Download Sensory stimulation (acupuncture) increases the release

Neuropeptides (1999) 33 (3), 244–250
© 1999 Harcourt Publishers Ltd
Sensory stimulation (acupuncture)
increases the release of calcitonin
gene-related peptide in the saliva of
xerostomia sufferers
I. Dawidson,1 B. Angmar-Månsson,1 M. Blom,1 E. Theodorsson,2 T. Lundeberg,3
1
Department of Cariology, Karolinska Institutet, Box 4064, 141 04 Huddinge, Sweden
Clinical Chemistry, University Hospital, Linköping, Sweden
3
Departments of Physiology and Pharmacology, and Surgery and Rehabilitation, Karolinska Institutet, 104 01 Stockholm, Sweden
2
Summary Over the last decade, several patients afflicted with xerostomia have been treated with acupuncture. Their
salivary flow rates increased significantly and the improvement lasted during a long observation period. We also found
that the release of several neuropeptides in the saliva of healthy subjects can be increased by acupuncture stimulation.
The concentration of vasoactive intestinal polypeptide increased significantly in the saliva of xerostomic patients after
acupuncture treatment. The release of the neuropeptide calcitonin gene-related peptide (CGRP) was investigated in
the saliva of xerostomic patients in order to elucidate further the mechanisms of the effect of sensory stimulation
(acupuncture) on the salivary secretion. CGRP-like immunoreactivity was measured with radioimmunoassay (RIA)
before and after a double series of acupuncture treatment, in stimulated saliva of 14 patients who suffered from
xerostomia. The results showed that the concentration of CGRP increased significantly (P<0.001) in the saliva of these
patients after the end of acupuncture treatment as compared to base-line levels. Taking into consideration the influence
of CGRP on the salivary flow, as well as its trophic effect, we concluded that the increased release of CGRP could be
one of the factors that affect positively the salivary flow rates of xerostomic patients who were treated with acupuncture.
INTRODUCTION
Severe xerostomia (dry mouth) is a well known symptom
of some systemic diseases, e.g. primary and secondary
Sjögren’s Syndrome, as well as an unwanted side-effect of
radiation treatment or medication. It causes many problems for the affected patient-difficulties in speaking, eating
and swallowing, rampant caries and infections of the oral
mucosa, inability to wear dentures, altered sense of taste,
and can be both physically and socially handicapping.1–5
Different kinds of techniques have been developed in
order to deal with xerostomia; stimulation of salivary
secretion by chewing gums or tablets, and saliva
substitutes ranging between plain water and ex tempore
Received 14 December 1998
Accepted 20 May 1999
Correspondence to: Irena Dawidson, Department of Cariology, Department
of Odontology, Karolinska Institutet, Box 4064, S-141 04 Huddinge, Sweden.
Tel: +46 8 728 8179; Fax: +46 746 7081, E-mail: [email protected]
concoctions that try to mimic natural saliva.2,3,6–9
Medications, such as pilocarpine and nicotinamide are
sometimes used.10–12 Battery operated devices applied to
the palate and tongue have also been tried.13,14 All these
methods have one thing in common: their alleviating
effect lasts only for a short period of time.2,3,6–14
Over the last decade we have treated patients with
xerostomia of different aetiologies with acupuncture. The
results showed that their salivary flow rates can be
increased with this method and that the improvement
often lasts for long periods; a year or longer.15–17
Acupuncture is a method for treating diseases and
their symptoms that has been used in China and other
eastern countries for several thousand years. Its use
spread to the western world in the 17th century and has
gained more and more approval as the mechanisms of its
function have been clarified by the modern science. The
influence of acupuncture on the nervous system has
been investigated in both animal and human studies. It
has been found that acupuncture stimulation affects the
244
Calcitonin gene-related peptide 245
Table 1 The patients involved in this study
Patient/Gender
Age
Time of xerostomia
Aetiology
1. female
2. male
3. female
4. female
5. male
6. female
7. male
8. female
9. female
10. male
11. female
12. male
13. male
14. female
57
71
38
63
55
54
45
54
57
37
63
54
65
66
2 years
10 years
10 years
10 years
1.5 years
8 years
4 months
9 months
13 months
17 months
8 months
13 months
2.5 years
2 years
Primary Sjögren’s Syndrome
Primary Sjögren’s Syndrome
Unknown
Primary Sjögren’s Syndrome
SLE
Primary Sjögren’s Syndrome
Radiation treatment
Radiation treatment
Radiation treatment
Radiation treatment
Radiation treatment
Radiation treatment
Radiation treatment
Radiation treatment
different parts of the nervous system; both the sensory
and motor system of the central nervous system (CNS),
as well as the autonomic nervous system (ANS).18–25
Several studies have been conducted on the effect of
acupuncture in the treatment of ischemic conditions,
and it has been shown that acupuncture stimulation
exerts its effect by increasing the release of neuropeptides from nerve endings.26–31 It has also been shown that
acupuncture can mimic muscle exercise.32,33
One of the neuropeptides that were affected by sensory stimulation was calcitonin gene-related peptide
(CGRP). This peptide consists of 37 amino acids, and it
wields a wide range of effects on gastrointestinal,
endocrine, cardiovascular and broncho-tracheal systems,
as well as on CNS and ANS. CGRP is involved in neurogenic inflammatory reactions and modulates response to
substance P (SP) in neurogenic inflammation; it causes
endogenous vasodilatation and acts by releasing an
endothelium derived relaxing factor (EDRF), but not
cyclic GMP. CGRP does not cause extravasation but
strongly potentiates the increase in extravasation produced by SP and potentiates the oedema induced by SP.
