A Galanin Receptor Subtype 1 Specific Agonist

Linda Lundstrom,1 Ulla Sollenberg,1 Ariel Brewer,2 Poli Francois Kouya,3 Kang Zheng,4

Xiao-Jun Xu,3 Xia Sheng,4 John K. Robinson,2 Zsuzsanna Wiesenfeld-Hallin,3

Zhi-Qing Xu,4 Tomas Hokfelt,4 Tamas Bartfai,5 and Ulo Langel1,6

(Accepted September 15, 2004)

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 culturedcells as well as in vivo. Displacement studies on cell membranes expressing hGalR1 or hGalR2show the presence of a high affinity binding site for M617 on GalR1 (Ki=0.23±.12 nM) while

lower affinity was seen towards GalR2 (Ki=5.71±1.28 nM) resulting in 25-fold specificity forGalR1. Activation of GalR1 upon stimulation with M617 is further confirmed by internali-zation of a GalR1-EGFP conjugate. Intracellular signaling studies show the ability of M617 toinhibit 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 onboth receptors studied after treatment with M617 in the presence of galanin. In noradrenergiclocus 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.) administra-tion of M617 dose-dependently stimulates food uptake in rats while, in contrast, M35completely 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 hyperexcitablilityinduced 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 forGalR1 and agonistic activity, both in vitro and in vivo.

KEY WORDS: cAMP production; chimeric peptide; feeding behavior; galanin; galanin receptor; inositolphosphate production; locus coeruleus; nociception; receptor internalization; receptor specificity.

INTRODUCTION

The neuropeptide galanin consists of 29 aminoacids (30 in human) and was originally isolated fromporcine upper intestine in 1983 (Tatemoto et al.1983). The N-terminus of galanin is associated withbiological activity of the peptide and is highly

Abbreviations: CHO, Chinese Hamster Ovary Cells; CS, condi-

tional stimulation; DMF, N,N-dimethylformamide; GalR1,

galanin receptor type 1; GalR2, galanin receptor type 2; HM,

HEPES-buffered magnesium solution; i.c.v., intracerebroventricu-

lar; i.p., intraperitoneal; i.t., intrathecal; IP, inositol phosphates;

LC, locus coeruleus; PBS, phosphate-buffered saline.

1 Department of Neurochemistry and Neurotoxicology, Stockholm

University, Stockholm, Sweden.2 Department of Psychology, SUNY at Stony Brook, Stony Brook,

NY, USA.3 Department of Laboratory Medicine, Division of Clinical Neu-

rophysiology, Karolinska Institutet, Stockholm, Sweden.4 Department of Neuroscience, Karolinska Institutet, Stockholm,

Sweden.5 Department of Neuropharmacology, The Scripps Research

Institute, La Jolla, CA, 92037, USA.6 Correspondence should be addressed to: Ulo Langel, Department

of Neurochemistry and Neurotoxicology, Stockholm University,

Stockholm, Sweden. Tel: +46-8-161793; Fax: +46-8-161371;

e-mail: ulo@neurochem.su.se

International Journal of Peptide Research and Therapeutics, Vol. 11, No. 1, March 2005 (� 2005), pp. 17–27

DOI: 10.1007/s10989-004-1717-z

171573-3149/05/0300–0017/0 � 2005 Springer Science+Business Media, Inc.

conserved across species. Galanin shows a wide-spread anatomical distribution throughout the cen-tral and peripheral nervous systems and coexists withother neuropeptides and/or classical transmitters inmany brain regions (Melander et al. 1986; Chan-Pa-lay 1988; Skofitsch et al. 1989). The galanin peptidemostly acts as an inhibitory, hyperpolarizing neuro-modulator and has been found to influence severalphysiological processes such as regulation of cogni-tion and memory (Wrenn and Crawley 2001),inducing food consumption (Crawley 1999), attenu-ating nociception (Xu et al. 2000), acting as an anti-convulsant (Mazarati et al. 2001), inhibiting insulinrelease (Sjoholm and Efendic 2001) and elevatinggrowth hormone levels (Ottlecz et al. 1988).

Three different receptors for galanin have beenidentified and cloned, Galanin R1 (Habert-Ortoliet al. 1994), Galanin R2 (Fathi et al. 1997; Smithet al. 1997; Wang et al. 1997a) and Galanin R3(Wang et al. 1997b, Smith et al. 1998). These receptorsubtypes possess only 40–50% sequence homologywithin species, but show a high degree of homologybetween species (Floren et al. 2000). The galaninreceptors are G-protein coupled and differ in signal-ing pathways, where GalR1 and GalR3 activate Gi/o

while GalR2 predominantly couples to Gq/11 (Wanget al. 1998, Branchek et al. 2000). All three receptorsdemonstrate high affinity for galanin but are distin-guishable by the maintained affinity of GalR2 andGalR3 for N-terminal deletions of the galanin pep-tide. Recently, Ignatov et al. reported on an addi-tional galanin receptor, galanin-receptor like(GalRL) receptor which shares 25–26% identity inprimary structure with the galanin receptor family.This receptor is weakly activated by galanin, sug-gesting an endogenous ligand, which shares sequencesimilarity with galanin (Ignatov et al. 2004).

