71

ORIGINAL ARTICLE

Immunohistochemical Detection of Serotonin in the Stomach of

Domestic Cat (Felis catus L.) (Carnivora: Felidae)

Rizza Mae L. Labadan, BS, Melanie Anne M. Atienza, BS,

Juan Carlos C. Dimayuga, BS and Rodel Jonathan S. Vitor II, DVM*

Biology Department, College of Science, De La Salle University, Manila, Philippines

ABSTRACT

Serotonin reactivity in the cardiac, fundic and pyloric regions of the stomach of 20 (ten male
and ten female) 2-year old domestic cats (Felis catus L.) was detected by immunohistochemistry.
Serotonin immunoreactivity was observed in the epithelium, basement membrane, parietal
cells, smooth muscle fibers of the lamina muscularis mucosae and lamina propria, blood vessels
in the lamina propria and tunica submucosa, myenteric plexus in the tunica sucmucosa, inner
circular and outer longitudinal layers of the tunica muscularis, and blood vessels in the tunica
serosa. Serotonin immunoreactive cells were distributed in the apical lining of the fundus
but were observed in the basal lining of the surfave epithelium in the cardia and pylorus.
Blood vessel reactivity suggests that serotonin can travel by blood to reach its target organs.
Serotonin immunoreactivity in the different structures of the stomach suggests its role in
regulating gastric secretions and motility.

Key words: 5-hydoxytryptamine, cat, Felis catus, immunohistochemistry, serotonin, stomach

Philipp. J. Vet. Med., 51(2): 71-78, 2014

INTRODUCTION
Serotonin (5-hydoxytryptamine) is
an extremely labile and fairly unstable
neurotransmitter of the body (Levin,
2004; Al-Tikriti et al., 2012). Over 95% of
serotonin is synthesized and secreted by the
enterochromaffin cells, the majority of which
is secreted during digestion (Saxena, 1995; Lai
et al., 2009). However, secretion of serotonin
is also affected and regulated by the pH level
in the gastrointestinal tract (GIT) (Kasacka
and Majewski, 2007). Some of the serotonin
secreted by the GIT are able to reach the target
organs through blood circulation (Vanhoutte,
1990). Serotonin is responsible for reducing
gastric acid secretion (Gesase, 2009; Lai et
al., 2009) and controlling gastric (Tamura et
al., 2006) and intestinal motility (Lai et al.,
2009), smooth muscle contraction (Ahlman
and Nilsson, 2001; Gesase, 2009), controlling
blood flow, mucus formation (Lai et al., 2009)
*FOR CORRESPONDENCE:
(email: rodel.vitor@dlsu.edu.ph)
and fluid secretion (Tamura et al., 2006; Lai et
al., 2009).
Distribution of serotonin immunoreactive
cells in the GIT has been studied in different
animals such as developing chicks of the
Gallus gallus domesticus species (Aksoy and
Cinar, 2009), mice (Kasacka and Majewski,
2007), rats (Fiorica-Howells et al., 2002; Lai
et al., 2009), guinea pigs (Fiorica-Howells et
al., 2000; Tamura et al., 2006), dogs (White
and Magee, 1958), pigs (Janssen et al., 2002),
horses (Fink et al., 2006), water buffalo (Lucini
et al., 1999) and humans (Gershon, 2004; Wade
et al., 2006). However, it has not yet been
demonstrated in the domestic cat (Felis catus
L.), specifically the stomach. Determining the
location of serotonin immunoreactive cells in
the domestic cat would allow comparison of
serotonin abundant sites with other species.
The results obtained from this study can be
used as reference for future researches on the
relationship of serotonin immunoreactive sites
with stomach activity.
72 LABADAN et al.
MATERIALS AND METHODS
Sections of the cardiac, fundic and pyloric
regions of the stomach were obtained from 20
(ten male and ten female) apparently healthy
2-year old domestic cats. The specimens
were collected from cats used for dissection
in comparative anatomy laboratory after
embalming. Tissue samples were fixed in 10%
phosphate buffered formalin solutions for 72