It also has a relaxant effect on vascular smooth muscle
and can increase the blood flow in the skin for several
hours. CGRP is a potent vasodilator, induces protracted
increase in microvascular blood flow, and potentiates the
increase in capillary permeability produced by platelet
activation factors. CGRP co-exists with SP in many primary afferents and monitors noxious events together
with SP. It plays a part in pain perception; potentiates the
hyperalgesia caused by SP and conveys nociceptive information to the first synapse in the spinal cord, where pain
is processed.26,34–36 CGRP induces a delayed (1–2 min)
increase in salivary secretion,35 and its concentration in
the saliva can be affected by pathology.37
© 1999 Harcourt Publishers Ltd
The salivary secretion is controlled by the ANS where
the transmitter substances are acetylcholine (Ach) and
noradrenaline (NA). The parasympathetic system is dominant and it induces a powerful increase of the salivary
secretion rates with low protein content. It causes contraction of myoepitelial cells and vasodilatation of capillaries
in the salivary glands, which leads to increased blood flow.
The sympathetic system has a more intermittent role;
when activated it causes a low, viscous salivary flow.38–43
The salivary secretion is also affected by other transmitters
apart from Ach and NA, neuropeptides, which can regulate the volume of the saliva produced by increasing the
blood flow in the salivary glands and affecting the secretion itself. They act beside or in co-operation with the classic neurotransmitters Ach and NA.44–49
In order to elucidate the mechanisms behind the effect
of acupuncture on salivary secretion in xerostomia sufferers, we conducted several studies concerning the connection between salivary secretion, neuropeptides and
acupuncture. We found that acupuncture stimulation
increases the blood flux in the skin over parotid gland in
patients with Sjögren’s syndrome.50
The neuropeptide concentration in saliva varies with
secretion rates51 and we have shown that the concentration of some neuropeptides can be increased in the saliva
of healthy subjects with acupuncture stimulation.52
In a recent study, we found that the concentration of
vasoactive intestinal polypeptide (VIP) increased after
the acupuncture treatment as compared with base line
levels, in the saliva of xerostomic patients.53 In this study,
we investigated the influence of acupuncture stimulation
on the concentration of CGRP in the saliva of xerostomia
suffers. We hypothesized that acupuncture stimulation
could increase the release of CGRP from the nerve endings of the autonomic nervous system by the way of
Neuropeptides (1999) 33(3), 244–250
246 Dawidson et al.
Table 2
The most commonly used acupuncture points
Skin
Innervation
Muscle
Innervation
m. levator labii
sup.
n. trigeminus,
r. ophtalmicus,
n. infraorbitale
n. facialis
m. masseter
n. trigeminus,
r. mandibularis
n. facialis,
r. mandibularis,
n. trigeminus
m. interosseus
dors. m. abduct.
pollicis,
m. lumbricale I
n. radialis,
r. superficialis
(C 6, 7, 8)
n. ulnaris
(C 8, Th 1),
n. medianus
(C 8, Th 1)
m. tibialis ant.
n. cutaneus lat.
