Research Article

Pharmacology Received: June 7, 2019

Accepted: July 31, 2019
DOI: 10.1159/000502471 Published online: September 4, 2019

The Presence of Calcitonin Gene-Related Peptide

and Its Receptors in Rat, Pig and Human Brain:
Species Differences in Calcitonin Gene-Related
Peptide Pharmacology
Karin Warfvinge b, c Lars Edvinsson b, c Darryl S. Pickering a Majid Sheykhzade a
       

a Department
of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of
Copenhagen, Copenhagen, Denmark; b Department of Clinical Sciences, Division of Experimental Vascular Research,
 

Lund University, Lund, Sweden; c Department of Clinical Experimental Research, Glostrup Research Institute,
 

Rigshospitalet Glostrup, Glostrup, Denmark

Keywords cortical neurons were positive for CGRP. Slender receptor

Binding affinity · Calcitonin gene-related peptide receptor ·
Gepants · Species differences · Brain
Abstract
Aim: The aim of present study is to investigate the binding
characteristics of non-peptide calcitonin gene-related pep-
tide (CGRP) receptor antagonists (i.e., gepants) in the brain
membranes of rat, pig and human. Methods: The interac-
tion of available gepants with the CGRP receptor was stud-
ied in the brain membranes of 3 different species using a
radioligand competitive binding assay. In addition, the dis-
tribution of CGRP and its receptor component receptor ac-
tivity modifying protein 1 (RAMP1) in rat cerebellum and
cortex was explored using immunohistochemistry. Results:
All gepants, except SB268262, displaced 100% of the radio-
ligand specific binding in the brain tissue of all 3 species
and showed highest affinity for CGRP receptors in human
brain as compared to rat and pig brain membranes. Fur-
thermore, radioligand binding studies revealed the pres-
ence of higher CGRP receptor density in human cerebellum
compared to human cortex. The morphology, size and den-
sity of CGRP immunoreactive cells suggest that all cerebral
immunoreactive fibres were found spanning through the
entire cortex. CGRP immunoreactivity was displayed in the
cell soma of cerebellar Purkinje cells and in large neurons
in the medial cerebellar nucleus. RAMP1 was found on the
surface of the Purkinje cells and in parallel fibres, indicating
presence in the granule cell axons. Conclusion: Cerebellum
and cerebral cortex are rich in CGRP and CGRP receptors,
which can be antagonized by gepants. However, all ge-
pants display higher affinity for human CGRP receptors as
compared to rat and pig CGRP receptors. Furthermore, hu-
man cerebellum seems to express higher density of CGRP
receptors. © 2019 S. Karger AG, Basel
Introduction
Calcitonin gene-related peptide (CGRP) is a 37-amino
acid neuropeptide and a potent vasodilator [1, 2] distrib-
uted both centrally and peripherally in the body [3–5].
CGRP encompasses the family of related hormones such
as calcitonin (CT), adrenomedullin (AM), intermedin
and amylin (AMY) [5, 6].

