Neuronal release of endogenous dopamine from corpus of guinea
pig stomach
Kazuko
Shichijo1,
Yasuko
Sakurai-Yamashita2,
Ichiro
Sekine1, and
Kohtaro
Taniyama2
1 Department of Molecular
Pathology, Atomic Bomb Disease Institute, and
2 Department of Pharmacology
II, Nagasaki University School of Medicine, Nagasaki 852, Japan
 |
ABSTRACT |
Neuronal release
of endogenous dopamine was identified in mucosa-free preparations
(muscle layer including intramural plexus) from guinea pig stomach
corpus by measuring tissue dopamine content and dopamine release and by
immunohistochemical methods using a dopamine antiserum. Dopamine
content in mucosa-free preparations of guinea pig gastric corpus was
one-tenth of norepinephrine content. Electrical transmural stimulation
of mucosa-free preparations of gastric corpus increased the release of
endogenous dopamine in a frequency-dependent (3-20 Hz) manner. The
stimulated release of dopamine was prevented by either removal of
external Ca2+ or treatment with
tetrodotoxin. Dopamine-immunopositive nerve fibers surrounding choline
acetyltransferase-immunopositive ganglion cells were seen in the
myenteric plexus of whole mount preparations of gastric corpus even
after bilateral transection of the splanchnic nerve proximal to the
junction with the vagal nerve (section of nerves between the celiac
ganglion and stomach). Domperidone and sulpiride potentiated the
stimulated release of acetylcholine and reversed the dopamine-induced
inhibition of acetylcholine release from mucosa-free preparations.
These results indicate that dopamine is physiologically released from
neurons and from possible dopaminergic nerve terminals and regulates
cholinergic neuronal activity in the corpus of guinea pig stomach.
peripheral endogenous dopamine release; acetylcholine release; peripheral dopamine-immunopositive neurons; calcium-dependent dopamine
release; tetrodotoxin-sensitive dopamine release
 |
INTRODUCTION |
THERE IS EXTENSIVE evidence that dopamine plays a role
in tissues of the gastrointestinal tract, particularly in the stomach (13-15, 21, 28, 35, 36). Dopamine seems to regulate microvascular circulation in the mucosa (14, 15) and decreases gastric motility (13,
21, 28, 35, 36), and dopamine receptor antagonists stimulate gastric
motility (25, 36). However, the localization of dopamine in the stomach
is not clear.
It is well known that dopamine is a neurotransmitter in the central
nervous system. In the peripheral tissues, small intensely fluorescent
(SIF) cells in the superior cervical ganglia contain dopamine, and they
appear to function as interneurons (9). The presence of peripheral
dopaminergic neurons other than SIF cells has been proposed in some
tissues (4-7, 18, 19); however, the criteria for dopamine as a
neurotransmitter in these tissues have not been fulfilled. The gastric
tissues are innervated by a high density of catecholaminergic neurons
(10, 12), which contain considerable amounts of norepinephrine (NE) and
smaller amounts of dopamine (7, 8). It is not clear whether
dopamine exists solely as a precursor of NE or as a bioactive substance in its own right in gastric tissues. In this study, we report data to
support the idea that dopamine is released from neurons, possibly
dopaminergic neurons, in guinea pig stomach corpus and regulates
activities of cholinergic neurons through dopamine receptors.
 |
MATERIALS AND METHODS |
Measurements of dopamine and NE in the tissues.
