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JARO 01: 082088 (2000)DOI: 10.1007/s101620010007

Pharmacological Characterization of the CGRP Receptor in

the Lateral Line Organ of Xenopus laevis

G.P. BAILEY1,2 AND W.F. SEWELL1,3

1Eaton-Peabody Laboratory of Auditory Physiology, Massachusetts Eye and Ear Infirmar y, Boston, MA 02114

2Department of Pathology, Boston University School of Medicine, Boston, MA 02115 USA

3Department of Otolaryngology and the Program in Neuroscience, Harvard Medical School, Boston, MA 02115 USA

Received: 24 February 2000; Accepted: 30 March 2000; Online publication: 13 July 2000

ABSTRACT CGRP family, is the most likely endogenous peptidemediating these effects.

Keywords: cochlea, vestibular, hair cell, efferents,Calcitonin gene-related peptide (CGRP) is a neuro-CGRP, metamorphictransmitter candidate colocalized with acetylcholine

in efferent fibers innervating hair cell organs. We haveused the Xenopus laevislateral line organ to investigatethe responses of a hair cell organ to the CGRP familyof peptides. Two isoforms of CGRP, r-CGRP and r- INTRODUCTIONCGRP, and a human analog of -CGRP, h(Tyr) CGRP, produced dose-dependent increases in afferent

Calcitonin gene-related peptide (CGRP) is an efferentnerve fiber discharge rate with EC50 values of approxi-

neurotransmitter candidate in hair cell organs. This

mately 1 M. Rate increases were 31.2, 18.9, and peptide is immunolocalized in cholinergic efferent10.3%, respectively. The peptide fragment rCGRP837fibers innervating hair cells of the vestibular system

a selective CGRP1 receptor antagonist, competitively(Tanaka et al. 1989a, b; Wackym et al. 1990, 1993;

inhibited the response to r-CGRP. Diacetoamido-Ohno et al. 1993; Scarfone et al. 1996), organ of Corti

methyl cysteine CGRP (r[Cys(ACM)2,7]CGRP), a(Lu et al. 1987; Takeda et al. 1987; Sliwinska-Kowalska

CGRP2 agonist, did not change discharge rate. Ratet al. 1989; Kuriyama et al. 1990; Ohno et al. 1993;

amylin did not increase rate until very high concentra-Safieddine et al. 1997; Reuss et al. 1999), and the

tions, and then the change was less than 7%. Rat adre-lateral line organ (Adams et al. 1987). The strongestnomedullin produced no increase in rate. Responsescase for its role as a neurotransmitter comes fromto r-CGRP developed after metamorphosis. No

work in the lateral line organ, where CGRP suppresseschange in spontaneous discharge rate was observed

responses to mechanical stimulation by around 10 dBuntil postmetamorphic day 6, and then it was only a

while increasing spontaneous discharge rate (Baileyfraction of the maximal response. This response pro-

and Sewell, 2000). A CGRP-like increase in spontane-gressively increased until postmetamorphic day 28, ous rate is also seen with electrical stimulation of effer-

when it reached its maximal value. The most straight-ent fibers even when the cholinergic component of

forward interpretation of our results is that the effectefferent stimulation has been blocked (Sewell andof CGRP is mediated by the CGRP1 receptor and thatStarr 1991). In this report, we examine the pharmacol-CGRP, of the peptides presently known to exist in theogy of the CGRP family of peptides and characterizethe CGRP receptor present at this synapse.

CGRP is a member of a family of structurally relatedCorrespondence to: Dr. William F. Sewell Eaton-Peabody Laboratory peptides that are biologically cross-reactive (Muff et al. Massachusetts Eye and Ear Infirmary 243 Charles Street Boston,

1995; Bell and McDermott 1996; Wimalawansa 1996,MA 02114. Telephone: (617) 573-3156; fax: (617) 720-4408; e-mail:[email protected] 1997; Poyner 1997; van Rossum 1997; Martinez and

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BAILEY AND SEWELL: Pharmacology of CGRP Receptors 83

Cuttitta 1998 for reviews). Other members of this fam- surface up on a piece of moistened filter paper. Theskin was rinsed with an artificial perilymph solutionily include calcitonin, amylin, and adrenomedullin.

Although there is appreciable sequence homology containing sodium chloride (120 mM), potassiumchloride (3.5 mM), calcium chloride (1.5 mM), andbetween CGRP and amylin, CGRP has less homology

to adrenomedullin and calcitonin. However, there is glucose (5.5 mM), buffered with HEPES (20 mM) andadjusted to pH 7.5 with sodium hydroxide (total Na+significant conservation of secondary structure within

this family. All have in common an N-terminally- 130 mM). Perfusion of the inner surface of the skinallowed relatively rapid diffusion (within 20 s) to thelocated disulfide ring, an amphipathic -helix, and an

amidated C-terminus. These regions are important for basolateral surface of the sensory epithelium.The nerve branch innervating the middle-lateralbiological activity, and this level of similarity explains

their biological and potential immunohistochemical row of stitches was dissected from the inner surfaceof the skin. Extracellular recordings were made usingcross-reactivity.