© 2019 S. Karger AG, Basel Majid Sheykhzade, PhD

Department of Drug Design and Pharmacology
Faculty of Health and Medical Sciences, University of Copenhagen
E-Mail karger@karger.com
Universitetsparken 2, DK–2100 Copenhagen Ø (Denmark)
www.karger.com/pha E-Mail mash @ sund.ku.dk
 The functional G-protein-linked CGRP receptor is
made of 3 key components: calcitonin receptor-like re-
ceptor (CLR), a specific protein called receptor activity
modifying protein 1 (RAMP1) and receptor component
protein required for signal transduction [7]. Three
RAMPs (RAMP1, RAMP2 and RAMP3) have been iden-
tified as chaperones, which are responsible for the trans-
location of CLR from cytosol to the plasma membrane
and for the insertion of CLR into the plasma membrane.
CGRP receptor requires association of CLR and RAMP1,
while AM receptors are generated by association of
RAMP2 or RAMP3 with CLR [8]. In addition, the amino
acid residue Trp74 of human RAMP1 is a key determi-
nant of the species selectivity and is responsible for high
affinity binding of small molecule CGRP receptor antag-
onists (the so-called gepants) to human CGRP receptor
(human-CLR/human-RAMP1) [9]. The N-terminals of
RAMP1 and CLR form a binding pocket for CGRP. The
C-terminal of CGRP interacts first with the extracellular
N-terminal regions of CLR and RAMP1 and then, the N-
terminal of the peptide interacts with the adjacent mem-
brane regions of CLR leading to receptor activation and
subsequent increase in cAMP levels [10]. Gepants inter-
act with a hydrophobic binding pocket formed by CLR
and RAMP1, and block the initial CGRP binding event
and subsequent receptor activation [10, 11]. Recently, Li-
ang et al. [12] have described the structure of CGRP re-
ceptor in more detail and showed that the gepant binding
site was located closely to the extracellular N-terminal do-
main.
Clinical studies have demonstrated a central role for
CGRP and activation of its receptor in migraine pathol-
ogy [13]. The gepants have been shown to be effective in
aborting the acute phase of migraine attacks [14]. Un-
fortunately, due to liver toxicity upon repeated expo-
sures of telcagepant, the development was temporarily
halted. However, novel non-liver toxic gepants have
now passed phase III (aterogepant, rimegepant, ubroge-
pant; see for review [15]). Antibodies against CGRP and
the CGRP receptor are now approved for prevention in
chronic migraine and in frequent episodic migraine
[16], providing new prophylactic tools for migraine pre-
vention.
Current views suggest that these antagonistic drugs act
on sites outside the blood-brain barrier [13], but the CNS
is rich in expression of CGRP and CGRP receptors [17].
The present study explores the binding characteristics of
gepants in cortex and cerebellum of rat, pig and man. In
addition, the distribution of CGRP and RAMP1 is visual-
ized using immunohistochemistry.
2 Pharmacology
DOI: 10.1159/000502471
Material and Methods
Animals’ Tissue
All animal procedures were carried out in accordance with na-
tional laws and guidelines. Male piglets (26 days old) were used in
this study. The piglets, kept in the animal facility at the School of
Veterinary Medicine and Animal Science (Faculty of Health and
Medical Sciences), were euthanized with Zoletil mix (1 mg/kg, Vir-
bac, Carros, France) combined with an overdose of pentobarbital
(200 mg/mL, Glostrup apotek, Denmark) via a catheterized ear
vein. The piglet brains were removed from the skull and kept frozen
at –80 ° C. Adult male Wistar rats (Taconic Europe, Denmark) were
housed in our local animal facility (Faculty of Health and Medical
Sciences) in a temperature (22–23 ° C) and humidity-controlled en-
vironment with a 12-h light and 12-h dark cycle and ad libitum ac-
cess to standard chow and tap water. The rats were euthanized by
CO2 inhalation followed by decapitation. The rat brains were care-
fully dissected, cut sagittally in the midline and placed in 4% para-
formaldehyde for 4 h, followed by incubation overnight in Sö-
rensen’s phosphate buffer (pH 7.2) containing sucrose (10% and
then 25%). Thereafter, the tissue was embedded in Yazulla embed-
ding medium (30% egg albumin, 3% gelatin) and cryo-sectioned at
12 µm. The sections were stored at –20 ° C until use.     
