Galanin inhibition of voltage‐dependent Ca2+ influx in rat cultured myenteric neurons is mediated by galanin receptor 1

Galanin activates three receptors, the galanin receptor 1 (GalR1), GalR2, and GalR3. In the gastrointestinal tract, GalR1 mediates the galanin inhibition of cholinergic transmission to the longitudinal muscle and reduction of peristalsis efficiency in the small intestine. Galanin has also been shown to inhibit depolarization‐evoked Ca²⁺ increases in cultured myenteric neurons. Because GalR1 immunoreactivity is localized to cholinergic myenteric neurons, we hypothesized that this inhibitory action of galanin on myenteric neurons is mediated by GalR1. We investigated the effect of galanin 1‐16, which has high affinity for GalR1 and GalR2, in the presence or absence of the selective GalR1 antagonist, RWJ‐57408, and of galanin 2‐11, which has high affinity for GalR2 and GalR3, on Ca²⁺ influx through voltage‐dependent Ca²⁺ channels in cultured myenteric neurons. Myenteric neurons were loaded with fluo‐4 and depolarized by high K⁺ concentration to activate voltage‐dependent Ca²⁺ channels. Intracellular Ca²⁺ levels were quantified with confocal microscopy. Galanin 1‐16 (0.01–1 μM) inhibited the depolarization‐evoked Ca²⁺ increase in a dose‐dependent manner with an EC₅₀ of 0.172 μM. The selective GalR1 antagonist, RWJ‐57408 (10 μM), blocked the galanin 1‐16 (1 μM)‐mediated inhibition of voltage‐dependent Ca²⁺ channel. By contrast, the GalR2/GalR3 agonist, galanin 2‐11 did not affect the K⁺‐evoked Ca²⁺ influx in myenteric neurons. GalR1 immunoreactivity was localized solely to myenteric neurons in culture, as previously observed in intact tissue. These findings indicate that the inhibition of depolarization‐evoked Ca²⁺ influx in myenteric neurons in culture is mediated by GalR1 and confirm the presence of functional GalR1 in the myenteric plexus. This is consonant with the hypothesis that GalR1 mediates galanin inhibition of transmitter release from myenteric neurons. © 2008 Wiley‐Liss, Inc.     

A novel GalR2-specific peptide agonist

The galanin peptide family and its three receptors have with compelling evidence been implicated in several high-order physiological disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family. In the current study we present data on receptor binding and functional response for a novel galanin receptor type 2 (GalR2) selective chimeric peptide, M1145 [(RG)₂-N-galanin(2-13)-VL-(P)₃-(AL)₂-A-amide]. The M1145 peptide shows more than 90-fold higher affinity for GalR2 over GalR1 and a 76-fold higher affinity over GalR3. Furthermore, the peptide yields an agonistic effect in vitro, seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family and opens new possibilities to apply the galanin system as a putative drug target.     

Distribution and differential regulation of galanin receptor subtypes in rat brain: effects of seizure activity

Galanin, acting at the GalR1-3 subtypes of galanin receptors, is involved in the regulation of cognition, mood, feeding, seizure activity and pain. The understanding of galanin's effects in molecular and cellular terms has been hampered by the lack of receptor subtype selective ligands and antibodies. Previous in situ hybridization data showed that GalR1 and GalR2 receptors are abundant in the rat brain, while the distribution of GalR3 is contradictory and most studies demonstrated a low expression levels in the rat brain. The distribution of galanin receptor subtypes at protein level is unknown. In the present study, we report the regional distribution of the galanin receptors: GalR1 and non-GalR1 receptors, using a recently synthesized high affinity GalR2/3 selective ligand, galanin (2-11), and galanin (1-29), as competitors, in saturating (125)I-galanin membrane binding assay. We show that paraventricular nucleus (PVN) express predominantly GalR1, whereas areas like the dorsal raphe nucleus (DRN), hippocampus and amygdala express both the GalR1 and non-GalR1 receptors. We speculate that the GalR2/3 binding sites detected by galanin (2-11) binding in our study probably represent mostly GalR2 receptors. In addition, we show regionally specific and subtype specific regulation of galanin receptors. Status epilepticus (SE), known to deplete galanin from axonal projections of locus coeruleus and septum/diagonal band neurons in the hippocampus and to induce galanin expression in a subset of hippocampal cells, down regulates GalR2 receptor mRNA and proteins by 30% without altering the GalR1 receptors.     

