Acta Physiol Scand 1998, 162, 517±522
Effects of physical activity and acupuncture on calcitonin
gene-related peptide immunoreactivity in different parts of
the rat brain and in cerebrospinal ¯uid, serum and urine
Y . W Y O N , 1 M . H A M M A R , 1 E . T H E O D O R S S O N 2 and T . L U N D E B E R G 3
1 Department of Health and Environment; Obstetrics and Gynaecology, University Hospital, Link
oping, Sweden
2 Department of Clinical Chemistry, Faculty of Health Sciences, University Hospital, Linkoping, Sweden
3 Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
ABSTRACT
Calcitonin gene-related peptide (CGRP) is a very potent vasodilator in the nervous system, and maybe
involved in hot flushes experienced by most women around menopause. Flushing post-menopausal
women had higher urinary excretion of CGRP before than after successful treatment of their flushes
with acupuncture.The prevalence of vasomotor symptoms is lower in physically active women. In a
rat model we therefore intended to assess whether acupuncture and exercise affected CGRP
concentrations in different parts of the brain and peripherally. The aim of the study was to elucidate
the short- and long-term effects of exercise and acupuncture on CGRP concentrations in the nervous
system of normal adult rats. In a rat model, we examined the effects of single interventions and longterm treatment with physical exercise and manual or electro-acupuncture on CGRP concentrations in
urine, cerebrospinal fluid and serum and different parts of the brain. In all compartments studied, but
significantly only in the cerebrospinal fluid, CGRP increased after a single session of physical exercise
or electro-acupuncture. Manual acupuncture did not change CGRP concentrations in any
compartment. Rats had the highest concentrations of CGRP in the pituitary and hypothalamus but the
concentrations did not differ significantly between control rats and those subjected to long-term
treatment with manual or electro-acupuncture or running rats. Rats treated with electro-acupuncture
had twice the CGRP concentration in the frontal cortex compared to control rats, albeit the difference
did not reach statistical significance. Evidently manual and electro-acupuncture have different effects,
whereas electro-acupuncture and physical exercise have more similar effects on CGRP production
and/or release. To elucidate the role of CGRP in vasomotor symptoms, further studies with older
flushing rats should be performed.
Key words acupuncture, calcitonin gene-related peptide, nervous system, physical activity, rat
Received 26 March 1997, accepted 22 October 1997
Around menopause most women suffer from vasomotor
symptoms (Hammar et al. 1984, Berg et al. 1988). During
¯ushes pulse rate, cutaneous temperature and serum LH
increase and central temperature decreases (Meldrum
et al. 1981, Tataryn et al. 1981, Rebar & Spitzer 1987,
Freedman & Woodward 1992). These phenomena could
be elicited by a sudden resetting of the thermoregulatory
centre (Meldrum et al. 1981). Vasomotor symptoms
correlate in time with the decreased ovarian production
of oestrogen, although neither low oestrogens nor elevated LH is the direct cause of the ¯ushes (Sturdee &
Brincat 1988). Both the potent vasodilator calcitonin
gene-related peptide (CGRP) and hypothalamic opioids
have been suggested to be involved in these symptoms
(cf. Shoupe & Lobo 1987, Valentini et al. 1996).
Hypothalamic b-endorphins affect LH pulsatility
(Grossman 1987) and thermoregulation (Widdowson
et al. 1983, Spencer et al. 1990). Intracerebral injection
of opioids increased the thermoregulatory set point
(Widdowson et al. 1983, Spencer et al. 1990). Sex steroids seem to stimulate the production of b-endorphin
in the hypothalamus (Cumming & Wheeler 1987,
Shoupe & Lobo 1987). b-endorphins were lower in
cerebrospinal ¯uid from post-menopausal women than
Correspondence: Yvonne Wyon, Department of Health and Environment; Obstetrics and Gynaecology, Faculty of Health Sciences, University
Hospital of LinkoÈping, S-581 85 LinkoÈping, Sweden.
Ó 1998 Scandinavian Physiological Society
517
Calcitonin gene-related peptide in the rat brain
Y Wyon et al.
in women of fertile age (Nappi et al. 1990), and hypothalamic b-endorphin activity increased in post-menopausal women given oestrogens (Melis et al. 1984).
Thus, the low central b-endorphin activity after
menopause may explain elevated levels of LH and
lability in thermoregulation. Oestrogen treatment increases central opioid activity (Shoupe & Lobo 1987),
which may cause the negative feedback effect of oestrogens on LH, and the decrease in vasomotor symptoms. Physical activity and acupuncture also increase
the production and release of b-endorphin (Cumming
& Wheeler 1987, Hoffman et al. 1990) and have been
associated with lower prevalence of vasomotor symptoms (Hammar et al. 1990, Wyon et al. 1995).