Human Tissue
Post-mortem (within 24 h of death) human cerebellum and
cortex specimens were obtained from Department of Pathology,
Lund University hospital, Sweden (3 females and 2 males, age span
67–88 years). The experiments were approved by the Regional Eth-
ical Review Board in Lund, Sweden (LU- 818-01).
Radioligand Binding
Crude synaptosomal (P2) membranes were prepared by stan-
dard methods [18]. Five piglet brains, 20 rat brains and 5 post mor-
tem human brain tissues were used to make P2 membranes. P2
membranes made from the brains of each species were pooled,
aliquoted and stored frozen at –20 ° C. For use in binding assays,
an aliquot of membranes was thawed and washed 4 times by cen-
trifugation 48,000 × g, 15 min at 4 ° C with Ultra-Turrax resuspen-
sion in 25 mL ice-cold hypotonic buffer (5 mmol/L Tris-HCl pH
7.4 at room temp). Membranes were then re-suspended in the as-
say buffer (50 mmol/L Tris-HCl pH 7.4 at room temp). Binding
assays were conducted in triplicate in siliconized (Sigmacote®, Sig-
ma-Aldrich Chemical Co., St. Louis, MO, USA) 12 × 75 mm glass
tubes in a final assay volume of 250 µL containing 25–100 µg P2
membrane protein and 10–40 pmol/L radiolabel. Rat or human
(125I)-αCGRP (2,200 Ci/mmol; PerkinElmer, Waltham, MA, USA)
was used as the radiolabel and nonspecific binding defined as re-
maining binding in the presence of either 10 µmol/L MK3207 or
1 µmol/L BIBN4096BS. Siliconized pipette tips were used for dis-
pensing radioligand. In competition experiments, stock ligand so-
lutions were diluted into assay buffer and tested at 12–16 concen-
trations in the range 1 pmol/L–0.5 mmol/L. Assay tubes were in-
cubated 2 h at room temperature followed by rapid filtration on
polyethyleneimine-treated GF/C type glass fibre filters (filter #626,
VWR, Denmark) using 12-well Millipore filtration manifolds
(Millipore, Darmstadt, Germany). Filters were rinsed twice with
3 mL ice-cold assay buffer (pH 7.4 at 4 ° C). Filters were transferred
to polypropylene tubes and counted in a Wallac γ-counter (Perki-
nElmer) for 1 min at 84% counting efficiency.
Warfvinge/Edvinsson/Pickering/
Sheykhzade
 Immunohistochemistry
Sagittal sections of the rat brain (containing cerebrum, cerebel-
lum and brainstem) were washed in phosphate buffered saline
(PBS) containing 0.25% Triton-X (PBS-T) for 15 min followed by
application of the primary antibody CGRP (B47–1, Rabbit, 1:
1,500, Europroxima) or RAMP1 (RAMP1 844, Goat, 1:100, Merck
and Co., Inc., USA). The samples were incubated overnight at 4 ° C
in moisturized incubation chambers. On the next day, the sections
were washed twice in PBS-T for 15 min prior to incubation with
secondary antibodies (FITC, anti-rabbit, 1: 100, Cayman Chemi-
cal, Ann Arbor, MI or Alexa 488, anti-goat, 1:100, Invitrogen, La
Jolla, CA, USA) for 1 h at room temperature. Finally, the sections
were washed 2 × 15 min and mounted with Vectashield mounting
medium containing 4’,6-diamidino-2-phenylindole (Vector Labo-
ratories, Burlingame CA, USA). Specificity of RAMP1 antibodies
have been demonstrated in-house [19], where the specificity of the
antibodies was confirmed by Western blot in HEK293 cells stably
expressing the CGRP receptor. Pre-absorption controls have also
been performed with the CGRP and RAMP1 antibodies [19]. The
omission of primary antibodies served as negative controls. The
sections were examined in an epifluorescence microscope (Nikon
80i, Tokyo, Japan) and a Nikon DS-2MV camera. The resulting
photos were processed using Adobe Photoshop CS3 (version 0.0
Adobe Systems, Mountain View, CA, USA).
Drugs, Chemicals and Solutions
Rat-αCGRP, human-αCGRP, rat-AMY (8–37), FITC-rat-
αCGRP, linear analogues of CGRP and C-terminally truncated
fragments of CGRP were all ordered from BACHEM (GmbH, Rhe-
in, Germany). BIBN4096BS (Olcegepant) [20], MK0974 (Telcage-
pant) [21], MK3207 [22] and BMS927711 (Rimegepant) were or-
dered from MedChem Express (MCE, Monmouth Junction, USA).
SB268262 [23] was purchased from TOCRIS (Minneapolis, MN,
USA). All gepants were dissolved in DMSO. CGRP, rat-AMY, lin-
ear analogues and C-terminal fragments of CGRP were dissolved
in distilled H2O. Stock solutions of the drugs were stored at –20 ° C
    