The lack of potent and selective non-peptidergicagonists and antagonists acting on the galaninreceptors has been a limiting factor in pharmaco-

logical characterization of the galanin receptors,leaving chimeric ligands and galanin analogues as theonly available tools, besides galanin, for activation ofthe receptors. The chimeric ligands consist of the N-terminal fragment (1–13) of galanin, linked to frag-ments of other bioactive peptides and subsequentlydisplay high affinity for the receptors, though none ofthem are subtype specific (Floren et al. 2000). Today,the most widely used chimeric ligands are M35 (Kasket al. 1995) and M40 (Bartfai et al. 1993) which ex-hibit partial agonistic effects on the receptors in vitro,while antagonistic properties have been observed invivo (Floren et al. 2000). For subtype specific recog-nition of the galanin receptors there exist a couple ofgalanin analogues with specificity for GalR2 such asGal(2–11) (AR-M1896) (Liu et al. 2001), Gal(2–29)(Wang et al. 1997a), D-Trp2-galanin (Smith et al.1997), while specific ligands for GalR1 remain elusive.

In this study, we present a novel chimeric galaninreceptor ligand named M617 with 25-fold subtypespecificity for GalR1 in comparison to GalR2. Inculture cell, we investigate the effect of M617 onGalR1 internalization and signal transductionthrough GalR1 and GalR2. We further examine theactivity of the ligand in noradrenergic locus coeruleus(LC) neurons and its effects in vivo on food con-sumption in rats and in nociception with a spinalflexor reflex model.

MATERIALS AND METHODS

Peptide Synthesis

The peptides (cf. Table I) were synthesized in a stepwise

manner in a 0.1 mmol scale on an automated peptide synthesizer

(Applied Biosystems, Model 431A) using the t-Boc solid-phase

peptide synthesis strategy. tert-Butyloxycarbonyl amino acids

(Neosystem, Strasbourg, France) were coupled as hydroxybenzo-

triazole (HOBt) esters to a P-methylbenzylhydrylamine (MBHA)

resin (Neosystem, Strasbourg, France) to obtain C-terminally ami-

dated peptides. Deprotection of the formyl protecting group on

Table I. Peptide Sequence and Ki Value for Displacement of Porcine-[125I]-Galanin on Bowes Melanoma Cells Expressing hGalR1 or CHOCells Stably Transfected with hGalR2 for the Peptides Used in the Study

Peptide Sequence Ki (nM) KihGalR2/Ki

hGalR1

hGalR1 hGalR2

rGal GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-amide 1.38±0.05 1.54±0.35 1.11M617 Gal(1–13)-Gln14-bradykinin(2–9)-amide; GWTLNSAGYLLGPQPPGFSPFR 0.23±0.12 5.71±1.28 24.8M35 Gal(1–13)-bradykinin(2–9)-amide; GWTLNSAGYLLGPPPGFSPFR 0.11* 1.95* 17.4

rGal (Rat Galanin). Data is mean±SEM of three independent experiments performed in duplicate.*(Borowsky, B et al. (1998)), hGalR1 and hGalR2 stably transfected in LMKT- and CHO cells, respectively.

18 Lundstrom et al.

tryptophan was carried out in 20% piperidine in DMF for 1 h, and

deprotection of the dinitrophenyl group on histidine was carried out

by treatment of 20% thiophenol in DMF for 1 h. The peptide was

finally cleaved from the resin using liquid HF at 0�C for 1 h in the

presence of P-cresol and P-thiocresol (1:1). The peptide was purified

by HPLC (Nucleosil 120-3 C-18 RP-HPLC column), and the

molecular weight was determined by MALDI-TOF mass spec-

trometry (Voyager-DE STR, Applied Biosystems, Framingham,

USA).

Construction of the EGFP-tagged GALR1 Receptor

For construction of GALR1-EGFP, the cDNA of the rat

GALR1 gene was amplified by RT-PCR, and HindIII and KpnI

were added to 5¢ and 3¢ primers, respectively, in order to facilitate

cloning into the pEGFPN1 vector from HindIII and KpnI sites

(Clontech Laboratories, Palo Alto, CA, USA). Plasmid DNA was

prepared using standard techniques. The full open reading frame

was sequenced to verify the integrity of the GALR1-EGFP fusion

prior to use.

