Differential expression of α-CGRP and β-CGRP by primary sensory neurons and enteric autonomic neurons of the rat

DIFFERENTIAL /KGRP

BY PRIMARY

ENTERIC P. K. Q.

M.

R.

and

IIcpartmcnts

A.

S. G. AMAKA.: S. R.

SPOICES.*

J. M.

AND

NEURONS

NEURONS

GHATEI.*

A.

S. KANSI-,*

OF cc-CGRP

SENSORY

AUTONOMIC

MLJI IW:RRY,*

HAMID.+

A.

EXPRESSION

AND

OF THE

P. M.

JOSI~S,*

BYRRIK,*

A.

RAT M.

S. LIGOV.$

PII RSOh.*

J. M. Par Alit

BLOOM*

of *Medicme.

tHistochem,strg and ~Chcmical Pathology. Royal Poctgradu.ttc Mcd~c:~l School, Flammersmith ljospital. Du C’ane Road. London W 12 OHS. I1.K. ol’ Molecular Nourohiology, Yale Unlvcrsity School of Mcdlcinc. Cedar Street. N\;cM lla~cn. C‘T 06510, CJ.S.A.

:Sectlon

Abstracts -C~prcss~on of the calcitonin gene-related peptide. r-calcitonln gene-related peptide (C‘GRP). and the homologous /I-CGRP were compared ,n sensor! and cntcrlc nerves of the rat. Ann1>vh of (‘GRP-like immunor~activit4 by cation exchange chromatography and radiotmmunoassaq hhoued that in the dorwl root $snpha, dorsal spinal cord and in those peripheral tisSt,es Lchere CGRP-like tnmunorcactiwty is primarily localircd to sensory libra. r-CGRP concentra,,ons wcrc three to 51, tl,nc\ grcatcr than /j-CARP concentrations. In the intcst,nc. however. /I-CARP conct‘ntrxtions ~L’I-c’up to w\cn times greatt‘r than r-C‘GRP concentrations. OnI) /I-CGRP HX detcc,cd ,n the Intcstincs t>f capuiclntreated rats. Northcm blot and in .vi/rr hyhridimtlon to r-CARPand /I-CARP-spwilic probes ahwbced that \hhile both x-CGRP and [I-CGRP mcsscngcr rihonucleic acids occurred in the dorsal Iroot ganglia. onl) /bCGRP messcngc~- rlbonuclcic ;tc~d occurred in the intestine. v,here It \\;I\ localized to t’nttxic IICUI-ens. Rcccptor binding sites on mt’mbranes of rat heart and colon had approumatcl> cqwl atlimtw 1;,1- r-CCiRP and /i-CGRP. The two pcptidcs wrc cquipotent in increasing the rate and force of atrul contraction\ hul r-CARP W:I\ slightly (2.6 time<) more potent than /I-<‘GRP 111rclawng colonic \moc)th musck!

Antlscra

raised

ncuropcplidc scripta

01‘

the

the

rcacti\ity

I I1

the

brain.

scnsorq

arc

tisbuc5.‘i

In

vasculaturc.

capzalcin. l,.lturc,i” C’GRI’-1~1

widely

man>

tract

and

in

indicating ,:I,‘, “‘y’44i4~ also

these

that

they

unatfcctcd

r-CGRP.’ (/j-CGRP)

the

containing

The

shin.

arc absent

after

OI with

sensory

in

houc~er.

intrinsic capsaicin

cntcric trcat-

and

this

residue

sensory

that

raised

against

x-CARP

not

/I-CARP

iwlatcd

in

the

be established

ha\c

expressed

and

rcsiduc

l’ot- rat

structural

raiw\

ho-

the

dctcctcd

ncccssal-iI)

h! the

antlwra 01‘

/I-C‘(;Rf’ and

E:v,Ing hunx,n

of

cord“

poshi-

product

human

spinal

at

acid I-esiduc\.

been she\+ n to euprchs r-C‘C;RP.“’

expression

entails

pcptide

close

.4lthough

absence

Lvhcthcr

necessarily

/i-C‘GRP

is

pcptidc

to occ,~r ,n the

/I-C‘GRP

fl-om

cc‘ll lines

shwn

:I to

(mRN.4)

acid

C’GRPl.1

-C‘GRP

gene expression.

been

Sarcoma

all Y

amino

The

of

by the st,hst,-

;I $utamatc

been

and

encoding

honiolog!

onI>

of37

ganglia.

that

prccuI-sor

homologous

for

ribonucleic

ol’ r-CARP

the

r-CGRP

has prcviousl~

bility

arc

rat

from

;I lysine

from

structur:il

35 in the sequence

mology

gene

tbc

messenger

brain

has

In

gene

encodes

close

dilTcrs

/KGRP

tract.

nconatally

‘-

of

position

intestine. the

tution

peripheral

a separate

r-CGRP

bearing

immuno-

including

arc

and

peptidc

;iutononiic

rat

fibrcs

treated

to

In rat and man calcitonin

pathHays

respirator>

rats

In

used

tibres in

tissues

OCCUI-s nithin

nci-vc‘b \\hich remain n~cl,,,. o- 111 Ii i’i1’

Nerve

tran-

inotoncurons.

enteric

stomach.

pcnitalia,

dcplctcd

spinal

in

periphei-al

been

neuronal

distributed

oesophagus.

residue

CGRP-like

and

and

- IOI i Ia2II.‘I22:1 151’)4’

ncL,r(l,,~,c

have

of

in cranial

acid

b> altcrnati\e

in specific

neurons

CGRI’-I-1

urinary

37-amino

gcnc.

presence

(CGRP-LI)

markedly

the

cncodcd

calcitonin

demonstrate

within

against

x-CGRP.

production Mhcther

indcpcndcntl~

I, rcni;I,,,x

to

01 the rat /I-C‘(;RP of

the t\\o

the

predicted

(‘GRf’

in ditfcrcnt

gcnch

p~~pularion\

0 I. ne II ro n 5. Pharmacolo~ic;il and

/i-CARP

and

the

pcptides

exhibit

propertx\

but

wnip;rrisc~nz

01‘ human

rat

ha\e

iii-c

x-(‘GRP qualitati\el~ not

nccwwrilv

r-C‘(

indlcatcd

Gmil;ii-

iR 1’ that

bioi~>$c;il

cqulpotcnt.

