have an inhibitory effect on food intake under free-feeding conditions, yet the effects of estrogens on food-maintained operant responding have been studied to a much lesser extent and, thus, are not well understood. Therefore, the purpose of the present experiment was to use a behavioral economics paradigm to assess differences in demand elasticity between mice with knockout of the estrogen receptor subtype α, knockout of subtype β, and their wild type controls. The mice responded in a closed economy, and the price of food was increased by increasing the fixed-ratio response requirement every four sessions. Overall, we found that mice with the knockout of receptor subtype α had the most elastic demand functions. Therefore, under these conditions, estrogens increased food seeking via activation of the receptor subtype α. The results were inconsistent with those reported by previous studies that employed free-feeding conditions. PMID:25869426

High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell–like diffuse large B-cell lymphoma cells

PubMed Central

Mathews Griner, Lesley A.; Guha, Rajarshi; Shinn, Paul; Young, Ryan M.; Keller, Jonathan M.; Liu, Dongbo; Goldlust, Ian S.; Yasgar, Adam; McKnight, Crystal; Boxer, Matthew B.; Duveau, Damien Y.; Jiang, Jian-Kang; Michael, Sam; Mierzwa, Tim; Huang, Wenwei; Walsh, Martin J.; Mott, Bryan T.; Patel, Paresma; Leister, William; Maloney, David J.; Leclair, Christopher A.; Rai, Ganesha; Jadhav, Ajit; Peyser, Brian D.; Austin, Christopher P.; Martin, Scott E.; Simeonov, Anton; Ferrer, Marc; Staudt, Louis M.; Thomas, Craig J.

2014-01-01

The clinical development of drug combinations is typically achieved through trial-and-error or via insight gained through a detailed molecular understanding of dysregulated signaling pathways in a specific cancer type. Unbiased small-molecule combination (matrix) screening represents a high-throughput means to explore hundreds and even thousands of drug–drug pairs for potential investigation and translation. Here, we describe a high-throughput screening platform capable of testing compounds in pairwise matrix blocks for the rapid and systematic identification of synergistic, additive, and antagonistic drug combinations. We use this platform to define potential therapeutic combinations for the activated B-cell–like subtype (ABC) of diffuse large B-cell lymphoma (DLBCL). We identify drugs with synergy, additivity, and antagonism with the Bruton’s tyrosine kinase inhibitor ibrutinib, which targets the chronic active B-cell receptor signaling that characterizes ABC DLBCL. Ibrutinib interacted favorably with a wide range of compounds, including inhibitors of the PI3K-AKT-mammalian target of rapamycin signaling cascade, other B-cell receptor pathway inhibitors, Bcl-2 family inhibitors, and several components of chemotherapy that is the standard of care for DLBCL. PMID:24469833

Functional Incompatibility between the Generic NF-κB Motif and a Subtype-Specific Sp1III Element Drives the Formation of the HIV-1 Subtype C Viral Promoter

PubMed Central

Verma, Anjali; Rajagopalan, Pavithra; Lotke, Rishikesh; Varghese, Rebu; Selvam, Deepak; Kundu, Tapas K.

2016-01-01

ABSTRACT Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-κB binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-κB and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-κB motif binds NF-κB with an affinity 2-fold higher than that of the generic NF-κB site. Importantly, in the context of an infectious virus, the subtype-specific Sp1III motif demonstrates a profound loss of function in association with the generic NF-κB motif. An additional substitution of the Sp1III motif fully restores viral replication, suggesting that the subtype C-specific Sp1III has evolved to function with the variant, but not generic, NF-κB motif. A change of only two base pairs in the central NF-κB motif completely suppresses viral transcription from the provirus and converts the promoter into heterochromatin refractory to tumor necrosis factor alpha (TNF-α) induction. The present work represents the first demonstration of functional incompatibility between an otherwise functional NF-κB motif and a unique Sp1 site in the context of an HIV-1 promoter. Our work provides important leads as to the evolution of the HIV-1 subtype C viral promoter with relevance for gene expression regulation and viral latency. IMPORTANCE Subtype-specific genetic variations provide a powerful tool to examine how these variations offer a replication advantage to specific viral subtypes, if any. Only in subtype C of HIV-1 are two genetically distinct transcription factor binding sites positioned at the most critical location of the viral promoter. Since a single promoter regulates viral gene expression, the promoter variations can play a critical role in determining the replication fitness of the viral strains. Our work for the first time provides a scientific explanation for the presence of a unique NF-κB binding motif in subtype C, a major HIV-1 genetic family responsible for half of the global HIV-1 infections. The results offer compelling evidence that the subtype C viral promoter not only is stronger but also is endowed with a qualitative gain-of-function advantage. The genetically variant NF-κB and the Sp1III motifs may be respond differently to specific cell signal pathways, and these mechanisms must be examined. PMID:27194770

Convergent mechanisms underlying rapid antidepressant action

PubMed Central

Zanos, Panos; Thompson, Scott M.; Duman, Ronald S.; Zarate, Carlos A.; Gould, Todd D.

