| 1. |
- Alier, Kwai, et al.
(författare)
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Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellular basis for the anti- and pro-nociceptive actions of galanin
- 2008
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Ingår i: Pain. - : Ovid Technologies (Wolters Kluwer Health). - 0304-3959 .- 1872-6623. ; 137:1, s. 138-146
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Tidskriftsartikel (refereegranskat)abstract
- Galanin modulates spinal nociceptive processing by interacting with two receptors, GalR1 and GalR2. The underlying neurophysiological mechanisms were examined by whole-cell recording from identified neurons in the substantia gelatinosa of young adult rats. GalR1 was activated with a 'cocktail' containing the GalR1/2 agonist, AR-M 961 (0.5 mu M), in the presence of the GalR2 antagonist, M871 (1.0-2.5 mu M). GalR2 was activated with the selective agonist, AR-M 1896 (0.5-1.0 mu M). Application of the 'GalR1 agonist cocktail' often activated an inwardly-rectifying conductance in delay firing (excitatory) and tonically firing (inhibitory) neurons. This conductance was not activated by AR-M 1896 which instead decreased or increased an outwardly-rectifying conductance at voltages positive to -70 rnV. Despite this variability in its actions on current-voltage relationships, AR-M 1896 very consistently decreased membrane excitability, as measured by cumulative action potential latency in response to a depolarizing current ramp. This strong GalR2-mediated effect was seen in neurons where membrane conductance was decreased, and where membrane excitability might be predicted to increase. GalR2 was also located presynaptically, as AR-M 1896 increased the interevent interval of spontaneous EPSCs in both delay and tonic cells. By contrast, the 'GalR1 agonist cocktail' had little effect on spontaneous EPSCs, suggesting that presynaptic terminals do not express GalR1. These diverse actions of GalR1 and GalR2 activation on both inhibitory and excitatory neurons are discussed in relation to the known spinal antinociceptive and pro-nociceptive actions of galanin, to the possible association of GalR1 with the inhibitory G-protein, G(i/o) and to report that GalR2 activation suppresses Ca(2+) channel currents.
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| 2. |
- Eriksson Sollenberg, Ulla, 1977-
(författare)
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Characterization of galanin receptors using chimeric peptides and site-directed mutagenesis
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Galanin, a 29 (30) amino acid neuropeptide, is found throughout both the central and peripheral nervous systems. It signals via three receptors, GalR1-3, all belonging to the rhodopsin-like G-protein coupled receptors. Galanin and its receptors have been implicated in a vast variety of biological processes. To facilitate further characterization of the physiological/pathological roles of galanin, subtype selective ligands targeting the three receptors individually would be of great aid. In this thesis the main objective was to provide more information about galanin receptor-ligand interactions, primarily concerning GalR2 and 3. By using information gained from previously developed chimeric peptides, we designed and synthesized a novel peptide selective towards GalR2 (Paper I). This peptide, M871, binds GalR2 in an inhibitory manner, likely due to its truncated N-terminus and bulky character. In Paper II and IV we performed L-alanine mutagenesis assays of GalR2 and 3 respectively. By point substituting amino acid residues in the receptor sequence, we identified crucial pharmacophores for ligand binding, primarily in transmembrane regions 6 and 7. The targeted residues were selected based on knowledge concerning GalR1 and on conservation between the three receptors. For GalR3 we also conducted a computational docking assay. A homology model was first constructed using three crystallized structures of other receptors also belonging to the Rhodopsin family. Ligands galanin(2-6) and SNAP398299 were then docked to GalR3 in flexible mode. The docking resulted in characterization of GalR3-ligand interactions and conclude that this receptor display a relatively deep and narrow binding pocket. As a result of this, it was hypothesized that the C-terminus of ligands is of importance for GalR3 affinity. An L-alanine scan of ligand was performed (paper III), which confirmed this theory. In conclusion, our results give insights into galanin receptor-ligand interactions, information that is relevant for ligand design and drug development.
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| 3. |
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| 4. |
- Jimenez-Andrade, Juan Miguel, et al.