and dilutions were prepared just before experiments.
Data Analysis and Statistics
Radioligand competition binding curves were analyzed with
GraphPad Prism version 6.07 using the one-site Ki and the 4-pa-
rameter logistic equations. Results are given as mean ± SEM
(n  =  number of individual experiments). Differences between
mean values were analyzed by one way-ANOVA followed by
Bonferroni post hoc comparison test, accepting significance at
p < 0.05.
Results
Radioligand Binding
The radioligand binding studies showed that BIBN-
4096BS and MK3207 had similar and the highest affinity
in human brain. BIBN4096BS displayed the highest affin-
ity in human brain with approximately ten and hundred-
fold less affinity in rat and pig brain respectively. Further-
more, MK0974 displayed 200 and 1,000 fold higher affin-
Species Differences in CGRP
Pharmacology in Brain
ity in human brain compared to rat and pig brain
respectively (Fig. 1, Table 1). In general, the blockade of
the CGRP binding with the gepants was more potent in
human brain tissue as compared to rat and pig. Finally,
the results from radioligand binding studies showed a
greater amount of displacement of 125I-CGRP and spe-
cific binding in human cerebellum compared to the hu-
man cortex, suggesting a higher receptor density in hu-
man cerebellum (Fig. 1, Table 1). SB268262 showed low
affinity to CGRP receptors in pig and human brain (Ki
>0.5 mmol/L) but had relatively high affinity (Ki = 233
nmol/L) to CGRP receptors in rat brain brain (Fig. 2, Ta-
ble 1). SB268262 was able to displace only approximately
50% of the radioligand specific binding in rat brain. In
addition, the higher Hill-coefficients (nH ≈ 2) determined
in radioligand binding studies in human brain compared
to rat and pig brain for BIBN4096BS (Olcegepant),
MK3207 and BMS927711 (Rimegepant) suggest the pres-
ence of either several binding sites on the human CGRP
receptor or CGRP receptors at multiple affinity states in
human brain. However, there was no significant differ-
ence in Hill-coefficients for gepants and MK3207 when
comparing rat and pig brain (Fig. 2, Table 1).
Immunohistochemistry
Cerebral Cortex
The analysis of the density, size and morphology of the
CGRP immunoreactive cells in cortex indicate that near-
ly all cortical neurons were positive for CGRP (Fig.  3).
The immunoreactivity was primarily observed in the cell
soma, visualized as positive grains in the cytoplasm. The
neuronal processes showed very limited or no immuno-
reactivity. The CGRP immunoreactivity was confined to
cortical layers II-VI. No CGRP immunoreactivity was ob-
served in cortical layer I, since there were few/no neuro-
nal cell somas.
The thin, slender RAMP1 immunoreactive fibres were
found spanning through the entire cortex, and also tra-
versing through all cortex layers (Fig. 3). These horizontal
fibres were most obvious in layer I close to the brain sur-
face and in layer III. We observed no neuronal cell somas
that were positive for RAMP1.
Cerebellum
In the cerebellum, we observed spectacular CGRP im-
munoreactivity confined to the Purkinje cells in the cer-
ebellar lobules (Fig. 4). As described for cortex, the im-
munoreactivity was displayed as CGRP positive grains in
the cell soma, often close to the nucleus. The axons and
dendrites were not CGRP immunoreactive.
Pharmacology 3
DOI: 10.1159/000502471
 Color version available online
125