Specific receptors exist for each member of the a silver wire electrode (Sewell and Mroz 1987). Forthese experiments, activity from three adjacent stitchesCGRP family (Gorn et al. 1992; Kapas and Clark 1995;

Kapas et al. 1995; Aiyar et al. 1996; Muff et al. 1999). was monitored simultaneously to reduce the variabilityin discharge rate normally seen in spontaneous affer-Two subtypes of CGRP receptors (CGRP1 and CGRP2)

have been proposed based on pharmacological prop- ent fiber discharge over time. The observed monopha-sic action potentials with positive polarity wereerties (Chiba et al. 1989; Dennis et al. 1989, 1990;

Mimeault et al. 1991; Quirion et al. 1992; Poyner 1992, amplified 1000-fold and monitored on an oscilloscope.The signal-to-noise ratio was optimized by analog filter-1995; Bell and McDermott 1996; Wisskirchen et al.

1998). The CGRP1 receptor subtype is preferentially ing. A Schmitt trigger device was used to determinethe occurrence of action potentials, which wereactivated by-CGRP. The peptide fragment-CGRP8

37, which lacks the first 7 N-terminal amino acids, is a counted with a microprocessor. Throughout the exper-iments, the inner synaptic surface was perfused contin-competitive antagonist for the CGRP1 receptor, but

has no action on the CGRP2, calcitonin, amylin, or uously at a flow rate of 1 L/s. Peptides were delivereddirectly to the inner synaptic surface. Only those prep-adrenomedullin receptors. While there are no specific

antagonists for the CGRP2 receptor subtype, the linear arations maintaining a spontaneous discharge rategreater than 100 spikes/min were used. Only thoseanalog diacetoamidomethyl cysteine CGRP

(Cys[ACM]2,7CGRP), which lacks the N-terminal preparations that recovered completely after drugadministration were considered in the analysis.region disulfide linkage, has little or no agonist activity

at CGRP1 receptors, but is a full agonist with less To obtain the doseresponse relationships for eachpeptide, peptides were applied while monitoring spon-potency relative to -CGRP in activating CGRP2

receptors. taneous discharge rates in the afferent fibers. Peptides

were divided into three groups for testing. Group 1These findings raise two questions about the roleof CGRP as an efferent neurotransmitter. First, is the consisted of the CGRP family peptides r-CGRP, r-CGRP, and rAmylin. Group 2 consisted of rADM andpeptide present in efferent nerve terminals CGRP, or

someothermember oftheCGRP familywith biological the synthetic analogs r[Tyr]CGRP and r[Cy-s(ACM)2,7]CGRP. Group 3 consisted of the peptideand immunological cross-reactivity; and second, what

receptor mediates CGRPs action in hair cell organs? fragments r-CGRP837, rADM1150, and rAmylin837.The peptides were applied in random order withinTo approach these questions we have examined the

effects of these peptides, their agonists, and antago- each group. Each preparation was exposed to only onepeptide concentration. The preparation was allowednists on the spontaneous discharge rate in the Xenopus

lateral line organ. to reverse fully from each peptide application beforesubsequent applications were made. Each of theGroup 1 peptides were tested separately at high con-centrations (106 M). Sigmoidal curves were fit toMATERIALS AND METHODS

the data by a least-squares method. EC50 values weredetermined from the sigmoidal curve fits. Data wereIn experiments that examined the response to mem-

bers of the CGRP family, Xenopus laevisapproximately obtained from 60 preparations.In separate experiments, the -CGRP antagonist,2 cm from nose to vent were obtained from Nasco

(Fort Atkinson, WI) between postmetamorphic days r-CGRP837 (1 M), was perfused for15 minutes priorto and during the application of r-CGRP. Each prepa-3060 and housed at room temperature in deionized

water containing 1 mM added calcium chloride. Each ration was exposed to only one concentration of r-CGRP. The doseresponse for r-CGRP was deter-frog was anesthetized by chilling to near 0C, then

decapitated. A piece of skin containing the middle- mined (0.13 M) in the presence of r-CGRP837.Sigmoidal curves were fit to the data by a least-squareslateral row of stitches was removed and placed inner


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84 BAILEY AND SEWELL: Pharmacology of CGRP Receptors

method. EC50 values were determined from the sig-moidal curve fits. Data were obtained from 13 pre-parations.