‘-

196

P. K.

~CiLUEKKV

Whether independent a-CGRP and p-CGRP receptor systems exist or whether both peptides act through a single receptor type is not known. We have studied the expression of r-CGRP and ,&-CGRP in sensory and enteric nerves of the rat

using radioimmunoassay and high performance cation exchange chromatography to analyse CGRP-LI in tissues of normal and capsaicin-treated animals. Northern blot and in situ hybridization to r-CGRPand P-CGRP-specific probes were used to determine sites of synthesis of the peptides in the intestines and sensory ganglia. To see whether the peptides can act independently, we have compared their properties in receptor binding assays and isolated rat organ bioassays. EXPRRI~ENTAL

PROCEDURES

Pepiides

Synthetic rat a-CGRP was purchased from Bachem U.K. Rat /I-CGRP was synthesized according to the predicted structure’ by Dr F. Bellini, Institut Armand-Frappier, Quebec, Canada. Anim&

Tissues were taken from Wistar rats of either sex weighing 150-300 g. Animals undergoing capsaicin treatment received doses of capsaicin (50mg/kg weight, Fluka AG, Switzerland) on days 2 and 3 of postnatal life as previously described.43 Littermate controls received injections of vehicle solution only. Capsaicin-treated animals and controls were sacrificed at the age of 12 weeks.

et

ui.

Fractions were collected for intervais of I mm and aliquots of 200 ~1 assayed with each radioimmunoassay. Northern blor hybridization

Deoxyribonucleic acid (cDNA) probes complementary to 3’-non-coding sequences of a-CGRP and [f-CGRP mRNAs excised from SP6-4 expression vectors’ were iabelled with ‘lP by random hexanucleotide priming.8 Total cellular RNA was extracted from rat dorsal root ganglia and from rat intestinal longitudinal muscle layer stripped away to include the myenteric plexus3* by the guanidinium isothiocyanatei caesium chloride ultracentrifugation method.?’ Twenty micrograms total RNA were size-separated by electrophoresis on a 3(N-Mo~holino)propanesuiphoni~ acidagarose formaldehyde gei2’ and transferred by Northern blotting to a Hybond-N membrane (Amersham International). Hybridization was performed by incubation with labelled probes overnight at 42°C in 50mM phosphate buffer pH 6.8, containing 50% (v/v) formamide. 0.75 M sodium chloride, 75mM sodium citrate and IOOpgjml sonicated, denatured herring sperm DNA with Denhardt’s solution composing 25g/l bovine serum albumin. (BSA, Pentax fraction V, Sigma), 2.5 g/l Ficoli 400 (Pharmacia), 2.5 g/l poiyvinylpyrrolidone, (PVP) (Sigma) to reduce nonspecific binding. The membrane was then washed in 15 mM sodium chloride, 1.5 mM sodium citrate and 0. I g/l sodium dodecylsulphate (SDS) for 30min at 60°C as described elsewhere.26 Hybridization bands were visualized by exposing pre-flashed XAR-5 film (Kodak) to the filter for 14 days at -70°C with a fast tungstate intensifying screen (Ilford).

in situ hybridization Rat colonic tissue was fixed in 4Og/l paraformaldehyde for 4 h and then rised in 0.1 M uhosohate-buffered 0.15 M saline pH 7.4. Sections IOgm ihick’ were cut at -2OY.Z Complementary RNA (cRNA) probes for 3’non-coding Rudioimmunoassays seauences of a-CGRP and B-CGRP mRNA were labeiled For radioimmunoassay and cation exchange chrowiih “S and ‘2P using SP6 polymerase and labelled cytosine matography, CGRP-LI was extracted from fresh rat tissues by boiling in 0.5 M acetic acid as previously described.2829 triphosphate on linearized SP6-4 vectors.’ Control probes were prepared by labelling non-complementary (sense) Two CGRP radioimmunoassays were used. One (a-CGRP assay) was the same as described previously” and used an RNA from a-CGRP and b-CGRP coding sequences. Preparation of the cryostat sections and hybridization were antiserum (code CG7) raised in a rabbit immunized with carried out essentially as described by Hamid et a1.r6Briefly. synthetic rat a-CGRP’conjugated to bovine serum albumin sections were permeabilized with Triton X-100 and pro(BSA) by a glutaraldehyde reaction. This antiserum crossreacts with /I-CGRP by only 2% on relative molar basis.30 teinase K, and prehybridization carried out with 50% (v/v) formamide in 0.3 M sodium chloride, 30 mM sodium citrate The second assay (total-CGRP) used an antiserum (code for 30min at 37°C. Hybridization was carried out by CG3) raised in a rabbit immunized with synthetic rat diluting the relevant probe (2.-3 ng per section) in 250 mM G(-CGRP conjugated to RSA by a carbodiimide reaction. Tris-HCl buffer pH 7.5, containing 50% (v/v) formamide, This antiserum cross-reacts fullv with rat B-CGRP.) Anart 100 g/l dextran suiphate, 0.3 M sodium chloride, 30 mM from the antisera, ail reagents-and conditions used in* the two assays were identical to those previously deseribed.28,29 sodium citrate, 2.5 g/l BSA, 2.5 g/l Ficoil 400, 2.5 g/l PVP, 5 g/l sodium pyrophosphate, 5 g/l SDS and 250 pg/ml denaThe sensitivities of the radioimmunoassays were approximately 1 fmol and 2 fmol a-CGRP per assay tube re- tured salmon sperm DNA overnight at 42°C followed by washing in standard saline citrate at 45°C. finishing with spectively. 75 mM sodium chloride, 7.5 mM sodium citrate at 45’C for Cation exchange chromatography 30min. The slides were dipped in Ilford K5 emulsion, exposed for 3 days at 4°C and developed with Dl9 developer Acetic acid tissue extracts were de-salted for cation exchange chromatography on Sep-Pak reverse-phase car(Kodak). Specificity of hyb~~zation was checked by comof complementary probes to tridges (Waters Associates) from which CGRP-LI was parison with hybri~~tion eluted with a 60/40 (v/v) acetonitrile/water mixture containsections pre-treated with ribonuciease and with hybridization of non-complementary probes to normal sections. ing 0.1% by volume trifluoroacetic acid. The eluate was diluted with 4 volumes 50 mM 2(N-MorpholinokethaneReceptor binding assay sulphonic acid/sodium hydroxide buffer pH 6.0, containing 0.05% by volume Tween 80 (buffer A) before injection onto Rat tissue membranes for receptor binding experiments a Pharmacia Fast Protein Liquid Chromatography (FPLC) were prepared by homogenizing rat tissues in 50mM system equipped with Pharmacia Mono-S cation exchange Tris-HCI buffer pH 7.4, containing 0.32 M sucrose at 4°C column eluted at I ml/min with buffer A. Two minutes flOm1 per gram of tissue) using an Ultra-Turrax homofollowing injection of the sample a linear gradient of 50 min genizer. The homogenates were centrifuged at 1OOOgfor duration between elution with buffer A and elution with 2Omin and the supernatant decanted and retained. The buffer B (composition as for buffer A but containing also pellet was re-homonogenized and centrifuged according 0.5 M sodium chloride) was developed, after which the to the same procedure. The combined supernatants were column was eluted for a further IOmin with buffer B. then centrifuged at 50,OOOg for 20min and the pellets