100

Specific bound, %
75

hCGRP
50
BIBN4096BS
Amylin (8–37)
25 SB268262
MK0974

0 MK3207

–13 –12 –11 –10 –9 –8 –7 –6 –5 –4 –3

a Log (ligand), mol/L

100

80
Total bound, %

60
Fig. 1. Competition curves in human tis-
sues. Human cerebellum P2 membrane li- 40
gand binding (a) and a comparison of
CGRP competition curves between human Cortex Ki = 0.43 nmol/L
20
cerebellum and cortex (b). Each symbol 1 µmol/L BIBN (cortex)
represents the mean ± SEM of pooled, nor- Cerebellum Ki = 2.4 nmol/L
malized data from individual experiments 0
(n = 3–5) conducted in triplicate. CGRP, –11 –10 –9 –8 –7 –6 –5
calcitonin gene-related peptide; AMY, am- b Log (h-α CGRP), mol/L
ylin.

The RAMP1 staining was found on the surface of the
Purkinje cells, notably on the cell membrane, and in par-
allel fibres, indicating RAMP1 positivity in the granule
cell axons spreading within the molecular layer (Fig. 4).
Furthermore, slender positive processes were found with-
in the granular cell layer and in the white matter. In the
medial cerebellar nucleus, we found a tight mass of thin
slender RAMP1 immunoreactive processes. We found no
RAMP1 staining in the cell somas.
Discussion
We have here demonstrated a rich supply of CGRP and
CGRP receptors in both cerebral cortex and cerebellum
[17]. Overall, the expression pattern in different regions
of the rat cerebral cortex was similar and there was also
similar expression pattern in different parts of the rat cer-
ebellum, with cell bodies storing vesicular CGRP and fi-
bres that show immunoreactivity for the CGRP receptor
key element RAMP1. The role of cerebral cortex in mi-
graine is mostly linked to experimentally spreading de-
pression and to the aura in the fore-math of a migraine
attack. However, despite much research ever since the first
findings of the spreading depression phenomenon by
Leao [24] the more detailed molecular explanation is far
from known [25]. Understanding the role of CGRP in the
cerebellum during a migraine attack is a rather new sub-
ject. Cerebellar activity is increased during trigeminal no-
ciceptive input in several functional imaging studies [26,
27], suggesting that the cerebellum is more active during
a migraine attack than interictal [28, 29]. Other studies
have demonstrated a role of the cerebellum in human no-
ciception, which might modulate pain perception [30].

4 Pharmacology Warfvinge/Edvinsson/Pickering/
DOI: 10.1159/000502471 Sheykhzade
Table 1. Ligand affinities across species

Ligands Rat Pig Human

Ki, nmol/L nH Ki, nmol/L nH Ki, nmol/L nH

Rat-αCGRP 8.2±2.2a 0.86±0.02 8.0±2.5 1.16±0.16 2.30±0.75 0.94±0.05

Human-αCGRP 0.91±0.21 0.69±0.03 13.4±1.8 1.21±0.21 3.28±1.13 0.80±0.01
Cys(Et)2, 7α-hCGRP 5.0±0.8 0.73±0.04 15.1±2.5 0.98±0.07 nd nd
Cys(Acm)2, 7α-hCGRP 27.5±7.5 1.23±0.05 37.3±2.8 1.12±0.10 nd nd
Human-αCGRP (8–37) 6.4±1.3 1.19±0.09 30.2±1.5 1.16±0.03 nd nd
Rat-αCGRP (29–37) 8,980±1,600 1.12±0.08 >10,000 – >10,000 –
Rat-AMY (8–37) >10,000 – >10,000 – >10,000 –
FITC-rat-αCGRP >10,000 – >10,000 – >10,000 –
SB268262 233±38 0.91±0.21 >500,000 – >500,000 –
MK3207 17.2±2.1a 0.83±0.18** 129±42 0.85±0.05** 0.76±0.21 2.10±0.25
MK0974 410±37a 0.72±0.02 3,110±290 0.69±0.02 2.27±0.68 1.16±0.15
BIBN4096BS 1.43±0.11a 0.95±0.06* 35.3±1.9 0.69±0.04** 0.34±0.11 2.41±0.36
BMS927711a 534±150 1.22±0.12* 1,920±460 0.81±0.04** 1.28±0.09 2.19±0.35
125I-rat-αCGRP 0.29±0.03b 0.88±0.01 nd nd nd nd
125
I-human-αCGRP nd nd 0.576±0.072b 1.03±0.01 0.055c –
a
 Sheykhzade et al. [31], 2017.
b K from radioligand saturation binding experiments (n = 3–4).
d
c
 Kd taken from Henke et al. [42], 1987.
* p < 0.05 versus human.
** p < 0.01 versus human.
Ligand affinities are presented in human (cerebellum), rat and pig brain P2 membranes. Shown are mean ± SEM values of Ki (nmol/L)
and Hill coefficient (nH) from n = (3–5) individual experiments, each conducted in triplicate. Some data has been previously published
but is included here for comparison purposes.
CGRP, calcitonin gene-related peptide; AMY, amylin; nd, not determined.