Cell Culture

Bowes human melanoma cells (American Type Culture Col-

lection CRL-9607) were cultured in Eagle’s minimal essential

medium with Glutamax-1 supplemented with 10% foetal bovine

serum (FBS), 1% non-essential amino acids, 1% sodium pyruvate,

100 U ml)1 penicillin and 100 lg ml)1 streptomycin. Chinese

Hamster Ovary (CHO) K1 cells stably expressing human galanin

R2 (a gift from Kathryn A. Jones and Tiina P. Iismaa, Sydney

Australia) were cultured in Dulbecco’s modified essential medium

F-12 with Glutamax-1 supplemented with 10% FBS, 2 mM

L-glutamine, 100 U ml)1 penicillin and 100 lg ml)1 streptomycin.

HEK293 cells were grown in Dulbecco’s modified Eagle’s medium

(DMEM) supplemented with 10% FBS and were transfected using

1 lg of DNA employing Effectene Transfection Reagent kit (Qia-

gen, Hilden, Germany). To generate stable transfectants, the

transfected cells were selected in the presence of G418 at 500 lg/ml.

Clonal expression was examined initially by fluorescence micros-

copy, and clones for further study were selected and expanded. Cell

cultures were grown at 37�C in a 5% CO2 incubator, cell culture

reagents were purchased from Invitrogen (Stockholm, Sweden) and

cell plastics were from Corning (Lab-Design, Stockholm, Sweden).

Galanin Receptor Displacement Studies

Cells for receptor displacement studies were seeded in

100 mm dishes and cultured 3–4 days until confluent. Cell dishes

were washed and scraped into PBS and centrifuged at 4�C, 1000 ·g for 10 min. The pellet was resuspended in HM buffer (20 mM

HEPES, 5 mM MgCl2, pH 7.4) and incubated for 30 min on ice.

The cell mixture was centrifuged at 4�C, 1000 · g for 10 min and

the obtained pellet was resuspended in HM buffer supplemented

with 0.3% BSA and 1% protease inhibitor cocktail (SIGMA).

Displacement studies on cell membranes were performed in a final

volume of 400 ll, containing 0.016 nM porcine-[125I]-galanin

(Amersham Biosciences, Uppsala, Sweden), 20 lg cell membrane,

and various concentrations of peptide (10)6)10)11 M). Protein

concentration was determined according to Bradford (BioRad,

Stockholm, Sweden). Peptide solutions were made in assay buffer

using silanised (Dichlorodimethylsialne, SIGMA) tubes and pip-

ette tips. Samples were incubated at 37�C for 30 min, and the

displacement reaction was terminated by addition of ice cold HM

buffer before filtration over Whatman GF/C glass fiber filters

(Whatman International Ltd., Mainstone, UK), pre-soaked in

0.3% polyethylenimine solution (SIGMA). The retained radioac-

tivity on the filters was determined in a gamma counter (Packard

Instrument Company, Meriden, CT). Non-specific binding was

determined as the part of total binding that could be displaced

with 1 lM unlabeled galanin. IC50 values for the peptides were

calculated using Prism (GraphPad Software Inc., San Diego, CA,

USA) and converted into Ki values using the equation of Cheng–

Prusoff.

Analysis of GALR-EGFP Internalization

For internalization studies, the cells were treated with galanin

(0.1 lM) for 10 or 30 min at 37�C before fixation with 4% para-

formaldehyde for 20 min at 4�C. Images were acquired using a

laser scanning confocal system installed on a Nikon E-600 micro-

scope (Biorad Radiance Plus, Biorad). EGFP was excited using a

488 nm argon laser and detected with 530–560 nm band pass filter.

Images were collected and processed using Adobe Photoshop

(Adobe Systems, CA).

Cyclic AMP Formation

Determination of the cyclic AMP (cAMP) formation in Bowes

melanoma cells were performed with CatchPoint Cyclic-AMP

Fluorescent Assay Kit (Molecular Devices Corporation, CA,

USA). Bowes melanoma cells were seeded in a 96-well plate to a

density of 105 cells 24 h before experiment. Cells were washed once

with 200 ll Krebs Ringer bicarbonate buffer (KRBB) (0.49 mM

MgCl2, 4.56 mM KCl, 120 mM NaCl, 0.7 mM Na2HPO4, 1.5 mM

NaH2PO4, 10 mM glucose, 15 mM NaHCO3, pH 7.4) prior to

incubation with 100 ll 1 mM cAMP phosphodiesterase inhibitor,

3-isobutyl-1-methylxanthine (IBMX), for 10 min. Cells were stim-

ulated with 50 lM forskolin and various concentrations,

10)6)10)11 M, of peptide at 37�C for 15 min. The cells were lysed

with 50 ll lysis buffer (Molecular Devices Corporation, CA, USA)

at RT for 10 min and 40 ll of the final cell lysate was used for

determination of cAMP concentrations. Fluroescence was read

with a Gemini XS detection system (Molecular Devices Corpora-

tion, CA, USA). IC50 values were calculated using Prism (Graph-

Pad Software Inc., San Diego, CA). Protein concentration of the

samples was determined according to the method of Lowry (Bio-

Rad, Stockholm, Sweden).