Regulation of limbic status epilepticus by hippocampal galanin type 1 and type 2 receptors

It has been well established that galanin is a potent endogenous anticonvulsant peptide. However, the role of galanin receptor subtypes in mediating anticonvulsant effects of the peptide is poorly understood. Using pharmacological, transgenic and antisense approaches, we examined the involvement of galanin receptors GalR1 and GalR2 in regulating seizures and associated neuronal degenerative changes. In the rat model of status epilepticus (SE) induced by electrical stimulation of perforant path, in vivo uncoupling of G protein coupled receptors (GPCR) through intrahippocampal administration of pertussis toxin (PTX) facilitated the initiation of SE, and increased the severity of the established SE. Injection of a non-selective GalR1/GalR2 agonist galanin (1-29) and a preferential GalR2 agonist galanin (2-11) into the hippocampus of PTX-pretreated rats revealed that while during early phase of SE galanin inhibited seizures predominantly through GalR1, GalR2 mediated anticonvulsant effects of the peptide during advanced stage of SE. GalR1 knockout mice showed increased severity of both pilocarpine- and perforant path stimulation -induced SE, compared to wild type (WT) littermates. In GalR1 knockout animals SE led to more severe and wider-spread hippocampal injury, than in WT. Focal downregulation of GalR2, which had been achieved in rats by intrahippocampal infusion of anti-GalR2 peptide nucleic acid (PNA) antisense, significantly increased the severity of perforant path stimulation- induced SE. Downregulation of GalR2 led to mild injury to hilar interneurons and potentiated seizure-induced hippocampal damage. In conclusion, both GalR1 and GalR2 mediate anticonvulsant effects of galanin. GalR1 and GalR2 exhibit differential effects on the initiation and the maintenance phases of SE. Activation of both galanin receptor subtypes exerts neuroprotective effects under conditions of excitotoxic injury.     

Effects of intracerebroventricular galanin or a galanin receptor 2/3 agonist on the lesion induced by transient occlusion of the middle cerebral artery in female rats

Several studies have shown that injury to the central and peripheral nervous system can increase expression of galanin, a 29 amino acid neuropeptide. Moreover, there is evidence that galanin, especially through its galanin receptor 2 (GalR2) receptor, plays a neuroprotective role in different injury models. However, direct studies of a possible neuroprotective effect of galanin in experimental stroke models are lacking. Galanin, a GalR2/3 agonist or artificial CSF was continuously infused intracerebroventricularly (i.c.v.) in naïve female rats after a 60 min transient and focal occlusion of the middle cerebral artery. The animals were sacrificed, and the ischemic lesion was visualized using 2,3,5-triphenyltetrazolium hydrochloride (TTC) staining. The lesion was 98% larger after i.c.v. administration of the GalR2/3 agonist (2.4 nmol/day) seven days after occlusion compared to artificial CSF (p = 0.023). No statistically significant differences were found after seven days in the groups treated with galanin in three different concentrations (0.24, 2.4 and 24 nmol/day; p = 0.939, 0.715 and 0.977, respectively). There was no difference in the size of the ischemic lesions measured after three days in the galanin-treated group (2.4 nmol/d) compared to artificial CSF (p = 0.925). The present results show, surprisingly, that a GalR2/3 agonist doubled the size of the ischemic lesion. Whether this effect primarily reflects the properties of the current model, species, gender and/or the mode of galanin administration, e.g. causing desensitization, or whether galanin indeed lacks neuroprotective effect of its own, remains to be corroborated.     

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.     

Developing novel antiepileptic drugs: Characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models

The endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity. In this report, we show that NAX 5055 is active in three models of epilepsy: 1) the Frings audiogenic seizure-susceptible mouse, 2) the mouse corneal kindling model of partial epilepsy, and 3) the 6 Hz model of pharmacoresistant epilepsy. NAX 5055 was not active in the traditional maximal electroshock and subcutaneous pentylenetetrazol seizure models. Unlike most antiepileptic drugs, NAX 5055 showed high potency in the 6 Hz model of epilepsy across all three different stimulation currents; i.e., 22, 32 and 44 mA, suggesting a potential use in the treatment of pharmacoresistant epilepsy. Furthermore, NAX 5055 was found to be biologically active after intravenous, intraperitoneal, and subcutaneous administration, and efficacy was associated with a linear pharmacokinetic profile. The results of the present investigation suggest that NAX 5055 is a first-in-class neurotherapeutic for the treatment of epilepsy in patients refractory to currently approved antiepileptic drugs.     