r-CGRP

and /I-CGRP

in sensory and cntcric ncrvc\

resuspended m 50 mM Tris -HCI buffer pH 7.4. containing 0.2 M sodium chloride. This procedure was repeated twice and the protein content of the resuspended membranes assayed by a Coomassie blue dye method (Picrcc U.K. reagents). Membrane preparations were stored frozen at -20 C until use. Synthetic rat x-CGRP and p-CGRP were labelled with “‘1 using the chloramine-T method as for radioimlnunoassay.~~ “’ Specllic activity of the tracers. estimated by self displacement in the receptor assay. ranged from 20 50 Bq’fmol.

CGRP-LI

(pmol/cj)

60 IA

For receptor blnding experiments. membranes (50 200 /tg protein) were Incubated at 20 <‘ in 50 mM Tris-HCI huller pH 7.4 containing 0.2 M sodium chloride. 3 g, I BSA and 40 mg.l bacltracin with rad~olabellcd r-CGRP or /I-CGRP and \arnous concentrations of unlabelled z-CGRP or /j-CGRP in a total volume of 0.5 ml. After 60 min incubation uith gentle agitation. I ml Tris HCI buffer contain~ng 0.2 M sodium chloride at 4 C was added and bound and free ligand separated hq centrifugation at l2,OOOg for 3 min. 7 he pellets were washed with I .O ml Tris HCI. 0.2 M sodium chloride bulrer, recentrifuged. the supcrnatant rcmovcd and radioactivity in the pellets counted.

*

Isolated rat atria were suspended in 1Oml organ baths contaimng Ringer Locke solution at 30 C. aerated wth IOO”~, oxygen. Isotonic recordings were made under 0.5 g tensIon on a Washington 400 MD4C four channel chart recorder Segments of mid-colon (5 cm) were placed in IO ml organ baths containing Tyrode solution at 37 C aerated with 95’!0 0 z 5%) CO, Isotonic recordings were made under 0 5 g tensIon a\ for atria. In all experiments sqnthetlc peptides aere dissolved in the relevant solution and added to the organ bath in volumes up to 0.3 ml at 8 IO min dose

interv:tl\. RESllLTS

To determine the effects of capsaicin treatment on CGRP-LI in the rat alimentary tract. extracts 01 alimentary tract tissues from rats treated neonatally with capsaicin and littermate controls were assayed with the r-CGRP and total CGRP assays (Fig. 1). In the control animals, concentrations determined by the r-CGRP assay wcrc comparable to those reported previously.” Concentrations detected by the total CGRP assay in several regions of the small and large intestines and also in the pancreas were significantly higher than those detected by the r-CGRP assay. In capsaicin-treated animals, little or no CGRP-LI was detected by the r-CGRP assay in any region of the alimentary tract while substantial concentrations could still be detected by the total CGRP assay in all regions apart from the oesophagus and stomach. The reduction in total CGRP-LI seen in capzaicin-treated animals compared to controls. was similar in magnitude to the concentration ol C‘GRP-LI detected by the r-CGRP assay in control animals.

.I (pmol g wet wt) In the Fig. I. Concentrations of C alimentary tract and pancreas of normal control rats (stlppled bars) and rats treated neonatallq with capsaicln (open bars) determined by radioimmunoas~ay of tlssuc extract\ with the z-CGRP assay (panel A) and the total CGRP assa\ (panel B). Values are mean 3 S.E.M for fibe rats in each group. Asterisks (*) on capsaicin group bars Indicate mcitn\

significantly different from mean of correspondlnp control group (P < 0 05. Student’s r-test for unpaired data). A\terisks on total CGRP control group bars Indicate meanr significantly dllrerent from mean of corresponding r-C‘GRP control group (P < 0.05. Student‘s I-test for palred Jat;cI

as determined hy radio86 i 8%. respectively (mean + S.E.M.. four runs each). immunoassay Under the conditions employed. 90% of the rccovered r-CGRP immunoreactivity was elutcd in the period l9-24min. peak at 21 min. and 90”#,, of the recovered /I-CGRP immunoreactivity was elutcd in the period 17-33 min, peak at 79 min. When rat tissue extracts were run on the cation exchange column. peaks corresponding to r-CGRP and /I-CGRP could be identified both from their positions on the profile and from their immunoreactikc characteristics in the assays. Thus, assay of column fractions ulth the total CGRP assay produced profiles with peak\ corresponding to X-CGRP and /j-CGRP while the [I-CGRP peak was absent from profiles produced bb the r-CGRP assay (Fig. 2). The r-CGRP peak consistently

Recoveries from

the

of synthetic r-CGRP and /I-CGRP cation exchange column were 95 + h”/b and

appeared

larger

on

profiles

produced

by the total CGRP assay than on those produced the r-CGRP assay and for intestinal extract\ capsaicin-trcdted

rats

(Fig.