In experiments that studied the postmetamorphic(PM) response to CGRP, Xenopus laevis tadpoles wereobtained from Nasco at stage 65 (54 days), andhoused at room temperature in deionized water asabove. Frogs were allowed to grow until adult stage 66

(58 days), as determined by morphological stagingcriteria (Nieuwkoop and Faber 1956). Frogs were des-ignated PM day 0 on the day they were categorized asstage 66. Postmetamorphic responsiveness to r-CGRP(3 M) was determined by monitoring spontaneousdischarge rates during peptide application as above. FIG. 1. CGRP increased spontaneous discharge rate, an effect last-Response was recorded approximately every other day ing long after CGRP was washed out. Thepreparation was continually

perfusedwith a balancedsalt solution.CGRP wasadministered duringfor 33 days, then one time at day 72. One or two frogsthe time indicated by the solid bar.were used each day that the response to r-CGRP was

tested. Sigmoidal curves were fit to the data by a least-squares method. Data were obtained from 18preparations.

The peptide response of the preparation was evalu-ated by on-line computer analysis for all experiments.The average number of spikes that occurred between60 and 220 s after peptide injection determined maxi-mal response. Percent change in rate was calculatedby comparing the maximal response with the averagebaseline spontaneous rate of the preparation taken160 s prior to peptide injection. Sigmoidal curves weregraphically fit to the data using software by Kaleida-Graph, Synergy Software, Reading, PA.

All peptides, peptide fragments, and synthetic ana-logs were purchased from Peninsula Labs (Belmont,CA).

RESULTS

CGRP is more effective than calcitonin,adrenomedullin, or amylin

As previously described (Adams et al. 1987; Sewell andStarr 1991), one effect of r-CGRP on this preparation

FIG. 2. CGRP was more effective than amylin or adrenomedullinwas to increase spontaneous discharge rate. We used at producing a change in afferent nerve fiber discharge rate. Sigmoidalthis rate change as a measure of the functional curves were fit to the data for all peptides except adrenomedullin

(open diamonds) by a least-squares algorithm. Each peptide wasresponse to application of these peptides. Thistested at least three times at each concentration.increase was observed within tens of seconds of appli-

cation, and the rate could stay elevated for tens of

minutes after washing the peptide from the synapse.An example is shown in Figure 1.

Adrenomedullin (ADM) and amylin were relativelyWe found CGRP to be the most potent peptideineffective. Rat amylin, with an EC50 near 3 M, wasexamined. All CGRP isoforms and a synthetic analogless potent than r-CGRP and produced a maximalproduced dosedependent increases in spontaneousincrease in rate of only 7.1 1.2%. Rat ADM essentiallydischarge rate, with EC50 values of approximately 1had no biological effect at concentrations up to 10M (Fig. 2). The r-CGRP isoform was more effectiveM (Fig. 3). We have previously shown that salmonthan r-CGRP, and r(Tyr) CGRP was the least effec-calcitonin does not increase spontaneous dischargetive, producing increases (mean s.e.) of 31.2 4.9%,

18.9 5.3%, and 10.3 2.1% respectively. rate at this synapse (Adams et al. 1987).


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response produced by r-CGRP. The response to r-CGRP was inhibited competitively by pretreatment

with 1 M r-CGRP837. The inhibition shifted thedoseresponse curve to the right, changing the EC50from 1 to 4 M (Fig. 3).

The -CGRP isoform is thought to mediate itseffects through the CGRP2 receptor (Jansen 1992;Tomlinson and Poyner 1996; Yoshimoto et al. 1998).

Our results showed that r-CGRP was similar inpotency but 40% less effective than r-CGRP at increas-ing spontaneous rate. While no specific antagonist forthe CGRP2 receptor exists, we did examine the effectsof r[Cys(ACM)2,7]CGRP, a specific peptide agonistfor the CGRP2 receptor. This compound was inactive,producing no effect on the spontaneous discharge rateat doses up to 10 M (Fig. 3), indicating that theresponse to r-CGRP was not mediated by the CGRP2receptor but was more likely a result of cross-reactivity

with the CGRP1 receptor.

Responses to CGRP develop post-metamorphically

Early in our investigation we recognized that occasion-ally when new frogs were received they were not imme-diately responsive to r-CGRP. The response to r-CGRP gradually increased over the next few days to

weeks. To eliminate variability in the age of the frogsand determine the age most responsive to r-CGRP,

we raised tadpoles to determine the exact day theybecame adult stage 66 (PM day 0). Responses to r-CGRP (3 M) were recorded from PM day 0 throughPM day 72. Increases in spontaneous rate were not

apparent until PM day 6, and then at only a fraction ofthe maximal response. Activity continued to increaseuntil PM day 28, at which time it had plateaued withmaximal responses of a 28.4% increase in dischargerate (Fig. 4).