CGRP consists of 37 amino acids (Amara et al. 1982),
and is a very potent vasodilator in the skin and coronary
arteries (Brain et al. 1986, McEwan et al. 1986). Human
CGRP, which differs by four amino acids from the rat
CGRP, was characterized from the human calcitoningene by Morris et al. (1984). Using immunohistochemical techniques CGRP has been shown to be distributed
in the central as well as the peripheral nervous system
(Rosenfeld et al. 1983, Gibbins et al. 1985, Lundberg et al.
1985). CGRP produced a dose-dependent increase in
cutaneous blood ¯ow for up to 90 min, when given i.v.
to healthy male volunteers (Jernbeck et al. 1990). Infusion of CGRP also induced a ¯ush in the face, neck, arm
and upper trunk; i.e. similar to the localization of postmenopausal ¯ushes (Jernbeck et al. 1990). Except at high
doses the local vasodilation induced by CGRP was not
associated with a wheal and ¯are as seen with histamine,
substance P and VIP (Brain et al. 1986).
In post-menopausal women with vasomotor symptoms 8 weeks of electro-acupuncture treatment reduced
their ¯ushes and urinary excretion of CGRP by about
50% (Wyon et al. 1995). Electrostimulated acupuncture
is thought to induce some of its effects through increased production of hypothalamic b-endorphins.
Central opioids regulate the excitability of sensory
neurones at the spinal cord level. Collin et al. (1993)
have reported that opioids inhibit the release of CGRP
at the spinal cord level, which in turn may affect
vasomotion and vasomotor symptoms.
As CGRP administered i.v. induced ¯ushes similar to
post-menopausal vasomotor symptoms, CGRP might
be involved in the mechanisms causing such symptoms.
Also CGRP excreted in 24-h urine was decreased in
women relieved from their ¯ushes during acupuncture
treatment. Furthermore, serum concentrations of
CGRP were higher during, than between ¯ushes in
post-menopausal women (Chen et al. 1993, Valentini
et al. 1996). Such observations make this peptide interesting in studies of the pathophysiology behind postmenopausal vasomotor symptoms. Some data are still
con¯icting, however, like serum concentrations of
518
Acta Physiol Scand 1998, 162, 517±522
CGRP being higher in control women of fertile ages
than among post-menopausal ¯ushing women (Valentini et al. 1996). Furthermore, we do not know whether
treatments that may affect CGRP in¯uence CGRP in
the central or peripheral nervous system.
In order to study mechanisms behind the effects of
exercise and various modes of acupuncture used for
climacteric complaints on the nervous system we used a
rat model. The aim of the present study was to establish
the effects of these treatments on CGRP concentrations in the normal adult rat brain and in urine, serum
and cerebrospinal ¯uid.
M A T E R I AL S AN D M E T HO D S
In all 83 female Sprague Dawley rats (ALAB Sollentuna, Sweden) 200±220 g were used. Six rats were
housed per cage at 21 °C with water and food ad libitum
and a 12-h light/dark cycle separated from all other
animals until groups of animals of certain ages were
obtained.
Two groups of 10 rats each received sensory stimulation (manual acupuncture or electro-acupuncture)
for 30 min once only while another two groups were
treated with manual (eight rats) or electro-acupuncture
(10 rats) for 30 min twice weekly for 5 weeks.
Points chosen bilaterally for stimulation treatment
were BL11 (close to the shoulder point) and BL54
(close to the hip joint). Stimulation was initiated by
manual rotation of the needles (0.3 mm) after insertion
to depths of 0.5±0.7 cm at the points chosen and rotations were repeated for 10 s every 5 min.
Electrical stimulation was performed through all four
needles (BL11-BL11, BL54-BL54) connected in pairs to
an acupuncture pulse stimulator (B.V. Enraf-Nonius,
Delft, the Netherlands) producing bipolar square wave
pulses of 0.2-ms duration and 2-Hz frequency. The
current intensity was adjusted so that localized muscle
contractions were seen (Lundeberg et al. 1988).
During treatment the rats were anaesthetized with
chloral hydrate 0.36 g kg body wt given intraperitoneally.
Two groups of rats performed physical exercise
running for 1 h, one group once only (10 rats), the
other group for 1 h day)1 for 5 weeks (eight rats) before being killed. A group of eight rats that were denied
exercise served as controls.