Inositol Phosphate Accumulation

CHO cells stably expressing human GalR2 were seeded in 12-

well plates and cultured for two days in regular growth medium

before incubation with 1 lCi [3H]-myo-Inositol (Amersham, Upp-

sala, Sweden) in M-199 medium containing 100 U ml)1 penicillin

and 100 lg ml)1 streptomycin for 24 h. The cells were incubated

with M-199 medium for 10 min at 37�C and washed twice with

Hepes Krebs Ringer, HKR (5 mMHepes, 137 mMNaCl, 2.68 mM

KCl, 2.05 mM MgCl2, 1.8 mM CaCl2, 1 g/l glucose, pH 7.4) fol-

lowed by 10 min pre-incubation in 1 ml HKR buffer with 10 mM

LiCl at 37�C. Cells were stimulated with peptide (10)5–10)10 M) for

A Galanin Receptor Subtype 1 Specific Agonist 19

1 h at 37�C, before the reaction was terminated by addition of 200 llice cold perchloric acid followed by incubation on ice for 30 min.

The sample volume was neutralized to pH 7 with 340 ll 1.5 M

KOH, 75 mM Hepes before diluted with water. Anion exchange

chromatography of the samples was performed over 1 ml 50:50

Dowex (AG 1-X8 Resin, 200–400 mesh formate). The columns were

washed with 5 ml water before the inositol phosphates (IP) were

eluted with 5 ml of 0.1 M formic acid/1.2 M ammonium formate.

Radioactivity of the eluate was determined using scintillation

counting in a b-counter (Packard Instrument Company, Meriden,

CT), and each sample was normalized against the total count ob-

tained before anion exchange chromatography.

in vitro Whole Cell Recording

The slices were prepared as previously described (Xu et al.,

1994). Briefly, the rat was decapitated, and the brain was rapidly

dissected out. LC-containing horizontal slices (300)400 lm thick)

were prepared with a Vibratome in ice-cold artificial cerebrospi-

nal fluid (ACSF), saturated with 95% O2/5% CO2. The ACSF

contained in mM: 124 NaCl, 2.5 KCl, 1.3 MgSO4, 1.24 NaH2-

PO4, 2.4 CaCl2, 25 NaHCO3 and 10 glucose. Slices were then

transferred to a submerged chamber perfused with oxygenated

ACSF at 35)37�C. Slices remained in the chamber for 1.5–2 h

prior to recording. The LC could be identified in the transillu-

minated slice as a dark oval area on the lateral edge of the fourth

ventricle. Whole cell recordings were made with an EPC-9 double

patch-clamp amplifier controlled by PULS (HEAK). LC neurons

were identified on the basis of somata shape and position using

infrared video microscopy and differential interference contrast

optics as well as their distinctive discharge and membrane

properties (Williams et al. 1984). Neurons were routinely held

near their resting membrane potential. Drugs were applied by a

pressure-driven DAD-12 VC superfusion system (ALA) or bath

perfusion.

Feeding

Sprague-Dawley male rats, approximately 120 days old, were

housed individually in plastic tub cages in controlled humidity

and temperature vivarium, and were maintained on a 07.00 on/

19.00 off lighting schedule. Twenty-one rats served in the first

experiment that examined M617, and a second group of 21 rats

served in the experiment that looked at M35. Stereotaxic surgery

in the rats was conducted under ketamine (50 mg/kg i.p.) and

xylazine (10 mg/kg i.p.) anesthesia. Each subject was unilaterally

implanted into the lateral ventricle with a guide cannula made of

stainless-steel hypodermic tubing (24 gauge, 1.7 cm). The coordi-

nates were AP (from bregma) )1.0, DV )3.5, LAT + 1.0. All

cannula placements were verified histologically to be in the lateral

ventricle. For behavioral testing, M617 and M35 were dissolved in

saline solution. Each rat received 5 ll of M617, M35 or 5 ll ofsaline i.c.v. 10 min before the start of the feeding testing. The rats

were then placed in an empty plastic tub cage in the presence of

two Nabisco Nilla WafersTM cookies (Nabisco Brands, Inc. East

Hanover, N.J.; 5 wt: fat = 14%, carbohydrate = 79%, pro-

tein = 7%) soaked in 10 ml water, in a plastic weighing boat. The

sessions were 10 min in duration. All animals had been pre-ex-

posed to one cookie in the home-cage 24 h prior to the first

feeding testing session to overcome neophobia to the food. All

spillage was collected and included in the calculations. Activity

was also scored during the feeding testing session using a Digiscan

(BRS/LVE, Columbus, OH). The procedures were conducted in

accordance with the NIH Guide for the Care and Use of Labo-

ratory Animals (1985) and with the approval of the State Uni-

versity of New York at Stony Brook Institutional Animal Care

and Use Committee.