h, processed using the paraffin technique and
sectioned using an 820 rotatory microtome
(American Optical®, New York) at 5 μm. The
sections were mounted on ordinary glass slides
for Hematoxylin-Eosin (H&E) staining for
histologic observation of the stomach sections
or on FLEX IHC microscope slides (Dako,
Denmark) for immunohistochemistry. Tissue
processing and H&E staining were performed
at the Department of Basic Veterinary
Sciences, College of Veterinary Medicine,
University of the Philippines Los Baños while
immunohistochemistry was performed at the
Biology Department, College of Science, De La
Salle University.
Modified avidin-biotin peroxidise complex
(ABC) method (Hsu et al., 1981) was used to
detect serotonin immunoreactivity. Three
tissue sections per sample were deparaffinized
and washed with 0.1 M phosphate buffer
saline (PBS) at pH 7.6, seven times at 5 min
intervals. The sections were then incubated
with 20% normal goat serum (NGS) in 10 mM/l
tris buffer solution-ethylenediaminetetraacetic
acid (TBS-EDTA) for 30 min under room
temperature to reduce non-specific staining.
The sections were then incubated for 16 h
with polyclonal mouse anti-serotonin antibody
(Abcam, Cambridge) at 1:1000 dilution with
TBS-EDTA containing 5% NGS at 4ºC in a
moisture chamber. The sections were then
washed four times with TBS-EDTA at 5 min
intervals. Sections were then incubated with
biotinylated anti-mouse IgG at 1:500 dilution
(Vector Laboratories, California) in TBS-
EDTA containing 1.5% NGS for 90 min at room
temperature. Sections were then washed in
PBS three times at 5 min interval; incubated
with ABC (Vector Laboratories, California) for
60 min at room temperature; and then rinsed
with PBS three times at 5 min interval. The
staining of serotonin was done by incubating
the slides in imidazole-HCl buffer containing
0.05% 3,3’-diaminobenzidine for 15 min at room
temperature. The samples were then processed
according to the procedures described by Vitor
et al. (2013).
Some sections of the stomach were used as
negative controls. The sections were subjected
to the same procedure as the IHC sections;
however, the primary antibody (mouse anti-
human serotonin) was omitted.
The slides were examined using a light
microscope (Olympus CX21) (Olympus UK
Ltd., Hertfordshire) to determine the serotonin-
immunoreactive structures. Reactions were
recorded as positive (+) or negative (-).
Photomicrographs of representative sections
were taken using a light microscope with a
digital camera (Olympus SP-350) (Olympus
UK Ltd., Hertfordshire).
RESULTS AND DISCUSSION
Serotonin immunoreactivity was observed
in the tunica mucosa, tunica submucosa, tunica
muscularis and tunica serosa of the cardia,
fundus and pylorus of the stomach, of the
domestic cat (Table). There were no significant
differences in serotonin immunoreactivity in
the different regions of the stomach except for
the epithelial cell serotonin immunoreactivity.
Moreover, no differences in serotonin
immunoreactivity between sexes were
observed.
In the tunica mucosa of the stomach,
immunoreactive cells were limited in the lamina
epithelialis mucosae, mucosal glands in the
lamina propria and in the lamina muscularis
mucosae. The simple columnar epithelium
of the lamina epithelialis mucosae showed
varying reactivity with serotonin. Among the
regions of the stomach, only the fundus showed
serotonin immunoreactivity in the apical lining
(Fig. 1A) while the cardia and pylorus showed
serotonin immunoreactivity in the basal
lining of the surface epithelium (Fig. 1B). The
presence of serotonin in different areas of the
lining epithelium can be due to the bipolarity
of cells that secrete this neurotransmitter
either through the basal or apical surface of
 IMMUNOHISTOCHEMISTRY OF SEROTONIN IN THE STOMACH OF CAT 73