Thus, both CNS regions appear to be involved in different
aspects of migraine attacks. In order to understand the
role of CGRP and to assess the affinity of known non-
peptide CGRP receptor antagonists (gepants) in brain, we
aimed to characterize the gepants’ pharmacology in the
rat, pig and man by 125I-CGRP competition binding assay.
In addition, the distribution and localization of CGRP and
RAMP1 was visualized using immunohistochemistry to
obtain understanding of the putative functional role of
CGRP and CGRP receptor antagonists. These studies in-
crease our knowledge about the localization of CGRP re-
ceptors in the subcellular key regions of brain and the ex-
istence of other binding sites and/or receptor subtypes in
these CNS regions, which is useful for identifying poten-
tial drug targets in the CNS and for future understanding
of the central role of CGRP in pain perception.
Antagonistic Effects of Gepants and CGRP Receptor
Binding
In general, there is good correlation between our re-
sults from 125I-CGRP competition binding experiments
using gepants in brain membrane preparations and our
previous results from myograph studies on isolated rat
mesenteric [31] and human subcutaneous arteries [32]
using the same antagonists showing similar affinity val-
ues (i.e., pA2) determined by the Schild plot method. In
addition, the affinity values determined in the current
study for gepants towards rodent (rat) and porcine CGRP
receptors are approximately 100–1,000 fold lower com-
pared to the human CGRP receptor. Previous studies
have shown that Trp-74, which is present only in human
RAMP1 together with Met-42 of human CLR, is indeed
forming the high affinity binding site for BIBN4096BS,
MK3207 and MK0974 [21, 22, 33, 34]. In our present
study, MK3207 displays relatively highest affinity for hu-
man CGRP receptors, medium affinity for rat CGRP re-
ceptors and lowest affinity for pig CGRP receptors, which
has previously been demonstrated [21, 22]. This is an im-
portant consideration for pre-clinical evaluations of fu-
ture CGRP receptor antagonists.
Gepants have the potential to be an important addition
to the pharmacological treatment of migraine. Randomized

Species Differences in CGRP Pharmacology 5

Pharmacology in Brain DOI: 10.1159/000502471
 Color version available online
100

75

Specific bound, %
50
hCGRP CYS (Et)
BIBN4096BS MK0974
25 CGRP (8–37) MK3207
Amylin (8–37) rCGRP
CYS (Acm) CGRP (29–37)
0 SB268262 FITC-rCGRP

–12 –11 –10 –9 –8 –7 –6 –5 –4 –3

a Log (ligand), mol/L

100

75
Specific bound, %

50
hCGRP CYS (Et)
BIBN4096BS MK0974
Fig. 2. Competition curves between spe- 25 CGRP (8–37) MK3207
cies. Ligand binding was examined in rat Amylin (8–37) rCGRP
(a) and pig (b) whole brain P2 membranes. CYS (Acm) CGRP (29–37)
Each symbol is mean ± SEM of pooled, 0 SB268262 FITC-rCGRP
normalized data from individual experi-
–12 –11 –10 –9 –8 –7 –6 –5 –4 –3
ments (n = 3–5) conducted in triplicate.
CGRP, calcitonin gene-related peptide; b Log (ligand), mol/L
AMY, amylin.