Flexor Reflex Model

The rats were anesthetized with chloral hydrate (300 mg/kg,

i.p.), ventilated and decerebrated by aspiration of the forebrain

and midbrain. The spinal cord was exposed by a laminectomy at

mid-thoracic level and sectioned at Th8-9. An i.t. catheter (PE 10)

was inserted caudal to the transection with its tip on the lumbar

enlargement (L4–5). The flexor reflex was elicited by supramaxi-

mal electric shocks applied to the sural nerve innervation area of

the left foot (0.5 ms, 1 mA, 1/min) that activated A- and C-fibers.

In all experiments, a conditioning stimulation (1 HZ, 20 s) of the

same intensity was used to elicit spinal cord hyperexcitability. The

flexor reflex was recorded as E.M.G. activity via stainless steel

needle electrodes inserted into the ipsilateral posterior biceps

femoris/semitendinosus muscles and was integrated over 2 s.

During the experiments the heart rate was monitored and rectal

temperature was maintained within normal range. Galanin (Ba-

chem, Bubenhoff, Switzerland) or M617 were dissolved in 0.9%

saline and injected i.t. in a volume of 10 ll followed by 10 llsaline flush.

Statistical Analysis

Statistical comparison on cAMP inhibition and IP produc-

tion after peptide stimulation was analyzed using ANOVA fol-

lowed by Dunnett’s test where galanin stimulation served as

control. On LC activity, statistical comparisons were performed

using Student’s t-test, and statistical differences were considered

significant at P<0.05. For the in vivomodels, the data were analyzed

using ANOVA followed by Fisher’s PLSD post hoc test or by paired

t-test.

RESULTS

Galanin Receptor Displacement Studies

Displacement studies of the M617 peptide wereperformed on Bowes melanoma cells endogenouslyexpressing hGalR1 and CHO cells stably transfectedwith hGalR2. Displacement by M617 at GalR1revealed a high affinity binding site with Ki=0.23±0.12 nM, while lower affinity was seen towardsGalR2, Ki=5.71±1.28 nM, showing a 25-foldincreased affinity for GalR1 (Table I; Fig. 1). In thesame experiments, porcine 125I-galanin was displacedby galanin on GalR1 and GalR2 with Ki=1.38±0.05 nM and 1.54±0.35 nM, respectively (Table I).Under these conditions, GalR1 shows even higheraffinity for M617, than for galanin itself.

20 Lundstrom et al.

Endocytosis of GALR1-EGFP

After expression of the GALR1-EGFP cDNA inHEK293 cells followed by fixation and imaging inconfocal microscope, a strong green fluorescent sig-nal was observed, mainly located at the plasmamembrane of the transfected cells (Fig. 2a). M617(100 – 1000 nM) application elicited a redistributionof GALR1-EGFP fluorescence from the plasmamembrane to the intracellular compartment(Fig. 2b). Internalization was detectable already after3–5 min, but appeared maximal within 10–15 min,and was maintained for at least 30 min.

Cyclic AMP Formation

Intracellular signaling by M617 via GalR1 wasanalyzed by cAMP inhibition following forskolinstimulation of Bowes melanoma cells endogenouslyexpressing GalR1. Treatment with 10 lM M617 for15 min resulted in a 57% reduction in cAMP levels ascompared to control cells, treated with forskolin inthe absence of peptide (Fig. 3a). The attenuation incAMP levels was diminished at 0.1 nM peptide, dis-playing an EC50=104±26.1 nM for M617 (Fig. 3a).In the same assay, galanin produced a 65% decreasein cAMP level, EC50=31.6±10.2 nM (Fig. 3a). Aminor right shift in the inhibition curve was seen forM617 compared to galanin (Fig. 3a), hence showingless intracellular activation through GalR1. Stimu-lation with M617 in the presence of 32 nM galaninwas not able to reverse the inhibitory effect of gala-nin, but rather producing a small additive effect onthe cAMP inhibition (significant for 0.1 lM M617,**P<0.01) (Fig 3b). The agonist-like effect of M617was seen at concentrations between 10 lM and 1 nM(Fig. 3b). In agreement with these observations, M35showed comparable agonistic effects (Fig 3b), where asignificant additive effect on galanin was observed athigher concentrations (10 lM; **P<0.01 and 0.1 nM;*P<0.05) (Fig. 3b).

Inositol Phosphate Accumulation

Signal transduction through GalR2 was examinedby assessing the ability to stimulate IP production inCHO cells expressing GalR2. Treatment with 10 lM

Fig. 1. Displacement studies of porcine-[125I]-galanin by M617 onmembranes from Bowes melanoma cells (GalR1) (d) or CHOGalR2 (s) cells. Receptor binding is presented as % of specificbinding. Data are from two representative experiments performedin duplicate, presented as mean±SEM.