For the running exercise the rats were placed in a
wheel (diameter 22 cm and width 9 cm) with a slight
resistance to rotation. Two groups of rats used as
controls without acupuncture or exercise were anaesthetized once (10 rats) or twice weekly for 5 weeks
(nine rats) in accordance with the schedule used for
repeated acupuncture treatments mentioned above.
The rats were killed immediately following the last
treatment by focused microwave irradiation (MWI)
Ó 1998 Scandinavian Physiological Society
Acta Physiol Scand 1998, 162, 517±522
during 2 s, using a microwave system (Metabostat,
Gerling Moore, CA, maximal power 5 KW, 2450 MHz)
specially built for this purpose. This method has been
found to allow a higher neuropeptide recovery than
decapitation (Bucinskaite et al. 1994). The brains were
quickly removed, dissected on dry ice into frontal
cortex, striatum, occipital cortex, hippocampus, pituitary and hypothalamus (Glowinsky & Iversen 1966),
weighed and stored at )80 °C until extraction.
The samples were cut into small pieces in the frozen
state, boiled for 10 min in 1 M acetic acid and then
homogenized. After centrifugation 1000 ´ g for 10
min, the supernatants were lyophilized and stored at
)40 °C before analysis.
Urine, serum and cerebrospinal ¯uid were collected
from the rats that had been treated only once with
acupuncture or had been made to run for 1 h and
analysed for CGRP. Urine, serum and cerebrospinal
¯uid collected were rapidly cooled and stored at
)80 °C until used.
Analysis of CGRP
Samples were extracted using a reverse-phase C18 cartridge (Sep Pak, Waters) and analysed for CGRP-like
immunoreactivity (CGRP-LI) using competitive radioimmunoassays (Theodorsson-Nordheim et al. 1987).
CGRP-like immunoreactivity (CGRP-LI) was analysed
using antiserum CGRPR8 raised against conjugated rat
CGRP. HPLC-puri®ed 125I-histidyl rat CGRP was used
as radioligand and rat CGRP as standard. The crossreactivity of the assay to SP, neurokinin A, neurokinin B,
neuropeptide K, gastrin, neurotensin, bombesin, neuropeptide Y and calcitonin was less than 0.01%. Crossreactivity towards islet amyloid polypeptide and adrenomedullin was below 0.1%. Cross-reactivity towards
human CGRP a and b was 93 and 24%, respectively,
and towards rat CGRP a and b 100 and 120%, respectively. Evidently the antiserum reacted more actively with rat CGRP b than with CGRP a, the peptide
which it was immunized against. Intra and interassay
coef®cients of variation were 8 and 14%, respectively.
Results are given as pmol L)1 urine, plasma or cerebrospinal ¯uid and as fmol g)1 tissue wet wt brain tissue.
Ethics. The study was approved by the local Ethical
Committee of the Karolinska Institutet.
Statistical methods. Comparisons between two groups
were performed with the Mann±Whitney U test and
student's t-test using MacIntosh Statview 4.1; Abacus
Concepts Inc. Multivariate analyses of variance and
multiple comparisons were performed using the
MGML module of SYSTAT for Windows version 5.02
(Systat, Inc. Evanston, Illinois). Multivariate ANOVA
was used for statistical analyses as the study involved
Ó 1998 Scandinavian Physiological Society
Y Wyon et al.
Calcitonin gene-related peptide in the rat brain
measurements of CGRP concentrations in samples
from three different body ¯uids using four different
treatments. Turkey's test was used for multiple comparisons and the exact P values are shown in the ®gures. P values less than 0.05 were considered signi®cant.
R E S UL TS
In the control rats that did not run, the highest concentrations of CGRP were found in the hypothalamus and
pituitary with signi®cantly lower concentrations in hippocampus, striatum and in the occipital and frontal
cortex (Table 1, Fig. 1). These relationships were the
same in the rats that had been anaesthetized (Table 1,
Fig. 2). The only cerebral region having a different
CGRP concentration in anaesthetized rats in comparison
with alert rats was the frontal cortex (Table 1), where the
CGRP concentration was higher in those anaesthetized.
Rats treated with electro-acupuncture had twice the
CGRP concentrations in the frontal cortex compared
to the controls, which was, however, non-signi®cant
with multivariate analysis (Table 1). In all other areas of
the brain CGRP concentrations were the same in
treated rats and controls (Table 1).
CGRP concentrations were about ®ve times higher
in cerebrospinal ¯uid than in serum or urine (Fig. 3).
After a single session of exercise or electro-acupuncture
the CGRP concentrations were signi®cantly increased
in cerebrospinal ¯uid, but not in urine or plasma. No
difference was observed in any compartment between
control rats and those that had been treated with
manual acupuncture (Fig. 3).