Adult guinea-pigs of either sex, weighing 300-500 g, were
separated into two groups. The first group was used as a control group,
and the second group was treated with a bilateral transection of the
splanchnic nerve proximal to the junction with the vagal nerve (section
of nerves between the celiac ganglion and stomach) (20), 7 days before
experiments were performed. The guinea pigs were killed by cervical
dislocation, and the stomach was immediately excised. Segments of
tissue were obtained from the stomach corpus in two groups of guinea
pigs. The gastric corpus was scraped with a glass slide and separated
into the mucosal layer and the muscle layer including the intramural
plexus (mucosa-free preparation). Each preparation was homogenized with
an ultrasonic probe homogenizer in 500 µl of solution containing 0.4 N HClO4, 5.3 mM
Na2S2O5, 1.4 mM EDTA, and 3,4-dihydroxybenzylamine (50 ng/ml) as the internal standard. Homogenates were centrifuged at 15,000 revolutions per minute
(4°C, 15 min), and isolation of dopamine and NE from the supernatant was accomplished by absorption with alumina (50 mg) in 2 ml
of 0.5 M tris(hydroxymethyl)aminomethane (Tris) · HCl buffer (pH 8.6). Dopamine and NE were then eluted by 150 µl 0.1 N
HClO4. A 25-µl aliquot of the
HClO4 eluent was injected into a
high-performance liquid chromatograph with an electrochemical detector
(HPLC-ECD).
Measurements of dopamine release.
The stomach corpus was cut into strips ~5 × 15 mm, in a
circular fashion. The strips were immediately separated into a mucosal layer and a muscle layer including the intramural plexus of the gastric
corpus. The preparation of muscle layer including the intramural plexus
was mounted in an apparatus and superfused at 37°C at a flow rate
of 1 ml/min with Krebs solution of the following composition (in mM):
118 NaCl, 4.8 KCl, 2.5 CaCl2, 1.19 MgSO4, 25.0 NaHCO3, 1.18 KH2PO4,
and 11 glucose, containing 0.1 mM pargyline and 0.01 mM ascorbic acid
and gassed with 95% O2-5%
CO2. Another preparation obtained
by the same procedure was frozen, cut into sections 20 µm thick on a
cryostat, and stained with hematoxylin and eosin for microscopic
examination. Experiments were started 60 min after the superfusion,
when the spontaneous release of dopamine had approached a plateau. The
preparations were stimulated by two parallel platinum electrodes at
parameters of 1-ms duration, 15-V intensity at various frequencies for
3 min at 70, 95, and 120 min after the superfusion. The superfusate was
continuously collected into a test tube containing 0.6 ml 1 N
HClO4, 0.5 ng 3,4-dihydroxybenzylamine as an internal standard, and 6 mg sodium metabisulfite every 5 min and was then adjusted to pH 8.6 with 2 M
Tris · HCl buffer. Dopamine in the superfusate was
absorbed with 50 mg of alumina and eluted with 250 µl of 0.1 M
HClO4. Dopamine in the eluates was
determined using an HPLC-ECD. As soon as the perfusion period ended,
the tissues were homogenized with an ultrasonic probe homogenizer, and
dopamine content in the tissues was determined as described above.
Measurement of ACh release.
The [3H]acetylcholine
(ACh) release experiments were carried out as described elsewhere (17).
The strips were immediately separated into two layers, the mucosal
layer and the muscle layer including the intramural plexus of the
gastric corpus, as described above. One preparation was used for the
release experiment and another was stained with hematoxylin and eosin
for microscopic examination. The muscle layer including the intramural
plexus of the gastric corpus was incubated with
[3H]choline (80 Ci/mmol) at a final concentration of 200 nM in oxygenated Krebs
solution at 37°C for 1 h. The preparations were washed in fresh
medium for 30 min and then mounted in an apparatus and superfused at
37°C at a flow rate of 0.6 ml/min with Krebs solution gassed with
95% O2-5%
CO2 containing 0.1 mM
hemicholinium-3. Experiments were started 60 min after the superfusion,
when the spontaneous release of 3H
had approached a plateau. The preparations were stimulated by two
parallel platinum electrodes at parameters of 1-ms duration, 15-V
intensity at 10 Hz for 1 min, at 30-min intervals. The superfusate was
collected every 3 min, and the radioactivity of the sample was
determined by counting in a liquid scintillation spectrometer. [3H]ACh of the total
radioactivity in superfusates was determined by electrophoresis (26).