Fig. 2. GALR1-EGFP is internalized after M617 treatment. (a) GALR1-EGFP is mainly located at the plasma membrane of the transfectedHEK293 cells. (b) Upon stimulation by M617, GALR1-EGFP is redistributed within 5–10 min from the plasma membrane to the cytoplasm.Bar equals 10 lm in both a and b.

A Galanin Receptor Subtype 1 Specific Agonist 21

of peptide, galanin or M617, stimulated IP produc-tion 5 times that of basal level (Fig. 4a). A some-what lower transduction was observed by M617compared to galanin, EC50=304.4±60.9 nM versus173.3±63.3 nM for galanin (Fig 4a). Stimulationwith M617 in the presence of 0.1 lM galanin showeda concentration dependent production of IP, in-creased production at high concentrations, whereasno change was observed at lower concentrations,compared to galanin alone (Fig. 4b). Similar ago-nistic effects were seen upon stimulation with M35 inthe presence of galanin (Fig. 4b).

Whole Cell Patch-clamp Recording from LC Slice

Preparation

Bath and micropipette application of M617 inhib-ited the spontaneous firing of LC neurons and causedhyperpolarization. Under whole cell patch-clampconditions, superfusion of M617 (100–1000 nM) pro-duced a reversible outward current, which lastedbetween 3 and 15 min in all tested LC neurons (n=12)(Fig. 5) with a peak value within 1–2 min. The M617-induced outward current was also seen in LC neurons(n=4) exposed to ACSF containing tetradotoxin

(TTX) low Ca2+ (0.25 mM), high Mg2+ (10 mM)(n=4) (data not shown). Thus, even when the afferentinput to the LC is blocked (via TTX Ca2+, highMg2+

concentration in the medium), M617 still exerted aneffect on LC neurons.

Feeding

As shown in Fig. 6, food consumption was sig-nificantly greater following i.c.v. administration ofM617 (F2,18=5.0, P<0.02). Fisher’s PLSD post hoctest revealed only the 20 lg (P<0.01) group as sig-nificantly different from the saline group (Fig. 6a).The saline versus the 10 lg group comparison wasP<0.06.While somewhat diminished, activity was notsignificantly altered at the P<0.05 level by M617administration (F2,18=2.2, P<0.13) (Fig. 6b).Administration of M35 produced no significant effecton food intake (F2,18=0.35) or activity (F2,18=0.39) (Fig. 6 c and d).

Flexor Reflex

The effect of M617 was examined over fourdoses, 66 ng, 660 ng, 6.6 lg and 66 lg. When

Fig. 3. Inhibition of forskolin stimulated cAMP production in Bowes melanoma cells. Cells were stimulated with 50 lM forskolin followedby stimulation with various concentrations of either galanin (s) or M617 (d) (a). Cells were further stimulated with peptides, M617 or M35 inthe presence of 32 nM (10)7.5 M) galanin (b). C=Control, forskolin stimulated cells in the absence of peptide. Data are presented as % ofcontrol, no peptide added and as mean ± SEM of three independent experiments performed in duplicate. Data are analyzed by one wayANOVA followed by Dunnett’s test, *P<0.05 and **P<0.01.

22 Lundstrom et al.

administered by itself, M617 produced an initialbrief (5–10 min) facilitation of the flexor reflexwhich was most clear at the lowest dose (Fig. 7). Noreflex depression was observed from M617 at thethree lower doses. At the highest dose tested in 3animals, 66 lg, M617 induced an irreversible total

reflex depression in one experiment and in the othertwo experiments it produced a strong increase inongoing activity, suggesting a non-specific, possiblytoxic, effect. I.t. galanin at 100 ng and 1 lg alsocaused an initial, brief facilitation of the flexor reflex(Fig. 7). This initial reflex facilitation was not sig-nificantly affected by pretreatment of M617 (Fig. 7),although an increase in galanin-induced facilitationwas seen in some experiments when combining highdoses. Conditioning stimulation (CS) of the C-fiberat 1 Hz for 20 stimulations produced a marked in-crease in spinal excitability (198.8±36.3% overbaseline for 4.9±1.4 min) (data not shown). Thisreflex facilitation was not affected by M617 at thelowest dose (Fig. 8). At two higher doses, C-fiberCS-induced facilitation was significantly reduced byM617 in the majority of the experiments (Fig. 8). I.t.galanin at 100 ng and 1 lg also reduced the C-fiberCS (Fig. 8). The effect of galanin was neither en-hanced nor antagonized by M617 at 660 ng and6.6 lg, respectively (Fig. 8).

Fig. 4. Inositol phosphate production in CHO GalR2 cells. Cells were pre-incubated with [3H]- myo-Inositol for 24 h before stimulation withpeptide, galanin (s) or M617 (d) at different concentrations (a). Cells were further stimulated with M617 or M35 in the presence of 0.1 lM(10)7M) galanin (b). Data are presented as % of control, no peptide added and mean±SEM of three data sets performed in duplicate. Dataanalysis by one way ANOVA did not reveal significant differences.