controlled trials evaluating the first generation of gepants
proved their efficacy and safety [35–37]. However, their de-
velopment was terminated early because of concerns of liv-
er toxicity. Rimegepant, ubrogepant and atogepant repre-
sent a second generation of gepants, chemically different
from the older ones. The first results from clinical studies
show that these compounds are effective and safe [38, 39].
As far as the SB268262 compound is concerned, a pre-
vious study demonstrated an inhibition of CGRP binding
and CGRP-mediated stimulation of adenylate cyclase
(AC) activity by the compound in SK-N-MC human neu-
roblastoma cell line [23]. The same authors determined
the antagonistic effect of SB-(+)-273779 (an optical iso-
mer of racemic compound SB-[±]-268262) on CGRP-
stimulated AC activity in SK-N-MC human neuroblas-
toma cells. The compound was without any antagonistic
effect at relatively low concentration (0.1 µmol/L) but dis-
played competitive antagonism at 1 µmol/L. However,
higher concentrations (3 and 10 µmol/L) of the com-
pound decreased the CGRP-induced maximum stimula-
tion by 30 and 60%, respectively. The effects of different
pre-incubation times with SB-(+)-273779 (3 µmol/L) on
the CGRP-stimulated AC activity were also determined
in SK-N-MC human neuroblastoma cells [23]. The com-
pound induced a parallel rightward shift of the log (CGRP
concentration)-response curve after 15–30 min pre-incu-
bation but prolonged pre-incubation (> 30 min) of the
cells with the compound (even after thorough washing)
resulted in a significant reduction in CGRP-induced
maximum effect, indicating an irreversible binding be-
haviour by the compound. In our radioligand competi-
tive binding experiments, SB268262 showed negligible
affinity to pig and human brain CGRP receptors (Ki >0.5
mmol/L) but had relatively high affinity (Ki = 233 nmol/L)

6 Pharmacology Warfvinge/Edvinsson/Pickering/
DOI: 10.1159/000502471 Sheykhzade

Color version available online
CGRP RAMP1
I
* ily members, there is still a vivid debate about the exis-
II
III
IV
50 µm 50 µm
Fig. 3. Distribution of CGRP and RAMP1 in rat cortex. The results
indicate that almost all cortical neurons were positive for CGRP.
The immunoreactivity was found mainly in the cell soma and vi-
sualized as positive grains in the cytoplasm (insert). Thin, slender
RAMP1 immunoreactive fibres were found spanning across the
entire cortex (insert) and also traversing through the cortical lay-
ers. These horizontal fibres were most obvious in layer I, close to
the brain surface (asterisk). CGRP, calcitonin gene-related pep-
tide; RAMP1, receptor activity modifying protein 1.
to rat brain CGRP receptors. Interestingly, SB268262 was
able to displace only approximately 50% of the radioli-
gand specific binding in rat brain, suggesting either sev-
eral subtypes of CGRP receptors or that the binding rep-
resents association with other subtypes of the CGRP fam-
ily of peptides in rat brain with one (or more) of these
being less sensitive to this ligand. Similarly, the Hill coef-
ficients of 2 for MK3207 (but not for MK0974), BIBN-
4086BS and BMS927711 perhaps suggest several binding
Species Differences in CGRP
Pharmacology in Brain
sites on the CGRP receptor (positive cooperativity) and
that CGRP receptor might exist in multiple affinity states
in human cerebellum [40–42]. Because of the complexity
of the receptor pharmacology of the CGRP peptide fam-
tence of different receptor subtypes for CGRP [43, 44]
perhaps with distinct physiological and pathophysiologi-
cal actions. Therefore, AMY1, AMY3, AM1 and AM2 re-
ceptors could generate the profile of CGRP receptor sub-
types displaying different affinities to the members of
CGRP peptide family [7].
The relatively higher Hill-coefficients observed for ge-
pants in human brain can therefore be attributed to the
presence of high density of receptors for other members of
CGRP peptide family (i.e., CT, AM and AMY receptors)
especially in cerebral arteries and in the neuronal network
of rat brain compared to the peripheral vascular tissue
where CT/AM/AMY receptor population and the neuro-
nal input seem to be sparse or insignificant (see for review
[43]). A recent study by Walker et al. [44] on primary rat
trigeminal ganglia demonstrated the presence of 2 CGRP-
like receptors: AMY1 receptor (CTR/RAMP1) and the
CGRP receptor (CLR/RAMP1). The AMY1 receptor is in-
teresting because a number of studies have shown that
AMY and CGRP are equally potent at this receptor, albeit
in transfected cells, indicating that RAMP1 is playing a
dominant role for high affinity binding. In isolated tissues,
however, the receptor phenotype is further complicated by
the presence of multiple key receptor components giving
rise to expression of several different receptor subtypes dis-
playing different affinities or potencies to the gepants (see
for review [43]). The expression level can also vary from
one tissue to another or between different species. In our
study, the results from radioligand binding experiments
showed similar inhibition pattern in the different species
examined (rat, pig and human). Furthermore, as expected,
the binding affinities of gepants were relatively higher in
human specimens as compared to rat and pig. Finally, the
results from radioligand binding studies showed a greater
displacement of 125I-CGRP and/or specific binding in hu-
man cerebellum compared to the human cortex, perhaps
suggesting the presence of a higher CGRP receptor density
in human cerebellum. To visualize the expression of CGRP
and the receptor component RAMP1, we used immuno-
histochemistry. The huge presence of CGRP and RAMP1
in cerebral cortex [17] and in cerebellum [17, 45, 46] has
previously been described. As has been shown in previous
reports and the present, there is a rich expression of CGRP
and CGRP receptor in the Purkinje cells suggesting a func-
tional role of CGRP in the cerebellum [47].
Pharmacology 7
DOI: 10.1159/000502471
 Color version available online
CGRP RAMP1