At the end of the experiment, the tissue was dissolved in toluene, and
the radioactivity was measured in a scintillation counter. The release
of 3H was represented as the
fractional rate, which was obtained by dividing the amount of
3H in the superfusate by the
amount of 3H in the tissue. The
3H content of the tissue at each
period was calculated by adding cumulatively the amount of each
fractional tissue efflux to the 3H
content of the tissue at the end of the experiments. From each of the
release values obtained by plotting the fractional release of
3H against time, the release of
3H evoked by stimulation in each
condition was calculated by subtracting basal release. The first
stimulated release in each case was identified as
S1 and the second as
S2, and the relationship between them was expressed as
S2/S1.
Data were analyzed by Dunnett's
t-test, and a
P value of <0.05 was considered
statistically significant.
Histochemical procedure.
Male guinea pigs weighing 200-300 g were separated into two
groups. The first group was the control group, and the second group was
treated with a bilateral transection of the splanchnic nerve proximal
to the junction with the vagal nerve (section of nerves between the
celiac ganglion and stomach) (20) 7 days before experiments, as in the
measurement of tissue catecholamine content. Two groups of guinea pigs
were anesthetized with an intraperitoneal injection of pentobarbital
(50 mg/kg) and perfused with isotonic saline followed by 2.5%
glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) containing 1%
sodium metabisulfite, through the left ventricle for 30 min at 4°C.
After perfusion, the stomach was removed and washed with 0.05 M Tris
buffer (pH 7.4) containing 1% sodium metabisulfite at 4°C. The
stomach corpus was excised and the mucosa was removed, and then whole
mount preparations of gastric corpus were prepared by dissecting the
overlaying circular muscle. Dopamine immunohistochemical studies were
performed according to the method of McRae-Degueurce and Geffard
(23). The whole mount preparations were incubated with
rabbit antidopamine polyclonal antibody (1:750) diluted in 0.05 M
Tris-buffered saline (pH 7.4) containing 1% sodium metabisulfite, 1%
bovine serum albumin (BSA), and 0.3% Triton X-100 for 48 h at 4°C.
Preparations were washed with 0.05 M Tris-buffered saline and then
stained using the avidin-biotin peroxidase system. The specificity of
dopamine antiserum was assessed by immunocytochemistry, in which
repeated absorptions of the serum with NE coupled to BSA did not alter
the immunocytochemical staining pattern of dopaminergic neurons of the
substantia nigra and of the A10 regions as well as dopaminergic nerve
terminals of the locus coeruleus, as noted by Yang et al. (37).
To establish the specificity of the procedure, the mucosa-free whole
mount preparations were incubated with the serum from unimmunized
rabbits or antidopamine serum preabsorbed with dopamine or NE.
For double staining of dopamine and choline acetyltransferase (CAT),
the stained preparations already labeled with avidin-biotin peroxidase
for dopamine were then incubated overnight at 4°C with rabbit
anti-CAT polyclonal antibody (1:500) diluted in 0.1 M
phosphate-buffered saline (pH 7.4) containing 3% goat serum,
followed by the alkaline phosphatase system.
For catecholamine histochemistry, the sucrose phosphate-glyoxylic acid
method was used, as described previously (10). The animals were
perfused through the left ventricle with 2% glyoxylic acid in 0.1 M
phosphate buffer containing 20% sucrose. The whole mount preparations
of gastric corpus were air-dried for 10 min and heated at 100°C for
4 min. Catecholamine fluorescence was analyzed by fluorescence
microscopy.
Drugs and chemicals.
The following substances were used: rabbit antidopamine polyclonal
antibody (AB122S) and rabbit anti-CAT polyclonal antibody (AB143)
(Chemicon International); avidin-biotin peroxidase system (Vector
Labs); alkaline phosphatase system (Cappel);
[3H]choline (80 Ci/mmol; New England Nuclear); pargyline, ascorbic acid,
3,4-dihydroxybenzylamine, hemicholinium-3, dopamine, and NE (Sigma);
domperidone and sulpiride (Research Biochemicals International); and
Soluene (Packard). Other chemicals used were of reagent grade.
 |
RESULTS |
Dopamine and NE contents in gastric corpus.