Galanin (2-11) binds to GalR3 in transfected cell lines: limitations for pharmacological definition of receptor subtypes

The neuropeptide galanin regulates a variety of physiological and pathophysiological processes through three G protein coupled receptors, GalR1, GalR2, and GalR3. The studies on galanin receptor subtype specific effects have been hampered by the lack of high affinity subtype selective antagonist and/or agonist to any of these three galanin receptor subtypes. Since its recent introduction in 2003, galanin (2-11) has been widely used as a GalR2 selective agonist in several in vitro and in vivo studies. In the present paper, we demonstrate that galanin (2-11) binds to rat GalR3 receptors in transfected cell lines with a similar affinity as it binds to GalR2. As none of the available antagonists are galanin receptor subtype selective, as shown here for M35 and M40, more work is needed to confirm whether a galanin (2-11) effect is GalR2 mediated and there is an urgent need for high affinity galanin receptor subtype selective ligands. For now one needs to interpret the data obtained at lower galanin (2-11) concentrations as effects mediated by non-GalR1 type galanin receptors, i.e., GalR2 and/or GalR3.     

Characterization of GalR1, GalR2, and GalR3 immunoreactivity in catecholaminergic nuclei of the mouse brain

The distribution of immunoreactivity for the three identified neuropeptide galanin receptors, GalR1, GalR2, and GalR3, was determined in areas of the mouse brain involved in drug addiction, including the ventral tegmental area (VTA), substantia nigra (SN), nucleus accumbens (NA), and locus coeruleus (LC). All three galanin receptors are found in the VTA, SN, NA, and LC; however, GalR1 protein is most highly represented in the VTA, NA, and SN, suggesting that GalR1 may play a predominant role in galanin-mediated regulation of dopamine neurotransmission. GalR1 and GalR3 protein levels are high in the LC, suggesting that these isoforms may be important for galanin-mediated regulation of noradrenergic transmission during opiate withdrawal. Although the distribution of GalR1, GalR2, and GalR3 largely recapitulates the pattern of galanin binding throughout the brain, some discrepancies exist, suggesting that another galanin receptor(s) may be present in some brain areas. Overall, GalR1, GalR2, and GalR3 are distributed widely throughout the brain, correlate with widespread galanin binding, and colocalize with tyrosine hydroxylase in catecholaminergic brain areas.     

The effects of systemic glucose concentration on brain metabolism following repeated brain ischemia

Since systemic glucose concentration is an important determinant of ischemic brain metabolism in neonates, we sought to determine if the systemic glucose concentration influences brain metabolic alterations following repeated partial ischemia. A group of hyperglycemic pigles (n = 12) were compared to a group of modestly hypoglycemic piglets (n = 12) using in vivo²H and ³¹P magnetic resonance spectroscopy to simultaneously measure cerebral blood flow and phosphorylated metabolites before, during and 30 min after two 10-min episodes of ischemia (i.e. Recovery 1 and 2). For both groups, β-ATP levels at Recovery 1 and 2 were lower than Control (91 ± 11and83 ± 15% of Control, respectively for both groups combined, P = 0.002 vs Control). Inorganic phosphorus was elevated in hyperglycemic piglets at Recovery 1 and 2 (117 ± 15and118 ± 10% of Control). In contrast, in modestly hypoglycemic piglets inorganic phosphorus progressively rose from Recovery 1 (131 ± 24% of Control) to Recovery 2 (149 ± 37% of Control), and differed from the hyperglycemic group (P = 0.02). These changes did not correlate with post-ischemic cerebral blood flow, cerebral O₂ delivery or cerebral glucose delivery. In both groups phosphocreatine and intracellular pH returned to Control values during Recovery 1 and 2. The progressive increase in inorganic phosphorus post-ischemia in hypoglycemic piglets suggests that modest hypoglycemia during and following repeated partial ischemia adversely affects immediate brain metabolic recovery.     

Homodimerization and internalization of galanin type 1 receptor in living CHO cells

Galanin is a 29- to 30-aa-long neuropeptide affecting feeding, cognitive, and sexual behavior. It exerts its effects through galanin receptors 1, 2 and 3, which are all seven transmembrane domain G-protein coupled receptors (GPCRs). The GPCRs have been shown to function as monomers, homodimers, heterodimers and oligomers. In this study, we examined the extent of galanin receptor 1 (GalR1) dimerization and internalization in living CHO cells using fluorescence resonance energy transfer (FRET) and time lapse confocal imaging. Ratio imaging analysis and emission spectral analysis revealed substantial homodimerization of GalR1. In addition, internalization of GalR1 after 1h of agonist stimulation with the GalR1 agonist galanin (1-29) was observed with time lapse fluorescence imaging, whereas stimulation with the GalR2 specific agonist galanin (2-11) did not lead to internalization. Treatment of GalR1 transfected cells with the non-selective adenylyl cyclase activator forskolin influenced the rate of internalization when administered together with galanin (1-29). These results indicate that GalR1 can act as a dimer on the cell surface and that receptor desensitization and internalization was observed after stimulation with the agonist galanin (1-29). Western blots further confirm the FRET data that GalR1-XFP dimerizes and can be detected in the cell as a monomer or dimer using antibodies to XFP. Internalization and dimerization of GalR1 is shown, contributing to the regulation of galanergic signaling.     