**

* *
**
Fig. 4. Distribution of CGRP and RAMP1 WM WM
in rat cerebellum. CGRP immunoreactivity
was displayed as positive grains in the Pur-
kinje cell soma (arrow), often close to the
nucleus. Fibbers were not CGRP immuno- 100 µm 100 µm
reactive. RAMP1 staining was found on the
surface of the Purkinje cells (arrow) and in
*
fibres parallel to the cerebellar surface (as- ** *
terisk), indicating RAMP1 positivity in the **
granule cell axons spreading within the
molecular layer. Furthermore, slender pos-
itive processes were found within the gran-
ular cell layer (2 asterisks) and the white
matter. Arrow – Purkinje cells, asterisk –
molecular layer, 2 asterisks – granular lay-
er. CGRP, calcitonin gene-related peptide; 25 µm 25 µm
RAMP1, receptor activity modifying pro-
tein 1; WM, white matter.

Conclusions
In summary, our main findings are as follows: (i)
CGRP and CGRP receptor are present in the cortex and
cerebellum, (ii) binding studies suggest higher density
of human CGRP receptors in cerebellum as compared
to cortex, and (iii) gepants display higher affinity for hu-
man CGRP receptors as compared to rat and pig CGRP
receptors. The huge presence of CGRP and the CGRP
receptor component RAMP1 suggest an important role
for the peptide in CNS function. Overall, migraine
headache depends on the peripheral sensitization of tri-
geminal ganglion neurons and central sensitization of
second-order thalamic neurons, in which CGRP plays
an important modulatory role [13–15, 27, 29, 35, 39,
48]. Sensitization likely has a central and peripheral
component. A recent PET study using CGRP PET trac-
er (11C) MK-4232 as a validated ligand for the CGRP
receptor showed very low (< 10%) occupancy of the
brain CGRP receptor by telcagepant at therapeutic dos-
es [49], indicating that occupancy of central CGRP re-
ceptors is not required for the therapeutic effect of tel-
cagepant, but the central occupancy that could occur
with higher doses may confer some additional thera-
peutic benefit. Since migraine pain seems to involve
both central and peripheral nociceptive mechanisms,
future work should preferably focus on understanding
the role of the CGRP family of peptides and their recep-
tors in different CNS regions and their contribution to
migraine attacks.
Acknowledgements
The financial support by Lundbeck Foundation (Denmark)
and the Swedish Research council grant no VR5958 (Sweden) are
acknowledged.
Disclosure Statement
The authors declare that they have no conflicts of interest to
disclose.
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