Dopamine content was 32.9 ± 1.9 ng/g wet wt
(n = 5) in the mucosal layer and 95.8 ± 23.2 ng/g wet wt (n = 5) in the
muscle layer including the intramural plexus of the gastric corpus. NE content was 195 ± 22 ng/g wet wt
(n = 5) in the mucosal layer and 879 ± 77 ng/g wet wt (n = 5) in the muscle layer including the intramural plexus. The ratio
of dopamine to NE was 0.17 in the mucosal layer and 0.11 in the muscle
layer including intramural plexus.
Release of endogenous dopamine from the muscle layer including the
intramural plexus of the gastric corpus.
The spontaneous release of endogenous dopamine from the muscle layer
including the intramural plexus of the gastric corpus remained steady
during the course of each 140-min experiment. The amount of
spontaneously released dopamine was 0.122 ± 0.011 ng · min
1 · g
wet wt
1, and the fractional
rate (amount in superfusate/amount in tissue) of spontaneously released
dopamine was 0.00127 ± 0.00007/min (mean ± SE for 38 determinations).
Electrical transmural stimulation (15-V intensity, 1-ms pulse duration)
for 3 min increased the release of endogenous dopamine above the
spontaneous release noted just before the stimulation, in a frequency
(3-20 Hz)-dependent manner (Table 1),
although electrical stimulation at a frequency of 1 Hz did not
significantly increase the release of dopamine. The electrically (15-V
intensity, 1-ms pulse duration, at a frequency of 10 Hz) induced
increase in dopamine release was markedly reduced by either treatment
with tetrodotoxin (TTX) (Fig.
1A) or removal of
Ca2+ from the superfusion medium
(Fig. 1B).
Immunohistochemistry of dopamine in whole mount preparations from
the gastric corpus.
Immunostaining with specific antiserum against dopamine was studied in
whole mount preparations from guinea pig stomach corpus. In all 18 preparations studied, a network of dopamine-immunopositive fine
varicose fibers was found to surround the nonimmunoreactive ganglion
cells within the myenteric plexus of the gastric corpus (Fig.
2A).
A fairly dense immunostaining of nerve fibers was prominent in the
primary plexus running from ganglion to ganglion, without leaving the
plane of the plexus. Dopamine-immunopositive varicose nerve fibers were
also observed around the blood vessels (data not shown). The same
pattern of dopamine immunostaining was seen in parallel experiments
using the antidopamine serum adsorbed with NE, whereas no
immunostaining was seen when antidopamine serum adsorbed with dopamine
or nonimmune serum was used. The immunopositive nerve cell bodies (~1
cell per 5 ganglia) were seen in the periphery of the myenteric ganglia
(Fig. 2B). They were generally
spheroidal in shape and gave rise to two thick processes within the
myenteric plexus. The population of cell bodies immunoreactive to
dopamine was 0.19 ± 0.07 cells/ganglion (mean ± SE, 53 ganglia/6 preparations).

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Fig. 1.
Tetrodotoxin (TTX)-sensitive (A) and
external Ca2+-dependent
(B) release of endogenous dopamine
from mucosa-free preparations of corpus of guinea pig stomach.
A: TTX at 300 nM was added to
superfusion medium 15 min before and during electrical stimulation (ES)
(1-ms duration, frequency 10 Hz); n = 5 guinea pigs. B:
Ca2+-free Krebs medium containing
1 mM ethylene glycol-bis( -aminoethyl
ether)-N,N,N',N'-tetraacetic
acid was superfused 15 min before and during application of ES (1-ms
duration, frequency 10 Hz); n = 8 guinea pigs. Values are means ± SE. Significantly
different from spontaneous release value
(P < 0.05 by Dunnett's
t-test).
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Fig. 2.
Dopamine immunoreactivity in mucosa-free whole mount preparation of
corpus of guinea pig stomach. A:
dopamine-immunopositive nerve fiber networks.
B: dopamine-immunopositive neuronal
cell body (arrow). Inset: different
dopamine-immunopositive neuronal cell body (arrow) in same preparation.
Scale bar, 50 µm.
|
|
Tissue contents and immunohistochemistry of dopamine after bilateral
transection of the splanchnic nerve proximal to the junction with the
vagal nerve.