3E.

F).

the

total

hq ol

<‘<;RP

19X

I?

750 -A 500

K.

ML:LDI:KKY

CI

rd.

7 ‘i

-t

250

-

.J-l”+-J-

250

n

A-L

20 Retention

Time

90

60

(minutes)

Fig. 2. Representative cation exchange-radioimmunoassay profiles of CGRP-LI in extracts of normal rat dorsal root ganglia (A, B); normal rat colon (C, D) and colon from capsaicin-treated rats (E, F). Each pair of profiles is from the same set of column fractions assayed either with the total CGRP assay (A, C, E) or with the a-CGRP assay (B, D, F). Arrows indicate the position on the profiles corresponding to the peaks produced by synthetic rat a-CGRP and synthetic rat p-CGRP.

cation exchange profile showed a peak in the position corresponding to a-CGRP where none was present on the profile produced by the a-CGRP assay. The cc-CGRP assay detected some material at the beginning of the cation exchange run which did not react in the total CGRP assay. To confirm the identities of the immunoreactive peaks on the cation exchange profiles, fractions were individually re-chromatographed on Sephadex G-50 superfine gel permeation columns eluted as described previously.28,29 Material detected by the total CGRP assay in the position of p-CGRP on cation exchange profiles (Fig. 2A, C, E) remained as a single component coeluted with synthetic /I-CGRP on the gel permeation column. Likewise, material detected by the a-CGRP assay in the position corresponding to sc-CGRP (Fig. 2B, D) remained as a single component coeluted with synthetic u-CGRP. Material appearing in the position of a-CGRP but reacting only in the total CGRP assay (Fig. 2E) was eluted from the gel permeation column significantly later

than the a-CGRP and P-CGRP standards indicating that it was of smaller molecular size than authentic a-CGRP. Although gel permeation chromatography did not resolve a-CGRP-like material detected by the total CGRP assay on cation exchange profiles of extracts from normal rats (Fig. 2A, C) into two components, we presume that the presence of this small molecular form accounts for the larger size of the a-CGRP peak seen on cation exchange profiles when assayed with the total CGRP assay than with the a-CGRP assay and that resolution on the gel permeation column was not adequate to separate this component from authentic a-CGRP. Therefore, for the purpose of estimating relative concentrations of a-CGRP and fl-CGRP in tissue extracts (Table 1, column 3), authentic a-CGRP was defined as immunoreactivity detected by the a-CGRP assay and eluted from the cation exchange column in the period 19-24 min while authentic j?-CGRP was defined as immunoreactivity detected by the total CGRP assay and eluted from the cation exchange column in the

I.

Table

Analysis

of CARP-like

immunoreactivlty

I

111rat ~ISSW\

z

x-C<;RP-LI

Total

3 Ratlo

CGRP-LI

pmol g

pm01 g

540.5 * 70.4 520.4 + hS.5

14x. I i 90.0 574.7 * 30.0

0 29 0.25

10.1 6.3 5Y.X 2Y.7 24.7 6.0 9.6 IX.7 I .z 0.6 lY.7 X0X.5

12.6 & 2 0 75*0x 64.71 II 5 31.5 Il.7 2x.‘) * 7 x 14.4* I.$ s4.3 i 5.4 54.4 & 4. I 3x.4 z s 0 42.3 i 4 3 21.X * I.7 69X.3 _t 14x.4

0.31 0.27 0. I7 0.27 0 2x 0 52 7 I3 3.70 i x0 > 30 0. I5 0 3x

& * & i * * 2 * * & i _t

1.2 0.5 9.4 3.3 3 7 0.7 1.5 1.6 0.x 0 4 1.4 160.5

/I-C‘GRP

A‘GRP

<‘olumn\ I and 2 show ahsolutc concentrations of C
detected by r -C‘GRP and total CCiRP radioimmunoassays m rat tissues suhsequcntly \uhjected to cation exchange analysis. Valuer are m pmol;g wet ht. mean i S.E.M. for IO rata each. C‘olumn 3 shows the ratios of /LCGRP to r-CC;RP. calculated from rclatlvc cluantities of authentic /KGRP and z-CCRP recovered from the callon ertchanpc \ystcm for each tissue. Values arc means of two indepcndcnt experiments uvng pooled extracts from five animals in each cast’. All result\ arc for tissue\ taken from normal rats except capGcin ileum and c~,psaun colon uhlch are for tlrsucs taken Irom rats treated with capsaun as neonarc$. Rcco\ery of C‘GRP-LI from all cation exchange runs ranged from 71 to 104%,.

period

27 32 min. Gel permeation

ofmatcrial

detected by the x-CGRP

~olumc

the cation

of

exchange

chromatography

rats are shown in Fig. 2. CGRP-LI

assay in the void

sensory neurons of the dorsal root gangha consisted

profile

this comprised the two carhoxy-terminal previously

FC‘GRP

Cation used

to

Y-CGRP

exchange

radioimmunoassay

compare

the

and /KGRP

sensor)

with

tlssucs

relative

normal

capsaicln-treated

analysis was

concentrations

ih localized

the

and /I-CGRP of

colon. distinct peaks corresponding

ol

in the sensory ganglia and in

CGRP-LI

libres

primarily

fragments of

described.“’

tissues where r-CGRP

showed that

relative

primarily

to

concentrations

of

in intestinal

rats

and

and pancreatic

intestinal

rats. Normal

tissues

of

rat brain and thyroid

concentrations

of

CGRP-LI

in

the

(Fig.

IA,

dnd /I-CGRP

were apparent

tissues from

capsaicin-treated

the

predominant

and

little

(Fig.