(författare)
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Activation of peripheral galanin receptors : differential effects on nociception
- 2006
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Ingår i: Pharmacology, Biochemistry and Behavior. - 0091-3057 .- 1873-5177. ; 85:1, s. 273-280
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Tidskriftsartikel (refereegranskat)abstract
- Numerous reports suggest a significant role of peripheral galanin (GAL) in pain transmission; however, due to the lack of selective galanin receptor agonists and antagonists, the role of GAL receptors (GalR1-3) in pain transmission remains unclear. In this study, a new agonist, M617, that preferentially binds to GalR1, a GalR2 agonist (AR-M1896), and a GalR2 antagonist (M871) were tested in the periphery to elucidate the role of peripheral GalR1 and GalR2 in nociception. Ipsilateral, but not contralateral, hindpaw injection of M617 reduced capsaicin (CAP)-induced flinching by approximately 50%, suggesting that GalR1 activation produces anti-nociception. This anti-nociceptive effect was blocked by intraplantar injection of the non-selective GalR antagonist M35. In contrast ipsilateral, but not contralateral, intraplantar injection of GalR2 agonist AR-M1896 enhanced the CAP-induced nociception (1.7-fold). The GalR2 antagonist M871 blocked the pro-nociceptive effect of AR-M1896 in a dose-dependent manner. This antagonist had no effect on nociceptive behaviors induced by CAP alone. The data demonstrate that activation of peripheral GalR1 results in anti-nociception but activation of peripheral GalR2 produces pro-nociception. Thus, the use of these pharmacological tools may help to elucidate the contribution of GalR subtypes in nociceptive processing, identifying potential drug targets for the treatment of peripheral pain.
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| 5. |
- Kuteeva, Eugenia, et al.
(författare)
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Differential Role of Galanin Receptors in the Regulation of Depression-Like Behavior and Monoamine/Stress-Related Genes at the Cell Body Level
- 2008
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Ingår i: Neuropsychopharmacology. - : Springer Science and Business Media LLC. - 0893-133X .- 1740-634X. ; 33:11, s. 2573-2585
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Tidskriftsartikel (refereegranskat)abstract
- The present study on rat examined the role of galanin receptor subtypes in regulation of depression-like behavior as well as potential molecular mechanisms involved in the locus coeruleus (LC) and dorsal raphe (DR). The effect of intracerebroventricular (i.c.v.) infusion of galanin or galanin receptor GalR1- and GalR2-selective ligands was studied in the forced swim test, followed by quantitative in situ hybridization studies. Naive control, non-treated (swim control), saline-and fluoxetine-treated rats were used as controls in the behavioral and in situ hybridization studies. Subchronic treatment with fluoxetine reduced immobility and climbing time. Intracerebroventricular infusion of galanin, the GalR1 agonist M617 or the GalR2 antagonist M871 increased, while the GalR2(R3) agonist AR-M1896 decreased, immobility time compared to the aCSF-treated animals. Galanin also decreased the time of climbing. Galanin mRNA levels were upregulated by the combination of injection + swim stress in the saline-and the fluoxetine-treated groups in the LC, but not in the DR. Also tyrosine hydroxylase levels in the LC were increased following injection + swim stress in the saline-and fluoxetine-treated rats. Tryptophan hydroxylase 2 and serotonin transporter mRNAs were not significantly affected by any treatment. 5-HT(1A) mRNA levels were downregulated following i.c.v. galanin, M617 or AR-M1896 infusion. These results indicate a differential role of galanin receptor subtypes in depression-like behavior in rodents: GalR1 subtype may mediate 'prodepressive' and GalR2 'antidepressant' effects of galanin. Galanin has a role in behavioral adaptation to stressful events involving changes of molecules important for noradrenaline and/or serotonin transmission.
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| 6. |
- Lundström, Linda, et al.
(författare)
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A galanin receptor subtype 1 specific agonist
- 2005
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Ingår i: International Journal of Peptide Research and Therapeutics. - : Springer Science and Business Media LLC. - 1573-3904 .- 1573-3149. ; 11:1, s. 17-27
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Tidskriftsartikel (refereegranskat)abstract
- The chimeric peptide M617, galanin(1–13)-Gln14-bradykinin(2–9)amide, is a novel galanin receptor ligand with increased subtype specificity for GalR1 and agonistic activity in cultured cells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2 show the presence of a high affinity binding site for M617 on GalR1 (K i=0.23±.12 nM) while lower affinity was seen towards GalR2 (K i=5.71±1.28 nM) resulting in 25-fold specificity for GalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internalization of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 to inhibit forskolin stimulated cAMP formation with 57% and to produce a 5-fold increase in inositol phosphate (IP) accumulation. Agonistic effects on signal transduction are shown on both receptors studied after treatment with M617 in the presence of galanin. In noradrenergic locus coeruleus neurons, M617 induces an outward current even in the presence of TTX plus Ca2+, high Mg2+, suggesting a postsynaptic effect. Intracerebroventricular (i.c.v.) administration of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35 completely fails to affect the feeding behavior. Spinal cord flexor reflex is facilitated by intrathecal (i.t.) administration of M617 as well as galanin with no significant change upon pre-treatment with M617. M617 dose dependently antagonizes the spinal cord hyperexcitablility induced by C-fiber conditioning stimulus and does neither enhance nor antagonize the effect of galanin. These data demonstrate a novel galanin receptor ligand with subtype specificity for GalR1 and agonistic activity, both in vitro and in vivo.