Seven days after bilateral transection of the splanchnic nerve proximal
to the junction with the vagal nerve (section of nerves between the
celiac ganglion and stomach), dopamine content was 33.3 ± 6.8 ng/g
wet wt (n = 5) in the mucosal layer
and 81.9 ± 8.2 ng/g wet wt (n = 5)
in the muscle layer including the intramural plexus, indicating
that dopamine content in these layers was not altered by section of
nerves between the celiac ganglion and stomach.
In the nontreated animals, the major patterns of catecholaminergic
innervation in the stomach detected by fluorescence histochemistry were
as described by Furness and Costa (10). A network of fine varicose
fibers was found to surround the nonfluorescent ganglion cells of the
myenteric plexus (Fig.
3A).
After section of nerves between the celiac ganglion and stomach,
catecholamine fluorescence was virtually abolished from the tertiary
plexus and the muscle layers, and fewer fluorescent fibers were
observed in the myenteric plexus (Fig.
3B), whereas no conspicuous changes
in dopamine-immunopositive nerve fibers were seen in the myenteric
plexus of the gastric corpus (Fig.
3C). Double staining for dopamine
and CAT demonstrated that a network of dopamine-immunopositive fine
varicose fibers closely encircled the CAT-positive ganglion cell bodies
within the myenteric plexus, even after section of nerves between the celiac ganglion and stomach (Fig.
3C).

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Fig. 3.
Catecholamine histochemistry (A and
B) and double staining of dopamine
and choline acetyltransferase (CAT)
(C) within myenteric plexus in
mucosa-free whole mount preparation of corpus of guinea pig stomach.
A: normal animal.
B and
C: animal after bilateral transection
of splanchnic nerve proximal to junction with vagal nerve (section of
nerves between celiac ganglion and stomach).
C: dopamine-immunopositive fine
varicose fibers (brown) closely encircled CAT-immunopositive ganglion
cells (blue, arrows) in myenteric plexus. Scale bar, 50 µm.
|
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Effects of dopamine receptor antagonists and dopamine on the
electrically stimulated outflow of ACh.
Electrical transmural stimulation (15 V, 1 ms) at 10 Hz evoked release
of endogenous dopamine from preparations consisting of the outer muscle
layers and myenteric plexus. The possible effects of this endogenous
dopamine on the release of
[3H]ACh were examined
using specific dopamine receptor antagonists. Electrical transmural
stimulation (15 V, 1 ms) at 10 Hz for 1 min increased the outflow of
3H from the muscle layer including
intramural plexus preloaded with
[3H]choline.
Applications of D2 dopamine
receptor antagonists (sulpiride and domperidone) at concentrations of
100 nM to 1 µM and
2-adrenoceptor antagonist
(yohimbine) at concentrations of 10 nM to 1 µM significantly potentiated the electrically stimulated outflow of
3H (Fig.
4A),
with no effect on spontaneous outflow. Even in the presence of 0.1 µM
yohimbine, sulpiride and domperidone also significantly potentiated the
electrically stimulated outflow of
3H (Fig.
4B). Sulpiride and domperidone
reversed the inhibitory action of dopamine (1 µM) on the stimulated
outflow of 3H (Fig.
4C).

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Fig. 4.
Effects of dopamine receptor antagonists on electrically stimulated
release of
[3H]acetylcholine
(ACh) from mucosa-free preparation of corpus of guinea pig stomach.
A: potentiation of electrically
stimulated release of
[3H]ACh by yohimbine,
sulpiride, and domperidone. B:
potentiation of electrically stimulated release of
[3H]ACh in presence of
yohimbine (0.1 µM) by sulpiride and domperidone.
C: antagonism by sulpiride
and domperidone on inhibitory effect of dopamine on electrically
stimulated release of
[3H]ACh. Dopamine (1 µM), yohimbine, sulpiride, and domperidone were applied 2 min, 10 min, 10 min, and 10 min before and during electrical stimulation (1-ms
duration, frequency 10 Hz), respectively. Significantly
different from value in absence of antagonists (control) in
A and
B and value of dopamine effect in
absence of antagonists in C
(P < 0.05 by Dunnett's
t-test).