D I S C US S I O N
CGRP is a vasoactive peptide which was found to decrease in 24-h urine in post-menopausal women successfully treated with acupuncture for vasomotor
symptoms (Wyon et al. 1995). Serum concentrations of
CGRP were also found to be higher in serum during
¯ushes than in between (Chen et al. 1993, Valentini et al.
1996). Intravenous administration of CGRP to healthy
male volunteers made them ¯ush (Jernbeck et al. 1990).
The peptide increases noradrenergic sympathetic out¯ow (Fisher et al. 1983) leading to an increase in heart rate
and blood pressure, symptoms which are also associated
with the ¯ushing in menopausal women. Dennis et al.
(1990) found that CGRP increases rectal temperature,
suggesting a direct role in thermoregulation.
We have examined the effects of single sessions of
physical exercise, manual and electrostimulated acupuncture on the CGRP concentration in urine, serum
and cerebrospinal ¯uid in a rat model. In the cerebrospinal ¯uid, CGRP increased after a single session of
physical exercise and after electro-acupuncture, whereas
519
Calcitonin gene-related peptide in the rat brain
Y Wyon et al.
Acta Physiol Scand 1998, 162, 517±522
Table 1 CGRP in different parts of the CNS after different treatments
Control 1
n
FC
Mean
Median
OC
Mean
Median
ST
Mean
Median
HP
Mean
Median
P
Mean
Median
HY
Mean
Median
Manu-AP
El-AP
10
Running
Control 2
9
8
8
10
3.2 (0.7)
3 (3±4)
2.5 (0.5)
2.5 (2.0±3.0)
6.5 (4.4)
4.5 (3.0±12.0)
2.4 (2.2)
1.3 (0.7±4.3)
0.9 (1.1)
0.4 (0.2±1.6)
3.7 (1.4)
4.0 (2.8±5)
3.6 (0.5)
4.0 (3.0±4.0)
4.2 (0.4)
4.0 (4.0±4.0)
2.9 (0.6)
3.0 (2.5±3.0)
3.1 (1.0)
3.0 (2.5±3.5)
5.3 (3.6)
4.0 (3±7.8)
8.0 (4.8)
7.0 (4.5±10.5)
8.4 (3.5)
9.5 (6.0±11.0)
3.4 (1.5)
3.0 (2.0±4.5)
4.1 (1.0)
4.5 (3.0±5.0)
5.1 (1.9)
5.0 (3.75±6.5)
8.0 (4.3)
6.0 (5.0±11.5)
6.1 (2.3)
6.0 (5.0±8.0)
7.8 (2.6)
7.5 (6.0±9.5)
7.9 (3.8)
7.5 (5.0±10.5)
52.9 (19.4)
43 (38.8±70.8)
49.1 (22.7)
52.5 (36.0±61.0)
58.4 (23.0)
53.5 (39.0±74.0)
35.0 (5.6)
35.0 (31.5±39.0)
45.3 (19.7)
39.5 (34.5±54.0)
22.6 (5.6)
23 (19.5±24.8)
18.1 (4.0)
19.5 (14.5±21.0)
19.9 (4.0)
21.0 (16.0±22.0)
20.1 (4.6)
19.5 (16.5±23.0)
23.9 (10.6)
21.5 (16.0±30.5)
Treatments with repeated manual acupuncture (Manu-AP), electrostimulated acupuncture (El-AP) or physical exercise (running), (9±10 animals/group).
Control 1 were anaesthetized several times according to the same routines as the rats that were treated with Manual acupuncture and Electro-acupuncture.
Control 2 were awake but not running and values should be related to those in the group that had been running. Results in mean (‹ SD) and median
(+ lower to upper 25%). FC = Frontal cortex, OC = Occipital cortex, ST = Striatum, HP = Hippocampus, P = Pituitary, HY = Hypothalamus.
Figure 2 CGRP as fmol g
Figure 1 CGRP as fmol g
)1
in different parts of the CNS in eight
rats that had not been anaesthetized. Frontal c ˆ frontal cortex,
occipital c ˆ occipital cortex. Pituitary and hypothalamic
concentrations are signi®cantly different (P < 0.01) compared to all
other areas according to multivariate analyses of variance and multiple
comparisons. Boxes include 25th±75th percentiles with median
shown as a line. Whiskers show 10th and 90th percentiles.
manual acupuncture did not change the CGRP concentration in any compartment. Evidently manual and
electrostimulated acupuncture have different effects on
the CGRP concentration in the rat nervous system,
whereas electro-acupuncture and physical exercise have
more similar effects. Considering data saying that central b-endorphins regulate CGRP production (Collin
et al. 1993), this is in line with observations that electroacupuncture induces more profound relief of chronic
pain than manual acupuncture (Haker & Lundeberg
520
)1
in different parts of the CNS in eight
rats that were repeatedly anaesthetized. Frontal c ˆ frontal cortex,
occipital c ˆ occipital cortex. Pituitary and hypothalamic
concentrations are signi®cantly different (P < 0.01) compared to all
other areas according to multivariate analyses of variance and multiple
comparisons. Boxes include 25th±75th percentiles with median
shown as a line. Whiskers show 10th and 90th percentiles.