2018-01-01

Traditional pharmacological treatments for depression have a delayed therapeutic onset, ranging from several weeks to months, and there is a high percentage of individuals who never respond to treatment. In contrast, ketamine produces rapid-onset antidepressant, anti-suicidal and anti-anhedonic actions following a single administration to depressed patients. Proposed mechanisms of ketamine’s antidepressant action include N-methyl-D-aspartate receptor (NMDAR) modulation, GABAergic interneuron disinhibition, and direct actions of its hydroxynorketamine (HNK) metabolites. Downstream actions include activation of mechanistic target of rapamycin (mTOR), deactivation of glycogen synthase kinase-3 and eukaryotic elongation factor 2 (eEF2), enhanced brain-derived neurotrophic factor (BDNF) signaling, and activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs). These putative mechanisms of ketamine action are not mutually exclusive and may complement each other to induce potentiation of excitatory synapses in affective-regulating brain circuits, which results in amelioration of depression symptoms. We review these proposed mechanisms of ketamine action in the context of how such mechanisms are informing the development of novel putative rapid-acting antidepressant drugs. Such drugs that have undergoing pre-clinical, and in some cases clinical, testing include the muscarinic acetylcholine receptor antagonist scopolamine, GluN2B-NMDAR antagonists (i.e., CP-101,606, MK-0657), (2R,6R)-HNK, NMDAR glycine site modulators (i.e., 4-chlorokynurenine - pro-drug of the glycineB NMDAR antagonist 7-chlorokynurenic acid), NMDAR agonists (i.e. GLYX-13 (rapastinel)), metabotropic glutamate receptor 2/3 (mGluR2/3) antagonists, GABAA receptor modulators, and drugs acting on various serotonin receptor subtypes. These ongoing studies suggest that the future acute treatment of depression will typically occur within hours, rather than months, of treatment initiation. PMID:29516301

The potential role of myocardial serotonin receptor 2B expression in canine dilated cardiomyopathy.

PubMed

Fonfara, Sonja; Hetzel, Udo; Oyama, Mark A; Kipar, Anja

2014-03-01

Serotonin signalling in the heart is mediated by receptor subtype 2B (5-HTR2B). A contribution of serotonin to valvular disease has been reported, but myocardial expression of 5-HTR2B and its role in canine dilated cardiomyopathy (DCM) is not known. The aim of the present study was to investigate myocardial 5-HTR2B mRNA expression in dogs with DCM and to correlate results with expression of markers for inflammation and remodelling. Myocardial samples from eight healthy dogs, four dogs with DCM, five with cardiac diseases other than DCM and six with systemic non-cardiac diseases were investigated for 5-HTR2B mRNA expression using quantitative PCR (qPCR). The results were compared to mRNA expression of selected cytokines, matrix metalloproteinases (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP). Laser microdissection with subsequent qPCR and immunohistochemistry were employed to identify the cells expressing 5-HTR2B. The myocardium of control dogs showed constitutive 5-HTR2B mRNA expression. In dogs with DCM, 5-HTR2B mRNA values were significantly greater than in all other groups, with highest levels of expression in the left ventricle and right atrium. Myocytes were identified as the source of 5-HTR2B mRNA and protein. A significant positive correlation of 5-HTR2B mRNA with expression of several cytokines, MMPs and TIMPs was observed. The findings suggest that serotonin might play a role in normal cardiac structure and function and could contribute to myocardial remodelling and functional impairment in dogs with DCM. Copyright © 2013 Elsevier Ltd. All rights reserved.

Selective targeting of G-protein-coupled receptor subtypes with venom peptides.

PubMed

Näreoja, K; Näsman, J

2012-02-01

The G-protein-coupled receptor (GPCR) family is one of the largest gene superfamilies with approx. 370 members responding to endogenous ligands in humans and a roughly equal amount of receptors sensitive to external stimuli from the surrounding. A number of receptors from this superfamily are well recognized targets for medical treatment of various disease conditions, whereas for many others the potential medical benefit of interference is still obscure. A general problem associated with GPCR research and therapeutics is the insufficient specificity of available ligands to differentiate between closely homologous receptor subtypes. In this context, venom peptides could make a significant contribution to the development of more specific drugs. Venoms from certain animals specialized in biochemical hunting contain a mixture of molecules that are directed towards a variety of membrane proteins. Peptide toxins isolated from these mixtures usually exhibit high specificity for their targets. Muscarinic toxins found from mamba snakes attracted much attention during the 1990s. These are 65-66 amino acid long peptides with a structural three-finger folding similar to the α-neurotoxins and they target the muscarinic acetylcholine receptors in a subtype-selective manner. Recently, several members of the three-finger toxins from mamba snakes as well as conotoxins from marine cone snails have been shown to selectively interact with subtypes of adrenergic receptors. In this review, we will discuss the GPCR-directed peptide toxins found from different venoms and how some of these can be useful in exploring specific roles of receptor subtypes. © 2011 The Authors. Acta Physiologica © 2011 Scandinavian Physiological Society.

Type-3 ryanodine receptors mediate hypoxia-, but not neurotransmitter-induced calcium release and contraction in pulmonary artery smooth muscle cells.

PubMed

Zheng, Yun-Min; Wang, Qing-Song; Rathore, Rakesh; Zhang, Wan-Hui; Mazurkiewicz, Joseph E; Sorrentino, Vincenzo; Singer, Harold A; Kotlikoff, Michael I; Wang, Yong-Xiao

2005-04-01

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl- currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3-/-) mice, hypoxia-induced, but not submaximal noradrenaline-induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3-/- mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline- and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline-induced Ca2+ and contractile responses in PASMCs.