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 |
DISCUSSION |
The guinea pig gastric corpus contains measurable concentrations of
dopamine: the concentration in the outer muscle layers (which include
the intramural plexus) was higher than that in the mucosal layer. The
ratio of dopamine to NE in the muscle layer including myenteric plexus
was 0.11, similar to that in the smooth muscle/myenteric plexus
preparation of mouse stomach fundus (7). In the generally
noradrenergically innervated tissues, dopamine and NE are measurable in
a relatively constant ratio of dopamine to NE within a species, and a
high ratio of dopamine to NE in any tissue suggests that dopamine is
not only the precursor of NE but is also a bioactive substance (4, 19).
Thus dopamine contained in the muscle layer including the intramural
plexus of the gastric corpus is thought to act as a bioactive
substance, although in the stomach the abundance of catecholaminergic
innervations has been demonstrated by fluorescent glyoxylic acid
histochemistry (10, 12), and it is accepted that the neurons are
NE-containing sympathetic adrenergic neurons.
The amount of dopamine released spontaneously from the muscle layer
including intramural plexus of gastric corpus was approximately 0.13%
per minute of the content in the tissue. This value is similar to that
released from the central dopaminergic neurons (25, 34). Electrical
transmural stimulation increased the release of endogenous dopamine
above the spontaneous release from the muscle layer including
intramural plexus of gastric corpus. When the properties of the
stimulated release of dopamine were determined, the stimulated release
was found to be TTX sensitive and external Ca2+ dependent. TTX blocks
neuronal conduction (24), and external Ca2+ is necessary for the
exocytotic release of most neurotransmitters from nerve terminals (27).
Thus these findings support the concept that dopamine is released in
response to stimulation from a neuronal component of the corpus of the
guinea pig stomach. The possibility that stimulation of adrenergic
nerves results in the release of dopamine as well as NE cannot be
completely excluded.
Immunohistochemical studies using dopamine antibody demonstrated the
presence of dopamine-immunopositive varicose nerve fibers within the
myenteric plexus of the guinea pig stomach corpus. Dopamine-immunopositive nerve fibers were prominent in the primary plexus running from ganglion to ganglion without leaving the plane of
the plexus, particularly with no projection to the muscle layer, being
different in density within the myenteric plexus and in distribution of
nerve fibers from the findings demonstrated by catecholamine
fluorescence histochemistry in the present study and previously
(10, 12) and by immunohistochemistry using tyrosine hydroxylase
antibody (3, 22, 31). The neurons containing both tyrosine hydroxylase
and dopamine-
-hydroxylase reflect the majority of NE-containing
neurons, whereas the neurons containing tyrosine hydroxylase, but not
dopamine-
-hydroxylase, may be dopaminergic neurons. Dopaminergic
nerve fibers have been suggested to be present in the rat stomach,
based on the findings that the immunoreactive nerve fibers to tyrosine
hydroxylase, but not to dopamine-
-hydroxylase, were detected in the
myenteric plexus of the rat stomach by double staining for tyrosine
hydroxylase and dopamine-
-hydroxylase (3). After section of nerves
between the celiac ganglion and stomach, dopamine contents in the
mucosal layer and the muscle layer including intramural plexus were not altered, and no conspicuous changes in dopamine-immunopositive nerve
fibers in the plexus were seen. However, catecholamine fluorescence was
virtually abolished from muscle layers and fewer fluorescent fibers
were observed in the plexus. Gastric NE is contained mainly in
sympathetic fibers derived from the celiac and superior mesenteric ganglia (8, 20). If dopamine is a precursor of NE in the sympathetic
neurons, section of nerves between the celiac ganglion and stomach may
reduce gastric dopamine content and dopamine-immunopositive nerve
fibers. Double staining for dopamine and CAT demonstrated that a
network of dopamine-immunopositive fine varicose fibers closely
encircled the CAT-positive ganglion cell bodies within the myenteric
plexus, even after section of nerves between the celiac ganglion and
stomach. Thus dopaminergic nerve fibers innervate the myenteric plexus
of the guinea pig stomach, and the dopamine released from these nerve
terminals by nerve stimulation may act on the enteric cholinergic
neurons.
