Peripheral Delta Opioid Receptors Mediate Formoterol Anti-allodynic Effect in a Mouse Model of Neuropathic Pain.

Neuropathic pain is a challenging condition for which current therapies often remain unsatisfactory. Chronic administration of β2 adrenergic agonists, including formoterol currently used to treat asthma and chronic obstructive pulmonary disease, alleviates mechanical allodynia in the sciatic nerve cuff model of neuropathic pain. The limited clinical data currently available also suggest that formoterol would be a suitable candidate for drug repurposing. The antiallodynic action of β2 adrenergic agonists is known to require activation of the delta-opioid (DOP) receptor but better knowledge of the molecular mechanisms involved is necessary. Using a mouse line in which DOP receptors were selectively ablated in neurons expressing Nav1.8 sodium channels (DOP cKO), we showed that these DOP peripheral receptors were necessary for the antiallodynic action of the β2 adrenergic agonist formoterol in the cuff model. Using a knock-in mouse line expressing a fluorescent version of the DOP receptor fused with the enhanced green fluorescent protein (DOPeGFP), we established in a previous study, that mechanical allodynia is associated with a smaller percentage of DOPeGFP positive small peptidergic sensory neurons in dorsal root ganglia (DRG), with a reduced density of DOPeGFP positive free nerve endings in the skin and with increased DOPeGFP expression at the cell surface. Here, we showed that the density of DOPeGFP positive free nerve endings in the skin is partially restored and no increase in DOPeGFP translocation to the plasma membrane is observed in mice in which mechanical pain is alleviated upon chronic oral administration of formoterol. This study, therefore, extends our previous results by confirming that changes in the mechanical threshold are associated with changes in peripheral DOP profile. It also highlights the common impact on DOP receptors between serotonin noradrenaline reuptake inhibitors such as duloxetine and the β2 mimetic formoterol.journal article20192020 02 14importe

Brain Struct Funct

Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives

A Novel G Protein-Coupled Receptor of Schistosoma mansoni (SmGPR-3) Is Activated by Dopamine and Is Widely Expressed in the Nervous System

Schistosomes have a well developed nervous system that coordinates virtually every activity of the parasite and therefore is considered to be a promising target for chemotherapeutic intervention. Neurotransmitter receptors, in particular those involved in neuromuscular control, are proven drug targets in other helminths but very few of these receptors have been identified in schistosomes and little is known about their roles in the biology of the worm. Here we describe a novel Schistosoma mansoni G protein-coupled receptor (named SmGPR-3) that was cloned, expressed heterologously and shown to be activated by dopamine, a well established neurotransmitter of the schistosome nervous system. SmGPR-3 belongs to a new clade of “orphan” amine-like receptors that exist in schistosomes but not the mammalian host. Further analysis of the recombinant protein showed that SmGPR-3 can also be activated by other catecholamines, including the dopamine metabolite, epinine, and it has an unusual antagonist profile when compared to mammalian receptors. Confocal immunofluorescence experiments using a specific peptide antibody showed that SmGPR-3 is abundantly expressed in the nervous system of schistosomes, particularly in the main nerve cords and the peripheral innervation of the body wall muscles. In addition, we show that dopamine, epinine and other dopaminergic agents have strong effects on the motility of larval schistosomes in culture. Together, the results suggest that SmGPR-3 is an important neuronal receptor and is probably involved in the control of motor activity in schistosomes. We have conducted a first analysis of the structure of SmGPR-3 by means of homology modeling and virtual ligand-docking simulations. This investigation has identified potentially important differences between SmGPR-3 and host dopamine receptors that could be exploited to develop new, parasite-selective anti-schistosomal drugs

Silkworm expression system as a platform technology in life science

Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3–6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the “Silkroad,” we expect that the “Bioroad” from Asia to Europe will be established by the silkworm expression system

Chemogenomic Analysis of G-Protein Coupled Receptors and Their Ligands Deciphers Locks and Keys Governing Diverse Aspects of Signalling

Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs) is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI) is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic) or inhibitory (antagonistic) ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs) 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences

Shear resistance of ultra high performance fibre-reinforced concrete I-beams

FraMCoS, JEJU, COREE, REPUBLIQUE DE, 23-/05/2010 - 28/05/2010Ultra High Performance Fibre-Reinforced Concrete (UHPFRC) refers to materials with a cement matrix and a characteristic compressive strength in excess of 150 MPa, and containing steel fibres in order to achieve ductile behaviour under tension. Thanks to these outstanding properties thin and durable structural elements can be made. Shear verifications of structures made of UHPFRC are thus often critical. The possibility to add the stirrups force at yielding and the post-cracking fibres contribution in the theoretical estimate of the ultime shear resistance requires appropriate verification. In order to quantify the safety margin of shear design provisions, an experimental campaign has been carried out at the LCPC (French Public Works Research Institute). In a Four-Point Bending configuration, shear tests have been conducted on nine 3m-long I-shaped girders with varied types of shear reinforcement (stirrups, fibers and both) combined with longitudinal prestressing or passive reinforcement

Molecular components of tolerance to opiates in single hippocampal neurons

We examined the effect of acute and chronic opioid treatment on synaptic transmission and µ-opioid receptor (MOR) endocytosis in cultures of naïve rat hippocampal neurons. Opioid agonists that activate MOR inhibited synaptic transmission at inhibitory but not excitatory autapses. [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), morphine, and methadone were all effective at blocking inhibitory transmission. These same drugs also reduced the amplitude of voltage-dependent Ca2+ currents in inhibitory but not excitatory neurons. Chronic treatment with all three opioids reduced the subsequent effects of a challenge with either the same drug or one of the others in individual autaptic neurons. Chronic treatment with DAMGO or methadone produced internalization of enhanced yellow fluorescent protein-tagged MOR expressed in hippocampal neurons within hours, whereas morphine produced internalization much more slowly, even when accompanied by overexpression of beta -arrestin-2. We conclude that DAMGO, methadone, and morphine all produce tolerance in single hippocampal neurons. Morphine-induced tolerance does not necessarily seem to involve receptor endocytosis

Co-expression of mu and delta opioid receptors as receptor-G protein fusions enhances both mu and delta signalling via distinct mechanisms

Abstract Mu and delta opioid receptors were co-expressed as fusion proteins between a receptor and a pertussis insensitive mutant Galphai/o protein in HEK 293 cells. Signalling efficiency was then monitored following inactivation of endogenous Galphai/o proteins by pertussis toxin. Co-expression resulted in increased delta opioid signalling which was insensitive to the mu specific antagonist CTAP. Under these conditions, mu opioid signalling was also increased and insensitive to the delta specific antagonist Tic-deltorphin. In this latter case, however, no G protein activation was observed in the presence of the delta specific inverse agonist N,N(CH3)2-Dmt-Tic-NH2. When a mu opioid receptor fused to a non-functional Galpha subunit was co-expressed with the delta opioid receptor-Galpha protein fusion, delta opioid signalling was not affected whereas mu opioid signalling was restored. Altogether our results suggest that increased delta opioid signalling is due to enhanced delta opioid receptor coupling to its tethered Galpha subunit. On the other hand, our data indicate that increased mu opioid signalling requires an active conformation of the delta opioid receptor and also results in activation of the Galpha subunit fused the delta opioid recepto

Evidence for the presence of N-methyl-D-aspartate receptors in the ventral tegmental area of the rat: an electrophysiological in vitro study.

Extracellular recordings were obtained from spontaneously active, presumed dopaminergic neurons of the ventral tegmental area (VTA) of the rat in a slice preparation. Bath-applied N-methyl-D-aspartate (NMDA) (1-20 microM) activated all neurons tested (n = 36). This effect was clearly concentration-dependent (n = 14), quickly reversible and reproducible. No bursting type of discharge was observed during NMDA infusion. The NMDA receptor blocker DL-2-amino-5-phosphonovaleric acid (50 microM) reversibly antagonized the increase in cell firing produced with 10 microM NMDA by 83.5 +/- 3% (mean +/- S.E.M.) (n = 8, P less than 0.05). Lowering the Mg2+ concentration of the perfusion fluid to one-third of its normal value significantly enhanced the excitatory effect of 5 microM NMDA (n = 7, P less than 0.05), but not of 500 nM carbachol (n = 6). Finally, NMDA did not modify the sensitivity of dopaminergic autoreceptors of VTA neurons (n = 8), when compared to controls (n = 10). These observations strongly support the presence of specific NMDA receptors in the VTA.In VitroJournal ArticleSCOPUS: ar.jinfo:eu-repo/semantics/publishe