Ulla E. Sollenberg

Regulation of Kindling Epileptogenesis by Hippocampal Galanin Type 1 and Type 2 Receptors: The Effects of Subtype-Selective Agonists and the Role of G-Protein-Mediated Signaling

The search for antiepileptic drugs that are capable of blocking the progression of epilepsy (epileptogenesis) is an important problem of translational epilepsy research. The neuropeptide galanin effectively suppresses acute seizures. We examined the ability of hippocampal galanin receptor type 1 (GalR1) and type 2 (GalR2) to inhibit kindling epileptogenesis and studied signaling cascades that mediate their effects. Wistar rats received 24-h-long intrahippocampal infusion of a GalR1/2 agonist galanin(1-29), GalR1 agonist M617 [galanin(1-13)-Gln14-bradykinin(2-9)-amide], or GalR2 agonist galanin(2-11). The peptides were administered alone or combined with an inhibitor of Gi protein pertussis toxin (PTX), Gi-protein activated K+ channels (GIRK) inhibitor tertiapin Q (TPQ), Gq/11 protein inhibitor [d-Arg1,d-Trp5,7,9,Leu11]-substance P (dSP), or an inhibitor of intracellular Ca2+ release dantrolene. Sixteen hours into drug delivery, the animals were subjected to rapid kindling—60 electrical trains administered to ventral hippocampus every 5 min. M617 delayed epileptogenesis, whereas galanin(1-29) and galanin(2-11) completely prevented the occurrence of full kindled seizures. TPQ abolished anticonvulsant effect of M617 but not of galanin(2-11). PTX blocked anticonvulsant effects of M617 and inversed the action of galanin(1-29) and galanin(2-11) to proconvulsant. dSP and dantrolene did not modify seizure suppression through GalR1 and GalR2, but eliminated the proconvulsant effect of PTX + galanin(1-29) and PTX + galanin(2-11) combinations. We conclude that hippocampal GalR1 exert their disease-modifying effect through the Gi-GIRK pathway. GalR2 is antiepileptogenic through the Gi mechanism independent of GIRK. A secondary proconvulsant pathway coupled to GalR2 involves Gq/11 and intracellular Ca2+. The data are important for understanding endogenous mechanisms regulating epileptogenesis and for the development of novel antiepileptogenic drugs.

Differential Role of Galanin Receptors in the Regulation of Depression-Like Behavior and Monoamine/Stress-Related Genes at the Cell Body Level

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-HT1A 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.

M871—A Novel Peptide Antagonist Selectively Recognizing the Galanin Receptor Type 2

Galanin and its three receptors have been linked to a wide variety of physiological processes and are distributed in both the central and peripheral nervous systems. Further knowledge of the properties of galanin-activated signaling systems can best be obtained by the availability of peptide and non-peptide ligands that are selective for the different receptor subtypes. The current study describes binding and signaling data for the chimeric peptide, galanin-(2–13)-Glu-His-(Pro)3-(Ala-Leu)2-Ala-amide (M871). This compound binds to the galanin receptor type 2 with more than 30-fold higher affinity than to the galanin receptor type 1 and exhibits antagonist actions at galanin receptor type 2, blocking increased release of inositol phosphate produced by galanin in CHO cells. This peptide opens new possibilities for the study of galanin receptor physiology.

Selective stimulation of GalR1 and GalR2 in rat substantia gelatinosa reveals a cellular basis for the anti- and pro-nociceptive actions of galanin

&NA; 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 μM), in the presence of the GalR2 antagonist, M871 (1.0–2.5 μM). GalR2 was activated with the selective agonist, AR‐M 1896 (0.5–1.0 μM). 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 mV. 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, Gi/o and to report that GalR2 activation suppresses Ca2+ channel currents.

Multiple interaction sites of galnon trigger its biological effects

Galnon was first reported as a low molecular weight non-peptide agonist at galanin receptors [Saar et al. (2002) Proc. Natl. Acad. Sci. USA 99, 7136-7141]. Following its systemic administration, this synthetic ligand affected a range of important physiological processes including appetite, seizures and pain. Physiological activity of galnon could not be explained solely by the activation of the three known galanin receptors, GalR1, GalR2 and GalR3. Consequently, it was possible that galnon generates its manifold effects by interacting with other signaling pathway components, in addition to via GalR1-3. In this report, we establish that galnon: (i) can penetrate across the plasma membrane of cells, (ii) can activate intracellular G-proteins directly independent of receptor activation thereby triggering downstream signaling, (iii) demonstrates selectivity for different G-proteins, and (iiii) is a ligand to other G-protein coupled receptors (GPCRs) in addition to via GalR1-3. We conclude that galnon has multiple sites of interaction within the GPCR signaling cascade which mediate its physiological effects.

Galnon – a low-molecular weight ligand of the galanin receptors

Galnon is a low-molecular weight galanin receptor ligand, with affinity towards the three galanin receptors in the micromolar range. Galnon is of interest as a drug candidate due to its stability and ability to pass the blood-brain barrier. Like galanin, galnon has also been shown to affect various physiological functions; however, occasionally galanin and galnon act in opposing ways. Since its introduction in 2002, galnon has been characterized to inhibit seizures, decrease feeding behaviour, diminish physical signs of opiate withdrawal and to alleviate heat-hyperalgesic response to partial sciatic nerve injury. In this review, we will summarize what is known about galnon to date.

Activation of peripheral galanin receptors: differential effects on nociception.

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.

Molecular characterization of the ligand binding site of the human galanin receptor type 2, identifying subtype selective interactions

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.

Determining receptor–ligand interaction of human galanin receptor type 3

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 Tyr103(3.33) in transmembrane helix (TM) III, His251(6.51) in TM VI, Arg273(7.35) or His277(7.39) in TM VII, Phe263(6.63) or Tyr270(7.32) 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.