Table. Localization of 5-hydoxytryptamine or serotonin in different regions of the stomach in male and
female domestic cats.

Cardia Fundus Pylorus

Structure Male Female Male Female Male Female
n=10 n=10 n=10 n=10 n=10 n=10
Tunica mucosa
Lining epithelium
Apical lining - - + + - -
Cytoplasm - - - - - -
Nucleus - - - - - -
Basal lining + + - - + +
Lamina propria
Gastric glands + + + + + +
Mucous neck cells + + + + + +
Parietal cells + + + + + +
Chief cells - - - - - -
Smooth muscle fibers + + + + + +
Blood vessels + + + + + +
Muscularis mucosae + + + + + +
Tunica subucosa
Blood vessels + + + + + +
Meissner’s plexus + + + + + +
Tunica muscularis
Smooth muscle layer + + + + + +
Myenteric plexus + + + + + +
Tunica serosa
Blood vessels + + + + + +

the lining epithelium (Nilsson et al., 1998). The
presence of serotonin in the lining epithelium
is congruent with the findings in the stomach of
dog (White and Magee, 1958) and rat (Lai et al.,
2009), suggesting that serotonin is associated
with the inhibition of gastric acidity during
digestion (Wazna, 1978). This is supported by
the study of Chang et al. (1965) who observed
decreased gastric acid secretion in the stomach
of serotonin-treated albino rats.
The presence of serotonin in the lining
epithelium of the gastric glands (Fig. 2)
suggests that serotonin is involved in
gastric acid secretion inhibition and mucous
secretion (Zhou et al., 2008; Lai et al., 2009).
Furthermore, the presence of serotonin in
the gastric mucosa may also be important in
controlling gastric motility (Tamura et al.,
2006). Gastric acid secretion is inhibited when
food is gradually emptied into the stomach, via
hormonal control, or when there is distention
of the stomach (Sherwood, 2013). When
several endogenous hormones are released in
response to acidification (pH less than 3) of
the duodenum as food bolus passes from the
stomach to the intestine, gastric acid secretion
is greatly diminished (Canfield and Spencer,
1983). Cholecystokinin and gastric inhibitory
polypeptide inhibit both gastric acid and pepsin
secretion while secretin and serotonin inhibit
gastric acid secretion but stimulate pepsin
secretion (Wazna, 1978). Serotonin is secreted
by the enterochromaffin cells in the lining
epithelium to protect the mucosa against excess
gastric acid secretion (White and Magee, 1958;
Lai et al., 2009). It has also been observed that
the absence of serotonin in the gastric lining
epithelium significantly increased gastric acid
secretion that can lead to enterochromaffin cell
carcinoid (Ahlman and Hilsson, 2001).
Gastric gland cells reactive to serotonin
include mucous neck cells, parietal cells
and chief cells, which are distributed in the
lamina propria of the tunica mucosa (Fig. 3).
74 LABADAN et al.
Mucous neck cells secrete mucin, a substance
which protects the gastric mucosa from excess
gastric acid secretion. According to the studies
of Black et al. (1958) and White et al. (1985),
the secretion of mucin is due to the action
of serotonin and not due to the mechanical
stimulation from peristalsis or the rubbing
together of mucosal surfaces.
Serotonin is present in the acidophilic
parietal cells and absent in the basophilic chief
cells (Fig. 4). Parietal cells are responsible for
secretion of hydrochloric acid, which is the
main component of gastric juice (Eroschenko,
2008); thus, it is probable that serotonin is
involved in controlling the secretion of parietal
cells (Ormsbee and Fondacaro, 1995; Zhou et
al., 2008). On the other hand, chief cells are
responsible for the production of pepsinogen,
gastric lipase and chymosin in the wall of the
stomach (Eroschenko, 2008). Without further
studies to confirm the reason behind the
negative immunoreaction of the chief cells,
it can only be hypothesized that serotonin
receptors may not be expressed on the surface
of the chief cells and, thus, prevent ligand
binding.
The smooth muscle fibers of the lamina
muscularis mucosae of the tunica mucosa
(Fig. 2) as well as the different layers of the
tunica muscularis (Fig. 5) were observed to be
immunoreactive to serotonin. These muscle
layers of the different tunics of the stomach
wall are responsible for movement; thus, it can
be surmised that serotonin may be involved
in the peristalsis of the stomach during the
digestive process (Ahlman and Nilsson, 2001;
Zhou et al., 2008). However, in this study, it
was not determined whether reactivity to
serotonin is intracellular or extracellular.
In the tunica submucosa, serotonin
immunoreactivity was limited to the blood
vessels (Fig. 5) and Meissner’s plexus. (Fig. 6).
Reactivity of blood vessels has been postulated
to be a result of platelet secretion to prevent
lesions in the lining of blood vessels. The
more intense immunoreactivity of the veins
than in the artery is similar with the results
obtained by Linder et al. (2007). Without any
concrete reason for such immunoreactivity, it
can only be postulated that veins can function
as serotonin reservoir in the periphery. The
presence of serotonin in the Meissner’s plexus,
which is responsible for innervating the lining
epithelial cells as well as the smooth muscles
of the lamina muscularis mucosae, has been
hypothesized to play a role in gastric motility
(Lai et al., 2009).
The myenteric plexus located between the
two layers of the tunica muscularis showed
serotonin immunoreactivity (Fig. 7). Positive
reaction of nerve and nerve elements have been
seen in different animals such as dog (Black et
al., 1958), chick (Aksoy and Cinar, 2009) and
horse (Fink et al., 2006). These results suggest
neural transmission of serotonin to and from
other tissues and organs of the body (Griffith
and Burnstock, 1983).
Specific structures of the tunica serosa that
reacted with serotonin were the blood vessels
and lining epithelium (Fig. 8). To date, there is
no known specific function of serotonin in the
tunica serosa. Fink et al. (2006) hypothesized
that the tunica serosa epithelial cells may be
involved in the reuptake of serotonin.
In conclusion, the results of the study
showed that serotonin is present in the basal
lining of the gastric epithelial cells of the
cardiac and pyloric regions and apical lining of
the fundic epithelial cells, parietal cells, smooth
muscle fibers of the muscularis mucosae, lamina
propria and tunica muscularis, blood vessels
in the lamina propria, tunica submucosa and
tunica serosa, Meissner’s plexus in the tunica
submucosa, myenteric plexus in the tunica
muscularis, and the lining of the tunica serosa.
Serotonin immunoreactivity in the above-
mentioned structures of the stomach suggests
its possible role in regulating gastric functions
such as gastric acid and mucus secretion and
motility.
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