2E. F).

or

The

summarl/ed

of

all

in Table

1. column

the

the r-CGRP

tissue hut,

were

for tissues other

than pancreas. ileum and 1. columns

I ant1 2). For cation exchange analysis. a sufficient quantity of

each

type

of

tissue extract

loading so that approximately I.1 wus loaded detection

prepared

for

on each run. The effective threshold of

for assay of cation

approximately rcsentative

was

1-~3 pmol total CGRPexchange fractions

10 fmol CGRP-LI

cation

exchange

per fraction.

profiles

of

was Rcp-

from normal rat dorsal root ganglia, from normal colon and from

colonic

root ganglia. bladder

inant

and

similar. was

rat

tissues of capsaicin-treated

was

P-CGRP quantities

z-CARP

of /I-CGRP

form.

still

to

thyroid

mRNA

could gland.

ileal

but

where

lung?

prcdon-

z-C’GRP

were

/I-(.‘(;RP

and

colonic

rats.

authcnttc

no

jlgnificant

be detected.

has prcvlously

was present.

than _*-CGRP.

In

present.

heart. uas

howeccr.

capsaicin-treated

of z-CGRP

and

and /I-(‘GRP

dorsal spinal cord.

predominant from

from

h> calculating

rat tissues cxa~mincd. In

stomach.

the ratios

the

/I-CGRP

and

;I\

to authentic z-(‘GRP.

In the ileum and colon,

tissues

brain

The rc\ults

of both r-C‘<;RP

in all normal

skin,

/J-CGRP

CARP-LI

quantities

present

dorsal

calculated characterization

immunoreactivity”)

tratlony

colon. the difference was not great (Table

and /i-(‘GRP

were

runs were then normalilcd

Significant

assay in the corresponding

3. For each run. the

column

the ratio of authentic /I-CCiRP

radio-

in rat ti\sucs XC

2-C‘GRP

assay generally

WII,

C‘CiRP-I.1

cxchungc

described above (“Chromatographlc individual

rat

U;I\ cictected

cation

total C GRP than

of

r-C‘GRP

different tissues studied varied over a wide range. The detected higher conccn-

/I-CGRP

form

of authentic

from

the

to both r-CGRP

analyses of CGRP-LI

total quantities recovered

H). In normal

rats.

molecular

results

immunoassay

in

(Fig. ?C. D). In colonlc

no authentic

of CGRP-like

were also studied. Absolute

of x-CGRP

present

the

In normal prcvzncc

been demonstrated.

but at Ioucr

conccntratlon\

01

Fig. 3. (A) Northern blot hybridization 01 “P-labelled, GICGRP- and @-CGRP-specific cDNA probes (as indicated) to total cellular RNA extracted from rat dorsal root ganglia (lane 1) and from rat intestine (lane 2). (B) In situ hybridization autoradiograph of 35S-labelled, fl-CGRP-specific complementary RNA probe to section of rat colon showing labelling of neurons in the submucous plexus (arrowed). The section was counterstained with haemotoxylin. MG indicates mucosal glands (crypts); MM indicates muscutaris mucosae. (C) In situ hybridization autoradiograph of “P-labelled, p-CGRP-specific cRNA probe to section of rat colon showing intense labelling’of neurons in both submucous and myenteric plexuses (arrowed). Background labelfing in muscularis mucosae (MM) and muscularis externa (MP) was nonspecific (see Experimental Procedures). Scale bars = 25 ,~m. X0

r-CGRP NortIwrn

h/o/

and /I-CGRP

in sensory

and enteric

nerves

100

hyhridizurion

establish whether r-CGRP and [j-CGRP are synthesized locally within the sensory ganglia and enteric nerve plexuses, the occurrence of rl-CGRP and [j-CGRP mRNAs was studied in these tissues. Total cellular RNA from dorsal root ganglia and intestine was analysed by the technique of Northern blotting which enables detection of specific mRNA sequences. Northern blot hybridization of RNA cxtractcd from rat intestine revealed the presence of a single species of RNA hybridizing to the [I-CGRP probe. No r-CGRP RNA was detected in the intestint. Northern blot hybridization of RNA extracted from dorsal root ganglia showed the presence of one species of RNA hybridizing to the r-CGRP probe and one hybridizing to the /j-CGRP probe (Fig. 3A). To

50 ._? u ._SI a ._v k u

0

In .c’,tu hybridization experiments were used to determlnc the precise localization of p-CGRP mRNA within the intestinal wall. Hybridization of the ITS- and “P-labclled cRNA probes for /I-CGRP to rat colonic sections produced an intense. selective labelling, directly localized to the submucous and myentcric plexuses (Fig. 3B. C). Pretreatment of scctlonc with ribonuclease resulted in a substantial reduction of labelling. There was no specific labelling of either the submucous or myenteric plexuses when the r-(‘GRP cRNA probe was used nor when noncomplementary control probes were used. -9

-11

Incubation of radiolabelled r-CGRP or /I’-CGRP with r;lt heart and colon membranes resulted in blnding of 2&5’% of the tracer. Non-specific binding, defined as that remaining in the presence of a IOOO-fold molar excess of unlabelled ligand, was typically 3@~50% of total radioligand binding. This level of non-specific binding is comparable to that obscrvcd by others for binding of radiolabelled r-CGRP to rat heart membranes.” Dose-response curves for the displacement of radiolabelled rat r-CGRP and /I-CGRP from rat colonic membranes by unlabclled r-CGRP and [$-CGRP arc shown in Fig. 4. No consistent difference was observed in the ability of unlabelled sc-CGRP or fl-CGRP to displace clther radioligand from membrane binding sites. Similar- results were obtained using heart membranes. The concentrations of unlabelled peptides required to

Log Fig. 4. Displacement

concentration

Displacing Heart Colon

ligand:

of radlolabelled rat r-CGRP rat [K’GRP (loner panel). each at a concentration of 0.1 nM. from receptor binding sites on membranes of rat colon by increasing concentrations of unlabelled rat r-CGRP (open circles) and unlabelled rat [j-CGRP (closed circles). Each point IS the mean result of three independent experiments each performed in duplicate. Curves are drawn for displacement of %-CGRP tracer ligand by unlabelled r-CGRP and for displacement of [KGRP tracer ligand by unlabelled /KGRP.

produce r-CGRP

curves

half-maximal displacement of radiolabelled and /j-CGRP are given in Table 2.

The effects of r-CGRP and p-CGRP on spontaneous contractions of rat atrium and relaxation of

[‘Z51]~-CGRP z-CGRP

/I-CGRP

0.68 iO.08 0.66 f 0.09

Each value is mean f S.E.M.