(L 5, S 1,2)
n. peroneus
m. flexor
digitorium long.
m. tibialis post.
n. sapheneus
(L 3,4)
n. tibialis
(L 4, S 2, 3)
AP-Point
Location
Tissues
St 3
Juliao
When the eyes look straight
forwards, it is inferior to the
pupilla at the level of the lower
border of the alae nasi
One finger width anterior and
superior to the angle of lower
jaw, at the prominence of the m.
masseter during mastication
On the middle point of os
metacarpale II, on the
prominence of the 1st m. inter
ossei dorsales slightly towards
the side of the index.
10 cm below apex patellae, one
finger width lateral to the crista
anterior tibiae
10 cm above the highest point of
the malleolus medialis at the
posterior border of the tibia
St 6
Jiache
Li 4
Hegu
St 36
Zusanli
Sp 6
Sanyinjiao
excitation of sensory efferent nerve fibres, thus leading
to an increase of salivary flow.
MATERIALS AND METHODS
Patients
Out of 65 patients that were treated with acupuncture for
xerostomia, 17 were chosen to participate in this study
due to their ability to produce enough stimulated saliva
for the radioimmunoassay (RIA) analyses prior to the
start of the study. We were able to conduct RIA analyses
of CGRP on the saliva of 14 of these patients, six men
and eight women, aged 37–71, median age 56 years. The
periods of time that they have suffered from xerostomia
differed from a few months (after radiation treatment) to
several years for the SS patients. Patient data are shown
in Table 1. The experimental design was approved by the
ethics committee at the Huddinge Hospital.
Saliva collection
Using the standardized salivary flow test procedures
used at the department of Cariology,54 we collected paraffin-chewing stimulated saliva from the patients before
the beginning of the study and then after the acupuncture treatment was completed. The patients were
informed about the study in writing and were asked to
refrain from eating, drinking and smoking for at least 1 h
prior to each experiment. Each individual participating in
the study was tested at approximately the same time of
the day considering the differences in saliva production
during the day. The collected saliva was weighed in order
to obtain precise measurements (1 g was considered to
Neuropeptides (1999) 33(3), 244–250
respond to 1 ml). The saliva samples were collected in
small test tubes, containing 1 ml 1 M acetic acid in order
to neutralize the enzymes that would otherwise destroy
the neuropeptides. The samples were kept in ice during
the experiment and were frozen to –70°C immediately
after the end of each session, until the radioimmunoassay analyses (RIA) could be carried out at a later date.
Acupuncture treatment
The acupuncture treatment was performed by an experienced acupuncturist, and the same points were used as
in earlier studies when xerostomia was treated with sensory stimulation.15–17 The following points were used
bilaterally St3, St6 in the face, Li4 in the hands and St36
and Sp6 in the legs. They are described in detail Table 2.55
Disposable, Chinese needles were used, made of stainless
steel, Hwato 0.32 × 40 mm, and Cloud & Dragon, 0.30 ×
15, 30 and 40 mm. After standard disinfecting of the site,
the needles were inserted through the skin to the depth
of 5–10 mm, and manipulated until the needle sensation
(DeQi) was reached. DeQi is described as a feeling of
heaviness and ache, sometimes combined with the sensation of a current originating from the point where the
tip of the needle is placed.56 When that sensation was
achieved, the needles were left in situ, and not manipulated again unless a needle was displaced. All patients
received two series of acupuncture treatments – 24 sessions of 20 min each.
Peptide analyses
Samples from the patients’ saliva were extracted using a
reverse-phase C18 cartridge (Sep Pak, Waters) and
© 1999 Harcourt Publishers Ltd
Calcitonin gene-related peptide 247
Fig. 1 The concentration of CGRP in the saliva of xerostomic patients before and after acupuncture treatment (AP).
analysed using competitive radioimmunoassay (RIA).
Calcitonin gene-related peptide (CGRP-LI) was analysed
using antiserum CGRPR8 raised against conjugated rat
CGRP. HPLC-purified125I-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 human CGRP alpha and beta
was 93 and 24%, respectively, and toward rat CGRP
alpha and beta 100 and 120%, respectively. Intra- and
interassay coefficients of variation were 8 and 14%,
respectively.57,58 The lower detection limit in all extracted
samples was 0.1 fmol/mL for all peptide assessments.59
Statistical analyses
Wilcoxon’s test (paired two samples) was used in order to
compare the concentrations of CGRP in the saliva before
the beginning of the study and after the acupuncture
treatment was completed. A probability value of P<0.05
was considered significant.