Fig. 5. M617-induced outward current in a LC neuron in vitro.Under whole cell patch-clamp conditions, bath application ofM617 (1 lM) induces a reversible outward current on the LCneuron. The holding potential was )60 mV.

A Galanin Receptor Subtype 1 Specific Agonist 23

DISCUSSION

In this study, we present a novel chimericgalanin receptor ligand named M617, galanin(1–13)-Gln14-bradykinin(2–9)-amide, with subtype speci-

ficity for GalR1, agonistic properties on intra-cellular signaling and in the noradrenergic LC neu-rons as well as functional agonistic activity infeeding and in flexor reflex models in vivo. M617 hashigh sequence homology with the well established

Fig. 6. Food consumption in rats following i.c.v. administration of peptides. The effect of M617 and M35 (10 or 20 lg) on the consumptionof cookie mash (a, c) and activity (photobeam interruption) per session (b, d). Mean±SEM are presented. **P<0.01 by Fishers PLSD test.M617; N=8 for each group, M35; N=7 for each group.

Fig. 7. Facilitatory effect of M617, galanin and their combination on the flexor reflex. Data are expressed as mean±SEM and the number ofexperiments is shown in brackets. NS=no significant difference compared to galanin alone with paired t-test.

24 Lundstrom et al.

chimeric antagonist M35, galanin(1–13)-bradyki-nin(2–9)-amide, with Gln at position 14 as the onlymodification. We compared receptor binding proper-ties ofM617 on hGalR1 (naturally expressed in Bowesmelanoma cells) and hGalR2 (stably transfected inCHO cells). Our studies revealed a high affinity bind-ing site forM617 onGalR1, whereas lower affinity wasobserved for GalR2, resulting in a 25-fold specificityfor GalR1 as well as an increased affinity compared tothe endogenous ligand. Displacement studies with therelated chimeric peptide M35 have been performedpreviously for characterization of novel galaninreceptors, where M35 has shown a somewhat higheraffinity (a 2–18 fold specificity) for cells transfectedwith GalR1 compared to cells transfected with GalR2(Smith et al. 1997, Smith et al. 1998, Borowsky et al.1998). The increasedGalR1 subtype specificity seen byM617 in the present study therefore indicates theimportance of Gln14 in the sequence, although thenature of this phenomena is not clear yet (Table I). Itis noteworthy that Bowes cells do not express brady-kinin receptors, which consequently are unable tocontribute to the observed high affinity receptorbinding. Further supporting the effect of M617 onGalR1 receptors, we show a distinct internalization ofGalR1-EGFP receptors transfected to HEK 293 cellsafter treatment with M617.

Intracellularly, M617 showed a somewhat de-creased inhibition of the forskolin stimulated cAMPproduction compared to galanin, despite its in-creased GalR1 receptor affinity, presenting a lowersignal transduction of the chimera through GalR1(Fig. 2). In general, chimeric ligands have showncontradictory results in GalR1 signaling, behaving

as partial agonists in vitro while antagonizing theinhibitory effect of galanin in vivo (Floren et al. 2000).A concentration dependence has been reported on theinhibitory effect of galanin where, in Rin m5F cells(Kask et al. 1995) and hippocampal membranes(Valkna et al. 1995), M35 displays antagonisticproperties at low concentrations (<10 nM) and anagonistic effect at higher concentrations. In contrast tothese results, Heuillet et al. reported the inability ofM35, at concentrations 0.1–1 nM, to antagonize theinhibitory effect of galanin on forskolin stimulatedcAMP inhibition in Bowes melanoma cells (Heuilletet al. 1994). In agreement with the author we show anagonistic effect of M35 on Bowes cells, with a smalladditive effect at the higher concentrations (Fig. 2).Comparable to M35, M617 acted as an agonist in thepresence of galanin also with additive effects, althoughit was not concentration dependent, resulting in fullagonistic activity of the subtype specific ligand onsignaling via GalR1. Signal transduction throughGalR2 displayed a dose dependent agonistic effect forM617 which alone was able to stimulate the IP pro-duction to the same extent as galanin, despite the re-duced receptor binding seen for M617. Further wasthe highly related ligand M35 shown to act as anagonist onGalR2, which is in agreement with previousstudies made on CHO cells (Smith et al. 1997).

In the present report, we show agonistic activityfor both M617 and the related peptide M35 onGalR1 and GalR2 signaling, whereas the subtypespecific ligand M617 failed to increase the GalR1transduction despite enhanced receptor affinity, incomparison to galanin. Regardless of contradictoryobservations between receptor binding affinity and

Fig. 8. Effect of i.t. M617, galanin and combination of M617 plus galanin on reflex facilitation induced by C-fiber CS. Data are mean±SEMand the number of experiments is shown in brackets. **P<0.01 compared to control facilitation with paired t-test.