Dopamine-immunopositive nerve cell bodies with two long thick
projections were seen in the periphery of myenteric ganglia. Although
it was not possible to observe the full extent of the emergent
processes and therefore define their class exactly, the stained cells
with two long processes appear to resemble the pseudo-uniaxonal multiaxonal type II neurons in the intestine, according to the morphological features classified into eight types of enteric neurons
(33), whereas Dogiel type II-AH/type II neurons have been shown to be
absent in the stomach (32). It is difficult to interpret
characteristics of the dopamine-immunopositive nerve cell bodies,
because no tyrosine hydroxylase-immunopositive neurons have been
detected in the myenteric plexus of the guinea pig stomach (22, 29,
31), and in fact the nerve cell bodies immunonegative to tyrosine
hydroxylase and immunopositive to dopamine-
-hydroxylase have been
demonstrated (29, 31). Thus the present findings suggest the possible
presence of dopaminergic intrinsic neurons in the enteric nervous
system of the guinea pig stomach as interneurons or nonmotor sensory
neurons (30), although it cannot be excluded that the neurons may be
capable of taking up dopamine but may not synthesize it. In the guinea
pig small intestine, intrinsic amine-handling neurons have been
shown, although these cells did not contain tyrosine hydroxylase
and dopamine-
-hydroxylase (11).
The source of dopamine-immunoreactive nerve fibers within the myenteric
plexus is thus possibly 1) the
dopaminergic intrinsic neurons detected in the present study;
2) the vagal efferent, parasympathetic fibers originating from the dorsal nucleus of vagus
nerve, which contains neurons immunoreactive to tyrosine hydroxylase
but not to dopamine-
-hydroxylase (2);
3) the vagal afferent fibers of the
nodose ganglion projecting to the stomach, which exhibit tyrosine
hydroxylase immunoreactivity and aromatic L-amino acid decarboxylase
immunoreactivity but not dopamine-
-hydroxylase immunoreactivity
(16); and 4) a subset of celiac
ganglion sympathetic neurons, which contain neurons immunoreactive to
tyrosine hydroxylase but not to dopamine-
-hydroxylase.
Activation of D2 dopamine receptor
has been reported to inhibit the release of ACh from cholinergic
neurons in the stomach (17). Sulpiride and domperidone are
D2 dopamine receptor antagonists, although domperidone exerts an inhibitory effect on cholinesterase, and
both antagonists were found to potentiate the release of ACh stimulated
by electrical stimulation under conditions that stimulated the release
of endogenous dopamine. Thus the blockade of the
D2 dopamine receptor with
sulpiride or domperidone prevented the inhibitory effect of endogenous
dopamine released by electrical stimulation on the cholinergic neurons,
resulting in potentiation of the release of ACh. The dopamine-induced
inhibition of stimulated release of ACh was reversed by sulpiride and
domperidone, which is similar to the finding noted previously (17). As
well, activation of
2-adrenoceptor has been shown
to inhibit the release of ACh from the cholinergic neurons of the
stomach (1). The potentiating effects of
D2 dopamine receptor antagonists
were obtained even when the
2-adrenoceptor was blocked by
yohimbine. Thus the target of dopamine released from neurons may be the
cholinergic neurons, and dopamine inhibits cholinergic neuronal
activity.
Dopamine may be present in the corpus of the guinea pig stomach not
merely as a precursor of NE, but may act through specific dopamine
receptors as well. The present study using biochemical, physiological,
and immunohistochemical methods showed neuronal release of dopamine
from the gastric corpus and suggests that dopamine may play a
physiological role in the control of gastric motility through
modulation of cholinergic neuronal activity.
 |
ACKNOWLEDGEMENTS |
We thank M. Ohara for helpful comments.
 |
FOOTNOTES |
This work was supported by grants from the Ministry of Education,
Science, Sports, and Culture, Japan.
Address for reprint requests: K. Taniyama, Dept. of Pharmacology II,
Nagasaki Univ. School of Medicine, Nagasaki 852, Japan.
Received 4 October 1996; accepted in final form 17 July 1997.
 |
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