1990). Single treatments did not signi®cantly affect
peripheral CGRP release as measured in serum and
urine. The increase in cerebrospinal ¯uid CGRP concentrations after single therapy may be related to both
dorsal root ganglia and more central production. On
the other hand, the observation that CGRP tissue
concentrations did not change in any part of the brain
after chronic treatment, suggests that the increase in
cerebrospinal ¯uid CGRP mainly stems from dorsal
root ganglia. CGRP in peripheral compartments seems
mainly to stem from perivascular nerve endings, but
Ó 1998 Scandinavian Physiological Society
Acta Physiol Scand 1998, 162, 517±522
Y Wyon et al.
Calcitonin gene-related peptide in the rat brain
)1
Figure 3 Concentration of CGRP (pmol L ) in 24-h urine, plasma and cerebrospinal ¯uid (CSF) in non-treated control rats, and rats treated
with manual or electrostimulated acupuncture or running, respectively. Boxes include 25th±75th percentiles with median shown as a line.
Whiskers show 10th and 90th percentiles. Multivariate analyses showed that CGRP concentrations were higher in CSF than in urine and serum
in controls and all treatment groups (P < 0.001) and that CGRP concentrations in CSF were signi®cantly higher (P < 0.001) after electrostimulated acupuncture and running but not after manual acupuncture compared to controls. No therapy changed CGRP concentrations in
urine and serum.
may also come from other sources, e.g. the thyroid
gland. The fact that CGRP content changed most evidently in the cerebrospinal ¯uid but in parallel with
slight (but insigni®cant) changes in serum and urine
supports the theory that the increment was mainly
because of changes in the nervous system.
In the non-running control rats, the highest tissue
concentrations of CGRP were found in the hypothalamus and pituitary, with signi®cantly lower concentrations in the hippocampus, striatum and in the occipital
and frontal cortex. Neither chronic treatment with
acupuncture nor repeated exercise affected the CGRP
concentration in different parts of the CNS.
Electro-acupuncture over a period of 8 weeks decreased the urinary CGRP excretion in post-menopausal women with vasomotor symptoms. This was in
accordance with the observations that vasomotor
symptoms decreased among these women during
treatment and it could be speculated that CGRP at least
in part was responsible for the vasodilatation, the
subjective feeling of heat and increase in cutaneous
temperature during the ¯ushes.
The changes in CGRP concentration in cerebrospinal
¯uid induced by single events of electro-acupuncture and
by exercise in normal adult rats were actually in the opposite direction in the present study, than expected from
human data. We have clearly demonstrated, however,
that both electro-acupuncture and physical activity affect
CGRP turnover. It should be emphasized that these
measurements were performed in normal rats that do
not spontaneously ¯ush (Simpkins 1984), which could
account for the differences in the direction of changes
between the post-menopausal women and the rat model.
Furthermore, the measurements were performed after
Ó 1998 Scandinavian Physiological Society
single treatments in the rats and after many weeks of
therapy in the women. Unfortunately we did not perform
any measurements in serum, urine and CSF after chronic
treatment, whereas tissue measurements showed no or
minor changes in different parts of the brain.
These rats were normal adult females, which do not
¯ush spontaneously. Rats addicted to and abstinent
from opioids as well as older female rats have previously been found to have peripheral temperature ¯uctuations, whereas oophorectomized rats do not
(Simpkins 1984). A ¯ushing rat model should be
studied concerning effects of physical activity as well as
electro-acupuncture to permit deeper insight into the
role of CGRP in vasomotor instability and possible
effects of different treatment regimens.
In conclusion CGRP content in the cerebrospinal
¯uid was signi®cantly altered by physical activity and
electro-acupuncture in normal, adult rats. To elucidate
the role for CGRP in vasomotor symptoms further
studies with older ¯ushing rats should be performed
and are underway.
We are grateful to technician Maud Hoffstedt for skilful technical
assistance and Lion's Foundation for ®nancial support.
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Ó 1998 Scandinavian Physiological Society