! M)

(upper panel) and radiolabelled

Table 2. Concentrations of unlabelled a-CGRP and /I-CGRP (nM) required to produce half-maximal displacement of z-CGRP and /I-CGRP tracer ligands (0.1 nM) from membranes of rat heart and colon

Tracer ligand:

-7

0.86iO.03 0.77 * 0.21

for three duplicate

[“‘I]/KGRP z(-CGRP 0.67 10.24 0.74 * 0.09 experiments.

[KGRP 0.98 kO.36 0.66 * 0. IO

“, Force

Increased_,

I net-ea*e

Rate

60 -

)

(min

B / t t

/ I/

40 -

20 $4

b/

OI

1

1o-g

I-

7

lo-*

1o-g

10 -8

1o-g

lo-*

lo-’

,

1

CCRP

(molll)

Fig. 5. Dose-response curve for rat c(-CGRP (open circles) and rat P-CGRP (closed circles) causing (A) increased force and (B) increased rate of spontaneous contractions in rat isolated atrium and (C) relaxation of rat colonic longitudinal smooth muscle. Values are mean f S.E.M. from IO experiments (heart) and 16 experiments (colon).

rat colonic longitudinal muscle are shown in Fig. 5. The two peptides were equipotent in causing increased rate and force of atrial contractions. While both I-CGRP and /?-CGRP caused relaxation of colonic muscle, there was a modest but nevertheless significant difference in their relative potencies. a-CGRP being 2.6 times more potent than /j-CGRP, the 95% confidence limits being 2.c3.4. DISCUSSION

Our primary aim in this study was to compare the relative levels of expression of a-CGRP and b-CGRP in the sensory and enteric nervous systems of the rat. The use of rddioimmunoassays of differing specificity to analyse CGRP-LI in normal rat tissues has shown that in the intestine, the concentrations of CGRP-LI found depend very much on the epitopic specificity of the antiserum used. Furthermore, in rats treated neonatally with capsaicin the cr-CGRP assay detected little or no CGRP-LI in the intestine, while substantial quantities were still detected by the total CGRP assay in which /I-CGRP cross-reacts. Cation cxchange analysis showed that while CGRP-LI in the

sensory ganglia was composed prtmarily 01’r-C‘GRP. the intestines of normal rats contained substantial quantities of both cc-CGRP and P-CGRP. In the intestines of capsaicin-treated rats, /j-CtiRP was present but authentic cc-CGRP was not. A small quantity of immunoreactive material which behaved like a-CGRP on cation exchange chromatography was detected but this was of significantly smaller molecular size than !x-CGRP and did not react in the a-CGRP assay. This moiety could not be positively identified. The extrinsic site of synthesis of rntestinal cc-CGRP was confirmed by analysis of cr-CGRP and fi-CGRP mRNAs which showed that only /j-CGRP mRNA is present in the intestine where it is localized to enteric neurons. Both r-CGRP and /I-CGRP mRNAs were detected in the dorsal root ganglia. The specificity of capsaicin in causing permanent degeneration of a population of primary afferent neurons when administered to neonatal rats is well documented (see references 3, 9 and 31 for reviews) and enteric neurons are apparently unaffected by capsaicin, at least with respect to peptidc content.” WC have established. therefore that CGRPimmunoreactive autonomic neurons of the cnteric nervous system express fi-CGRP and that r-CGRP in the intestines of normal rats is prcscnt in the extrinsic capsaicin-sensitive sensory fibres. The rcsuits of experiments reported by Sternini c’f rrl.,*” in which radioimmunoassay and reverse-phase chromatography were used to analyse and compare CGRP-LI in the intestines of normal and capsaicintreated rats have suggested that the major molecular form of CGRP-LI is the same in both the extrinsic sensory and intrinsic ncrvcs. However, separation ol X-CGRP and /J-CGRP by reverse-phase criteria is minimal (P. K. Mulderry. unpublished observations), so failure to effectively separate r-CGRP and /j-CGRP may explain the apparent contrast vvith the results of the present study. Previous immunocytochcmical studies have cstablished that CGRP-immunorractive ncrvc librcs can bc found throughout the alimentary tract and pancreas while ncuronal cell bodies can bc stained in the Capsaicin treatment intestine. ih7iO.li.,~.‘I.??.~X.ll.l~iX.ll).JI or surgical lesion of the atrerent nerve supply lead to a reduction of CGRP-LI concentrations and a rcduclion in the number of CGRP-immunoreactivc times in all parts of the alimentary tract but do not affect the number of CGRP-immunoreactivc ncuronal cell Thus while bodies in the intestine. hii~.i~.~~.L~.~X.~~.~~~.~~ CGRP-immunorcactivc extrinsic sensory ner\cs arc present throughout the alimentary tract. (‘GRPimmunoreactivc enteric neurons are restricted to the small and large intestines. The results of the immunochcmical analysis in the present study correlate well with thcsc findings since the capsaicinresistant //-CGRP present in the intestine did not extend to the stomach or ocsophagus. In the present study. low concentrations of capsaicin-resistant CGRP-LI wcrc found In the pan-

crcas

to extrinsic

intrinsic

CGRP

ascertained

of

that

/i-C<;RP r-CGRP

and

p-CGRP

neurons

i\

associated

with

for

present

thcreforc.

forms

in that

of CGRP

In tissues

sensory

ganglia

RNAs

both

but

where CGRP-

tibrcs.

study.

To

produce

rat heart that

unlahelled

of binding

to heart

i.c.

unknown the method

of

indicating

by either pcptldcs

modest.

was

whether

this

nant but /i-CARP

could

of

that

arc transported

both

peptidcs

cell hodie\ Whether

to their r-CGRP

distinct

and

populations

cstahlishcd. hraln

of

or

thyroid

suggcstcd

that

cxpresscd rcmainz

unknown

so

cells

in

r-CGRP studies

of /i-CGRP

sonic

in scvcral

motoncurons

may

aim

cxpcriments

c\-

cranial

prefcren-

The the

present

rat

/i-CXiRP

can

colo@c;illy ditYcrcnt studies

another

the

WC could existence

find sites

th

with and

of rat

could

\

aluo\

in

r-CGRP

half-

r-CGRP

bc achic\cd nM. in

In to rat

that

rat

at 0.35

obtained

MI-

of any

significantly

1’1 I//. found

r-CGRP

ol

/j-CGRP.

of radiolabellcd

mcmhranex

their

pharma

via cxprcssion

r-CARP

Sigrijt

displacement

of

the

in the

uhich

is

prcscnt

has

system

;I

01‘ the

althou$

to he dctcrmincd

from

difrcrcnt

cflic,~c~c\

lebcl or from

by en/!