Short N-terminal galanin fragments are occurring naturally in vivo

The galanin family currently consists of four peptides, namely galanin, galanin-message associated peptide, galanin-like peptide and alarin. Unlike galanin that signals through three different G protein-coupled receptors; GAL₁, GAL₂, and GAL₃, binding at its N-terminal end, the cognate receptors for other members of the galanin family are currently unknown. Research using short N-terminal galanin fragments generated either by enzymatic cleavage or solid-phase synthesis has revealed differences in their receptor binding properties exerting numerous biological effects distinct from galanin(1-29) itself. Our studies on tissue extracts derived from rat small intestine and bovine gut using chromatographic techniques and sensitive galanin(1-16)-specific radioimmunoassay revealed the presence of immunoreactive compounds reacting with antiserum against galanin(1-16) distributed in distinct elution volumes. These results suggested a possible presence of short N-terminal galanin fragments also in vivo. Moreover, employing immunoaffinity chromatography and reverse-phase high performance liquid chromatography (HPLC) followed by mass spectrometry allowed specific enrichment of these immunoreactive compounds from rat tissues and identification of their molecular structure. Indeed, our study revealed presence of several distinct short N-terminal galanin sequences in rat tissue. To prove their receptor binding, four of the identified sequences were synthetized, namely, galanin(1-13), galanin(1-16), galanin(1-20), galanin(6-20), and tested on coronal rat brain sections competing with ¹²⁵I-labeled galanin(1-29). Our autoradiographs confirmed that galanin(1-13), galanin(1-16), and galanin(1-20) comprehensively displaced ¹²⁵I-galanin(1-29) but galanin(6-20) did not. Here we show, for the first time, that short N-terminal galanin fragments occur naturally in rat tissues and that similar or identical galanin sequences can be present also in tissues of other species.Biological significanceThis study is first to provide an evidence of the presence of short N-terminal galanin fragments in vivo in a biological system and provides further foundations for the previous studies using synthetized short N-terminal galanin fragments.     

Differential galanin receptor-1 and galanin expression by 5-HT neurons in dorsal raphé nucleus of rat and mouse: evidence for species-dependent modulation of serotonin transmission

Galanin and galanin receptors are widely expressed by neurons in rat brain that either synthesize/release and/or are responsive to, classical transmitters such as gamma-aminobutyric acid, acetylcholine, noradrenaline, histamine, dopamine and serotonin (5-hydroxytryptamine, 5-HT). The dorsal raphé nucleus (DRN) contains approximately 50% of the 5-HT neurons in the rat brain and a high percentage of these cells coexpress galanin and are responsive to exogenous galanin in vitro. However, the precise identity of the galanin receptor(s) present on these 5-HT neurons has not been previously established. Thus, the current study used a polyclonal antibody for the galanin receptor-1 (GalR1) to examine the possible expression of this receptor within the DRN of the rat and for comparative purposes also in the mouse. In the rat, intense GalR1-immunoreactivity (IR) was detected in a substantial population of 5-HT-immunoreactive neurons in the DRN, with prominent receptor immunostaining associated with soma and proximal dendrites. GalR1-IR was also observed in many cells within the adjacent median raphé nucleus. In mouse DRN, neurons exhibited similar levels and distribution of 5-HT-IR to that in the rat, but GalR1-IR was undetectable. Consistent with this, galanin and GalR1 mRNA were also undetectable in mouse DRN by in situ hybridization histochemistry, despite the detection of GalR1 mRNA (and GalR1-IR) in adjacent cells in the periaqueductal grey and other midbrain areas. 5-HT neuron activity in the DRN is primarily regulated via 5-HT1A autoreceptors, via inhibition of adenylate cyclase and activation of inward-rectifying K+ channels. Notably, the GalR1 receptor subtype signals via identical mechanisms and our findings establish that galanin modulates 5-HT neuron activity in the DRN of the rat via GalR1 (auto)receptors. However, these studies also identify important species differences in the relationship between midbrain galanin and 5-HT systems, which should prompt further investigations in relation to comparative human neurochemistry and which have implications for studies of animal models of relevant neurological conditions such as stress, anxiety and depression.     