A Galanin Receptor Subtype 1 Specific Agonist 25

intensity of intracellular G-protein signaling, M617shows a significant and consistent agonistic effecton the studied galanin receptors in vitro. In contrastto this novel chimeric galanin receptor ligand, theavailable chimeric peptides have shown partial ago-nistic properties in vitro and antagonistic effects whenadministrated in vivo, consequently increasing theexpectations on M617 for in vivo studies.

The present electrophysiological experimentssupport the suggestion that M617 acts on GalR1receptors. Thus, it has been shown that galanin and amixed Gal-R1/R2 agonist (AR-M961) but not aselective GalR2 agonist (AR-M1896) causes an out-ward current in LC neurons (Ma et al. 2001). Thiseffect is postsynaptic, since it can be seen after addi-tion of TTX to the medium and/or under conditionswith low Ca2+, high Mg2+ concentrations, which isan effect similar to the one shown for M617.

The involvement of the neuropeptide galanin inappetite and feeding behavior has been well estab-lished (Crawley 1999). Central administration ofgalanin induces feeding in rats, an effect that canbe reversed by co-administration with the chimericantagonists M40 and C7 (Crawley et al. 1993).Furthermore, the chimeric antagonists, includingM35, do not affect food consumption, whenadministered alone (Crawley et al. 1993, Abramovet al. 2004). In this study we report an increasedfood consumption following i.c.v. injection of M617but not after administration of M35. The inducedfeeding observed by M617 was dose-dependent andcomparable to the effect seen by galanin (Crawleyet al. 1993, Kyrkouli et al. 1990). The ability ofM617, but not the related peptide M35, to stimu-late feeding strongly confirms the agonistic effectsseen on galanin receptor signaling and the impor-tance of Gln14 to alter the effect of the chimera.From this study, it is not possible to establish thespecific role of GalR1 and GalR2 in mediatingfeeding behavior, but we verify their involvementand thus activation by the chimeric ligand M617.

Galanin is present in a population of smalldorsal root ganglion cells (Ch’ng et al. 1985, Sko-fitsch and Jacobowitz 1985) and exhibits bothexcitatory and inhibitory effects on nociception,although the inhibitory action appears to be dom-inant at the spinal level (Xu et al. 2000). In a flexorreflex model, galanin produces a biphasic dose-dependent effect, facilitation at low doses andinhibition at high doses (Wiesenfeld-Hallin et al.1989). In addition, galanin antagonizes the CS-in-duced reflex of unmyelinated afferents and by i.t.

applied excitatory neuropeptides such as substanceP and calcitonin gene-related peptide (Xu et al.1990, Xu et al. 1991). In previous studies we haveshown that several chimeric peptides, such as M15and M35, are able to block both the excitatory andinhibitory action of spinally delivered galanin,indicating the nature of these chimeric peptides asnon-selective galanin receptor antagonists. In con-trast, i.t. administrated M617 did not seem to re-duce the spinal effect of galanin, but ratherproduced inhibitory effect on C-fiber CS at highdoses, an effect that was known to be mediated bythe GalR1 receptor. Thus, these results support anagonistic property of M617 on GalR1 receptor inthe spinal cord. In addition, M617 also producedan initial excitatory effect which may be related toresidual activation of the GalR2 receptor (Liu andHokfelt 2002).

In conclusion, we report a novel chimeric gal-anin receptor ligand, M617, with increased speci-ficity for GalR1 and agonistic activity in vitro aswell as in vivo. Distinct internalization of a GalR1-EGFP full probe, agonistic activities on intracellularsignal transduction through GalR1 and GalR2 as wellas hyperpolarization and a postsynaptic outwardcurrent in LC neurons are shown for M617. Theagonistic activities in vitro are further strengthenedin vivo, where M617 induces feeding and exhibit bothexcitatory and inhibitory effects in the flexor reflexmodel in rats. Thus, the effects of M617 are in a waysimilar to galanin and in contrast to other chimericligands acting on the galanin receptors. This studytherefore provides evidence for a novel subtype spe-cific chimeric galanin receptor ligand, which is a usefultool for further characterizing the role of galaninreceptor subtypes in galaninergic signaling andbehavioral models as well as in development of im-proved subtype specific agonists and antagonists act-ing upon the galanin receptors.

ACKNOWLEDGMENTS

We thank Tiit Land for discussions, Erik Diurlin and Mikael

Johansson for undergraduate thesis work and Kathryn A. Jones

and Tiina P. Iismaa, Neurobiology Program, Garvan Institue of

Medical Research, Sydney, Australia for Chinese Hamster Ovary

cells stably transfected with Galanin Receptor 2. This work was

supported by grants from the The Swedish Research Council

(33X-14579–01A and 04X-2887), The Swedish Science Council

(07913, 12168), the U.S. National Institute on Aging (1RO3

AG21295–01 to J.K.R.), and Marianne and Marcus Wallenberg’s

Foundation.

26 Lundstrom et al.

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