111~s

~omc

well

;I\

prczcnt

in

function.

dilfcrent

hy

r-<‘(;RP The

results

cxperimcnts

/I-CGRP

arc

/I-CGRP.

as well

ilS ;I

lar from

probably as

ncurotransmittcr physiological r-CGRP,

Icntcric

ncrvou5

which the

suggest

rccclltor

handing \imil:tr.

lx

may

of

/i-C‘GRP.

hc as ;I ncur~,transmittsr

s;\steni.

and

and ThuS.

c\pcctcd IO act

or ncuromodIil~itor. function

C‘Y-

h;~\c hccn

r-(‘GRP

that

ma>

and

pi-cfcrcntiallv

functionall>

r-CGRP.

tn

dihtlnct

ncuron5

rcspecti\,cly.

of

,I

IL+O ~IO\CI!

and /i-(‘(;RP.

scllsor)

/I-CARP

bioassa)

<‘GRP-I

the

Furthcrmorc.

i.e.

neurons.

or

that

x-CGRP

genes.

popul~ilions. autonomic

shown comprisa

neuropcptides.

Idontifcd.

and

whether

or colon

on the binding

to

or

production

cvidcnce

hlnding

pr-csenc~ of unl;thcllcd c~mparahlc

r-CGRP

no

for

splcn~c

whether

study

nervous

homologous ncuronal

bloassaq

in the heart

rcccptor

membranes.” r;lt

and

compounds

atfinities study

mar~m;~l I‘rom

tlic

binding

indcpcndcntly

rcpresentr

gcncs.

01‘

ticsuc

act

dctcrminc

equivalent

liti-

ditfcrcnt

to

bioassay.

to receptor

cncodcd cntcric

receptor

the

was

occurI-c‘ncc

clasS

of‘

in clicitlny

<‘ON(‘I.I SKIN

press The

wcrc cntll-cl\

the tissue.

OIha\c

/i-CARP.

cxpr-css

results

of protcolysis

the

cxprcssion

expression

colon

in the

to he

cxclusivcly

the icvcl

hl

OI-

of the rcccp

clticicnt

at the receptor

unrclatcd

rates

bioassay

occupation

It remain5

diKcrcncc

is in

01‘ r-C‘C;RP

difYcrencc In potcnclcs

rat

III

whcthcl

in sirrc hybridization

of r-C’GRP

niiclei,

neurons

remains

express

Previous

motor

arc

the

the two pcptidcs

dilrcrcnt

actions

is equally

signiticant.

phenomenon

terminal\.

C<;RP-immtinorcactive

/I-U;RP. cccd~ that

/i-CGRP

it

the sensory

and peripheral

of sensory

Similarly.

populations

tiallh

central

from

suggesting

in

atEnIt> found

discrcpanq

of memhrancs The

The

was

this

of

of 5 nM

from some dilTcrencc

that

pcptidc

responhc.

spinal cord. heart. lung, bladder. skin and stomI I/ I’I~ )~~i ‘-~4:‘3, j’ r_(‘GRP WBs again predon,i_ ach. also be detected.

GIIISC

to

found

lower

than

on the rat atrium

equivalent.

t&o

The

incubation.

and /I’-CGRP

biological

dorsal

mcmhranes

of preparation

conditions

authors

significantly

a

it may result

but

thcsc

at a concentration

implying study.

cffcct on binding

howevcr,

2-CGRP

was required.

tars

the equivalent

membranes,

the present

but that some

also

both

it can bc

root

conclude.

express

of

/I-CGRP.

Messenger

prcdominatcs.

cells but

before

dorsal

wcrc WC

it is

investigation

is predominant, present.

of

that

and islet

express

from

ganglia.

that x-(‘GRP

cells

CGRP-LI also

root

sensory

nerves Further

islet

stud&

have shown

be necessary

r-CGRP

is

dorsal

will

whether

Analysis showed

sensory

ncurons.‘“.‘H42

pancreatic

Ll

histochemical

in the rat pancreas

locah~cd

not

Detailed

I ).

(I:ig.

CGRP-I,1

A parlia;I\ distinct 01‘ the

6. Costa

I. 8. 9. IO. 11. 12.

13.

14.

15. 16.

17. 18. 19

20

21

22

23. 24

25. 26. 27. 28.

29. 30.

31. 32. 33.

34.

35.