DISCUSSIONFIG. 3. (Upper panel) Responses to CGRP are shifted to the right bypreincubation of the preparation with the CGRP1 receptor antagonist,

The most straightforward interpretation of our resultsCGRP(837). (Lower panel) r[Cys (ACM)2,7]CGRP, a selective agonistfor the CGRP2 receptor, had no effect on discharge rate, indicating is that the effect of CGRP is mediated by the CGRP 1that the response seen with 3 M r-CGRP was likely due to cross- receptor, and that CGRP, of the peptides presentlyreactivity with the CGRP1 receptor. known to exist in the CGRP family, is the most likely

endogenous peptide mediating these effects. Calcito-nin, amylin, and adrenomedullin were ineffective at

Responses to CGRP are likely produced by theincreasing afferent discharge, suggesting that exoge-

CGRP1 receptor nously applied CGRP is not acting on calcitonin, adre-nomedullin, or amylin receptors. If receptors for otherThe truncatedpeptide fragment r-CGRP837 is a selec-

tive blocker for the CGRP1 receptor. We first studied members of the CGRP family do exist, their effectson the hair cell organ are likely different than thosewhether r-CGRP837 had any intrinsic biological activ-

ity and found that this antagonist had none at concen- for CGRP.In general, the effects on discharge rate for peptidestrations up to 30 M (data not shown). We then

studied the effect of r-CGRP837 on the excitatory within the CGRP family was closely related to their


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While amylin had a small effect on spontaneous rateat high concentrations, this action is likely mediatedthrough the CGRP receptor. Because in other systemssalmon calcitonin and amylin are equally effective onamylin receptors (Muff et al. 1999; Christopoulos etal. 1999), the biological inactivity of salmon calcitoninin our preparation suggests that an amylin receptor isnot mediating this increase in discharge rate.

The functional profile of CGRP activity in the lateralline organ indicates that responsiveness to the peptidedevelops during the first postmetamorphic month. Wedo not know specifically what developmental changesare responsible for this observation. Possibilitiesinclude activation of the CGRP receptor or an induc-

FIG. 4. The response to r-CGRP increased postmetamorphically.tion of the receptor components (Luebke et al. 1996;

The change in afferent discharge produced by 3 M r-CGRP wasMcLatchie et al. 1998) necessary for a functional recep-notapparentuntilpostmetamorphicday 6, andthen at only a fractiontor at this synapse. In the lateral line organ, efferentof the maximal response. This response progressively increased until

postmetamorphic day 28, when it began to plateau near its maximal activity is functional during the premetamorphic larvalvalue (28.4% change in rate). stages of development, (Shelton 1971), but it is during

metamorphic transformation that myelinated efferentfibers send branches to each neuromast (Shelton1970) and establish synaptic contact with receptor cellshomology to CGRP. The amino acid sequences of r-

CGRP, r-CGRP, and r(Tyr)CGRP differ by only (Fritzsch 1989; Hellmann and Fritzsch 1996). Post-metamorphically, myelinated efferent fiber diameters3%, and are approximately 82% homologous with the

CGRP sequence of the European green frog Rana ridi- increase from 0.31.2 to 24 m (Mohr and Gorner1996). It is possible that this temporal sequence hasbunda (Conlon et al. 1993). Rat amylin, which had

only slight activity, exhibits a 51% homology with frog some significance for CGRP receptor maturation.There are likely many biochemical and molecularCGRP. Rat adrenomedullin and salmon calcitonin,

which were inactive, have a 22% and 27% homology, events associated with these anatomical changes thatmay lead to an induction or activation of the CGRPrespectively, with frog CGRP.

The EC50 of 1 M for rat CGRP in the Xenopus receptor.In summary, the experiments presented here pro-lateral line preparation is 1030-fold higher than that

found in Xenopus oocytes (Luebke et al. 1996) and in vide further support for the hypothesis that CGRP is an

efferent peptide transmitter, characterize the receptortissue slices of frog adrenal gland (Esneu et al. 1994).This difference is likely attributable to the fact that, meditating that effect as the CGRP1 receptor, and pres-ent evidence for the developmental regulation ofin the lateral line preparation, CGRP must diffuse

through 2030 m of connective tissue to reach the this receptor.synapse, where it may encounter peptidases, uptake,and nonspecific binding. The EC50 for rat CGRP inour preparation is also 1001000-fold higher than in ACKNOWLEDGMENTSmammalian preparations (Ohhashi and Jacobowitz1985; Maton et al. 1990; Cox 1995; Sheykhzade and We wish to thank our colleague C. Brown for discussion andNyborg 1998; Wisskirchen et al. 1998, 1999), and may criticism. This work was supported by a grant from thereflect differences between amphibian and mamma- NIDCD.lian CGRP receptors.

The strongest evidence for our conclusion that

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