Antinociceptive roles of galanin receptor 1 in nucleus accumbens of rats in a model of neuropathic pain

It has been reported that galanin and its receptors might be involved in the modulation and transmission of nociception in the central nervous system. Our previous research has also demonstrated that galanin induces antinociception in the nucleus accumbens (NAc) of intact rats. However, the interaction between galanin and its receptors in the NAc and the underlying mechanism of suppressing pain transmission remain unclear. The present study seeks to determine the antinociception induced by galanin receptor (GalR)?1 stimulation in the NAc of rats with neuropathic pain. The left sciatic nerve of rats was ligated to mimic a neuropathic pain model. Western blots showed that the expression of GalR1 was significantly upregulated in the NAc of rats with neuropathic pain. Intra‐NAc injection of GalR1 agonist M617 induced a dose‐dependent increase in hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulations in rats with neuropathic pain. Also, the effect of M617 was attenuated by M35, a GalR1/2 antagonist; at the same time, M35 reduced the galanin‐induced antinociception, suggesting that GalR1 mediates antinociception induced by galanin in the NAc of rats with neuropathic pain. Furthermore, we found that M617‐induced antinociception in rats with neuropathic pain was stronger than the antinociception in intact rats. We also found that injections of M617 and galanin each induced significant increases in HWL, but the galanin‐induced antinociception was stronger than that of M617. All these results suggest that GalR1 plays an important role in antinociception and that other GalRs also are involved in pain modulation induced by galanin in the NAc of rats with neuropathic pain. © 2015 Wiley Periodicals, Inc.     

Influence of chronic administration of antidepressant drugs on mRNA for galanin, galanin receptors, and tyrosine hydroxylase in catecholaminergic and serotonergic cell-body regions in rat brain

Activity of locus coeruleus (LC) neurons and release of the peptide galanin (GAL), which is colocalized with norepinephrine (NE) in LC neurons, has been implicated in depression and, conversely, in antidepressant action. The present study examined the influence of chronic administration (for 14days, via subcutaneously-implanted minipump) of antidepressant (AD) drugs representing three different classes (tricyclic [desipramine], selective serotonin reuptake inhibitor [SSRI] [paroxetine], and monoamine oxidase inhibitor [MAOI] [phenelzine]) on mRNA for GAL, GAL receptors (GalR1, GalR2, and GalR3), and tyrosine hydroxylase (TH), the rate-limiting enzyme for NE synthesis, in four brain regions--LC, A1/C1, dorsal raphe (DRN), and ventral tegmentum (VTA) of rats. Consistent with previous findings that chronic administration of AD drugs decreases activity of LC neurons, administration of AD drugs reduced mRNA for both GAL and TH in LC neurons. GAL and TH mRNA in LC neurons was highly correlated. AD drugs also reduced GAL and TH mRNA in A1/C1 and VTA but effects were smaller than in LC. The largest change in mRNA for GAL receptors produced by AD administration was to decrease mRNA for GalR2 receptors in the VTA region. Also, mRNA for GalR2 and GalR3 receptors was significantly (positively) correlated in all three predominantly catecholaminergic brain regions (LC, A1/C1, and VTA). Relative to these three brain regions, unique effects were seen in the DRN region, with the SSRI elevating GAL mRNA and with mRNA for GalR1 and GalR3 being highly correlated in this brain region. The findings show that chronic administration of AD drugs, which produces effective antidepressant action, results in changes in mRNA for GAL, GAL receptors, and TH in brain regions that likely participate in depression and antidepressant effects.     

Antinociceptive effects of galanin in the rat tuberomammillary nucleus and the plasticity of galanin receptor 1 during hyperalgesia

Although the tuberomammillary nucleus (TM) is well defined in terms of anatomy and neurochemistry, little is known about its function in nociceptive modulation. There was an abundance of galanin-immunoreactive fibers in the TM, and galanin has been implicated in pain processing. The present study assessed the role of galanin in the modulation of nociception in the TM of rats. Intra-TM injection of galanin dose-dependently increased the hindpaw withdrawal latency of rats to a noxious thermal stimulus, indicating an antinociceptive role of galanin in the TM. The antinociceptive effect of galanin was blocked by a subsequent intra-TM injection of galantide, a putative galanin receptor antagonist, suggesting that the antinociceptive effect of galanin is mediated by galanin receptors. Moreover, there was abundant galanin receptor 1 (GalR1) in the TM, and the number of GalR1-positive neurons in the ipsilateral TM increased significantly after unilateral loose ligation of the sciatic nerve compared with the contralateral TM or the TM of intact rats. However, the number of GalR1-positive neurons was not significantly altered by carrageenan-induced inflammation, in either the ipsilateral or the contralateral TM. The results suggest that galanin and GalR1 in the TM may play important roles in pain regulation.