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| 7. |
- Lundström, Linda, et al.
(författare)
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Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions
- 2007
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Ingår i: Journal of Neurochemistry. - : Wiley. - 0022-3042 .- 1471-4159. ; 103:5, s. 1774-1784
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Tidskriftsartikel (refereegranskat)abstract
- To define the specific role of the galanin receptors when mediating the effect of galanin, effective tools for distinct activation and inhibition of the different receptor subtypes are required. Several of the physiological effects modulated by galanin are implicated to be mediated via the GalR2 subtype and have been distinguished from GalR1 effects by utilizing the Gal(2–11) peptide, recognizing only GalR2 and GalR3. In this study, we have performed a mutagenesis approach on the GalR2 subtype and present, for the first time, a molecular characterization of the interactions responsible for ligand binding and receptor activation at this receptor subtype. Our results identify four residues, His252 and His253 located in transmembrane domain 6 and Phe264 and Tyr271 in the extracellular loop 3, to be of great significance. We show evidence for the N-terminal tail of GalR2 to participate in ligand binding and that selective binding of Gal(2–11) includes interaction with the Ile256 residue, located at the very top of TM 6. In conclusion, we present a mutagenesis study on GalR2 and confer interactions responsible for ligand binding and receptor activation as well as selective recognition of the Gal(2–11) peptide at this receptor subtype. The presented observations could be of major importance for the design and development of new and improved peptide and non-peptide ligands, selectively activating the GalR2 subtype.
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| 8. |
- Runesson, Johan, 1980-, et al.
(författare)
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Determining receptor–ligand interaction of human galanin receptor type 3
- 2010
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Ingår i: Neurochemistry International. - : Elsevier BV. - 0197-0186 .- 1872-9754. ; 57:7, s. 804-811
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Tidskriftsartikel (refereegranskat)abstract
- Galanin is a neuropeptide found throughout the central and peripheral nervous systems of a wide range of species, ranging from human and mouse to frog and tuna. Galanin mediates its physiological roles through three receptors (GalR1–3), all members of the G-protein coupled receptor family. In mapping these roles, receptor subtype selective ligands are crucial tools. To facilitate the ligand design, data on receptor structure and interaction points are of great importance. The current study investigates the mechanism by which galanin interacts with GalR3. Mutated receptors were tested with competitive binding analysis in vitro. Our studies identify six mutagenic constructs that lost receptor affinity completely, despite being expressed at the cell surface. Mutations of the Tyr1033.33 in transmembrane helix (TM) III, His2516.51 in TM VI, Arg2737.35 or His2777.39 in TM VII, Phe2636.63 or Tyr2707.32 in the extracellular loop III all result in complete reduction of ligand binding. In addition, docking studies of an in silico model of GalR3 propose that four of the identified residues interact with pharmacophores situated within the galanin(2–6) sequence. This study provides novel insights into the interaction between ligands and GalR3 and highlights the requirement for correct design of targeting ligands.
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| 9. |
- Runesson, Johan, 1980-, et al.
(författare)
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Identification of important pharmacophores in the galanin receptor subtypes
- 2010
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The neuropeptide galanin family currently consists of four members, namely galanin, galanin-message-associated peptide (GMAP), galanin-like peptide (GALP) and alarin. Galanin has been shown to influence several physiological processes including cognition, affective behavior, nerve injury, Alzheimer’s disease, neuroregeneration, seizures, feeding, and hormone release. Galanin and galanin receptors (GalR1-3) show a widespread distribution throughout nervous systems where galanin co-localizes with several neuropeptides and/or classical neurotransmitters. Regionally specific expression of the galanin receptors suggests different physiological roles, a feature which mostly remains unexplored due to the lack of stable, selective and potent ligands acting on the galanin receptor subtypes. In order to design novel galanin receptor ligands with enhanced subtype selectivity we are aiming at determining the molecular interaction between the galanin peptide and the galanin receptor subtypes. By introducing site directed mutations to the extracellular loops and the external parts of the transmembrane domains in the galanin receptor subtypes we will characterize areas and single amino acids with significant importance for ligand binding to this receptor.
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| 10. |
- Runesson, Johan, 1980-, et al.
(författare)
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Twenty-Five Years of Galanin Research
- 2009
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Ingår i: Bioactive Peptides. - Boca Raton : CRC Press. - 9781420061147 - 9781420061161 ; , s. 237-260
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Bokkapitel (refereegranskat)
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