RESULTS
The concentrations of CGRP in the saliva of the patients
in this study were significantly higher after the end of
acupuncture treatment as compared to the base line levels, before the AP was started (P<0.001). The results are
visualized in Figure 1. HPLC showed a distinct
immunoreactive component with regard to CGRP, as
reflected by a main peak eluting in the position of the
corresponding synthetic peptide. No evidence was found
to indicate presence of multiple immunoreactive components (Fig. 2).
© 1999 Harcourt Publishers Ltd
Fig. 2 Reverse-phase HPLC of calcitonin gene-related peptide
immunoreactivity (CGRP-LI) in extracts from human saliva. An
arrow indicates the elution profile of CGRP. The CGRP-LI had its
mark peak eluting in the position of the corresponding synthetic
peptide.
DISCUSSION
In earlier studies, we found that acupuncture treatment
can increase the salivary secretion in patients who suffer
from xerostomia. The results of those studies showed
that the improved salivary flow rates persist during a
long period – a year or longer.15–17 In this study, we investigated the influence of sensory stimulation on the
release of CGRP in the saliva of xerostomic patients. We
found that the concentration of CGRP was significantly
increased in the stimulated saliva of those patients after
the end of acupuncture treatment as compared to the
levels before the start of the study. The salivary secretion
is controlled by the ANS, in complicated co-operation
with the sensory afferents and CNS. The afferent
impulses from the masticatory system activate the
Neuropeptides (1999) 33(3), 244–250
248 Dawidson et al.
parasympathetic and sympathetic systems of ANS, thus
inducing an increase in the salivary flow. The salivary
neurons in the lateral reticular formation of the lower
brain stem are activated by the stimulation of group II
and group II afferent nerve fibres of n. trigeminus.60
CGRP, one of the neurotransmitters found in the
nerve fibres of both the sensory and autonomic nervous
system, beside Ach and NA, is involved in salivary secretion.35,43,44,61–64 We have shown in earlier studies that the
concentration of CGRP in the saliva of healthy human
subjects vary with the stimulation of salivary flow
rates, and can be increased significantly by acupuncture
stimulation.51,52
We found in this study that the concentration of CGRP
can also be increased significantly by acupuncture stimulation in the saliva of xerostomic patients. We speculated
therefore that the mechanism behind this effect might be
the excitation of the afferent fibres by sensory stimulation. Upon stimulation the afferents release significant
amounts of CGRP locally. There is also a release of CGRP
from the motor efferents of the masticatory system,
which are activated by the afferent input.
This increased release of the neuropeptide CGRP could
be one of the factors that contribute to the salivary secretion. We have suggested in the preceding study that VIP
can act as a trophic factor on the salivary gland tissues.53
It is possible that CGRP has a similar function apart from
its influence on the salivary secretion rates. According to
Dalsgaard et al., CGRP has trophic influence on the
endothelial cells,65 and might positively affect the microcirculation in the capillary vessels. The salivary glands
contain considerable amounts of neurotrophins, which
exert profound trophic effect on the neurons of the ANS
and the sensory nervous system.
Acupuncture stimulation may produce a release of the
neurotrophins which affect the sensory and autonomic
NS by the way of inducing an increase in the production
of CGRP in the nerve cells and subsequent release of that
CGRP in target organs, in this case the salivary glands.
Such course might be the explanation of the long-term
effect of acupuncture treatment.66–71
We concluded that the increase of the release of CGRP
could be one of the factors that contribute to the
improvement of the salivary secretion rates in xerostomic
patients who were treated with acupuncture. The mechanisms behind the effect of the sensory stimulation might
be the activation of the nerve fibres of the systems
involved, namely sensory afferents as well as the motor
and efferent neurons, leading to an increased release of
the neuropeptide CGRP, which could have a dual effect.
A direct influence on the salivary flow rates, as well as a
long-term influence on the condition of the salivary
gland tissues through its trophic influence on the
endothelial cells of the blood vessels, resulting in
Neuropeptides (1999) 33(3), 244–250
improvement of the blood supply of the salivary
glands.
ACKNOWLEDGEMENT
The Swedish Dental Society, the Swedish Patent Revenue
Research Fund, the Gustav Vth 80-year Anniversary
Foundation, the Professor Nanna Schvartz Foundation
and the Swedish Society against Rheumatism (RMR)
supported this study.
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