M.. Furness J. 8. and Llewellyn-Smith I. J. (1987) Histochemistry of the enteric nervous system. In I’//~,.trt&~gj qfrhe Gusrroinfesfinal Tracr (ed. Johnson L. R.). 2nd edn, pp. I-40. Raven Press. New York. Ekblad E., Winther C., Ekman R., Hakanson R. and Sundler F. (1987) Projections of peptide-containing neurons in rat small intestine. Neuroscience 20, 169-188. Feinberg A. P. and Vogelstein B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Analyf. Biochem. 132, h-13. Fitzgerald M. (1983) Capsaicin and sensory neurons-a review. Pain 15, 109%130. Furness J. B.. Costa M., Gibbins I. L., Llewellyn-Smith I. J. and Oliver J. R. (1985) Neurochemically similar myenteric and submucous neurons directly traced to the mucosa of the small intestine. CeN Tiss. Re.r. 241, 155--163. Ghatei M. A., Gu J., Mulderry P. K., Blank M. A., Allen J. M., Morrison J. F. B., Polak J. M. and Bloom S. R. (1985) Calcitonin gene-related peptide (CGRP) in the female rat urogenital tract. Peptides 6, 809 815. Ghatei M. A., Mulderry P. K., McGregor G. P., Bishop A. E. and Polak J. M. (1986) Experimental investigation of the nature of the calcitonin gene-related peptide innervation of the rat gastrointestinal tract; comparison with other enteric neuropeptides. Can J. Physiol. Phurmuc. Suppl. p, 159. Gibbins I. L., Furness J. B.. Costa M., MacIntyre I., Hillyard C. J. and Girgis S. (1985) Co-localization of calcitonin gene-related peptide like immunoreactivity with substance P in cutaneous, vascular and visceral sensory neurons of guinea pigs. Neurosci. Lerr. 57, 125 130. Gibson S. J., Polak J. M.. Bloom S. R., Sabate 1. M., Mulderry P. K., Ghatei M. A., McGregor G. P., Morrison J. F. B., Kelly J. S., Evans R. M. and Rosenfeld M. G. (1984) Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and eight other species. J. Neurosci. 4, 3101&31 Il. Green T. and Dockray G. J. (1987) Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat. Neurosci. Le/[. 76, 151 ~156. Hamid Q,, Wharton J., Terenghi G., Hassall C. J. S., Aimi J., Taylor K. M., Nakazato H., Dixon J. E., Burnstock G. and Polak J. M. (1987) Localization of atria1 natriuretic peptide mRNA and immunoreactivity in rat and human atrial appendages. Proc. nam. Acud. Sri. U.S.A. 84, 676G-6764. Holman J. J., Craig R. K. and Marshall I. (1986) Human I- and p-CGRP and rat cr-CGRP are coronary vasodilators in the rat. Peptides 7, 231-235. Holzer P., Gamse R. and Lembeck F. (1980) Distribution of substance P in the rat gastrointestinal tract-lack of effect of capsaicin pretreatment. Eur. J. Pharmuc. 61, 303-307. Hoppcner J. W. M., Steenbergh P. H., Slebos R. J. C., Visser A., Lips C. J. M., Jansz H. S., Bechet J. M., Lenoir G. M., Born W., Haller-Brem S., Petermann J. B. and Fischer J. A. (1987) Expression of the second calcitoninicalcitonin gene-related peptide in Ewing Sarcoma cell lines. J. clin. Endocr. Metab. 64, 809-817. Ju G., Hokfelt T., Brodin E., Fahrenkrug J., Fischer J. A. Frey P., Elde R. P. and Brown J. C. (1987) Primary sensory neurons of the rat showing calcitonin gene-related peptide immunoreactivity and their relation to substance P, somatostatin, galanin, vasoactive intestinal polypeptide and cholecystokinin immunoreactive ganglion cells. Cell Tiss. Res. 247, 41 l-43 1. Lee Y.. Takami T., Kawai Y., Girgis S., Hillyard C. J., MacIntyre I., Emson P. C. and Tohyama M. (1985) Distribution of calcitonin gene-related peptide in the rat peripheral nervous system with reference to its co-existence with substance P. Neuroscience 15, 1227-1231. Lee Y., Shiotani Y., Hayashi N., Kamada T., Hillyard C. J., Girgis S. I., MacIntyre I. and Tohyama M. (1987) Distribution and origin of calcitonin gene-related peptide in the rat stomach and duodenum: an immunocytochemical analysis. J. Neural Transm. 68, l--14. Lehrach H.. Diamond D., Wozney J. M. and Boedtker H. (1977) RNA molecular weight determination by gel electrophoresis under denaturing conditions a critical re-examination. Biochemistry 16, 47433475 1. Lundberg J. M., France-Cereceda A., Hua X., Hokfelt T. and Fischer J. A. (1985) Co-existence of substance P and calcitonin gene-related peptide-like immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin. Eur. J. Pharmuc. 108, 315-319. Maniatis T., Fritsch E. F. and Sambrook J. (1982) Molecular Cloning: A Laboratory Manual. p. 196. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Op. cii., Ref. 25, pp. 326328. Mulderry P. K., Nicholl C. G., Ghatei M. A., Springall D. R., Polak J. M. and Bloom S. R. (1984) Distribution and possible dual role of CGRP-containing nerves in the skin of the rat. Regul. Pepf. 9, 341. Mulderry P. K., Ghatei M. A., Bishop A. E., Allen Y. S., Polak J. M. and Bloom S. R. (1985) Distribution and chromatographic characterisation of CGRP-like immunoreactivity in the brain and gut of the rat, Regul. Pepr. 12, 133m-143. Mulderry P. K., Ghatei M., A., Rodrigo J., Allen J. M., Rosenfeld M. G., Polak J. M. and Bloom S. R. (1985) Calcitonin gene-related peptide in cardiovascular tissues of the rat. Neuroscience 14, 947-954. Mulderry P. K., Ghatei M. A. and Bloom S. R. (1987) In oirro production and characterisation of low molecular weight forms of calcitonin gene-related peptide immunoreactivity from rat thyroid. Biochem. biophys. Res. Commun. 144, 8833890. Nagy J. I. (1982) Capsaicin a chemical probe for sensory neuron mechanisms. In Handbook of Psychophurmuco/ogy (eds Iversen L. L.. Iversen S. D. and Snvder S. H.), Vol. 15. DD. 185.-235. Plenum Press. New York. Paton W. D. M. and Zar M. A. (1968). The origin of acetylcholine released from guinea pig’intestine and longitundinal muscle strips. J. Physiol. Land. 194, 13-33. Petermann J. B., Born W., Chang J-Y. and Fischer J. A. (1987) Identification in the human central nervous system, pituitary and thyroid of a novel calcitonin gene-related peptide and partial amino acid sequence in the spinal cord. J. biol. Chem. 262, 542-545. Rodrigo J., Polak J. M., Fernandez L., Ghatei M. A., Mulderry P. K. and Bloom S. R. (1985) Calcitonin gene-related peptide-immunoreactivity sensory and motor nerves of the rat, cat and monkey esophagus. Gasfroenterology 88, 444451. Rosenfeld M. G., Mermod J. J., Amara S. G., Swanson L. W., Sawchenko P. E., Rivier J., Vale W. W. and Evans R. M. (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nafure 304, 1299135.

x-CGRP

and P-CARP

m wnsory

and cntcrlc ncrvt‘~

71)5