J. Biol. Macromol., 7 (3), 55-62 (2007)

Regular paper

Cells expressing GABA synthetic enzyme, glutamate decarboxylase, in stomach andintestine: RT-PCR and immunohistochemistry studies
Kanako Akamatsu1,†, Yumi Nakamura1, Hana Hayasaki2, Kiyohito Kanabara2, Kentaro Maemura2,
Yuchio Yanagawa3, Kunihiko Obata4, Masahito Watanabe2, Hiroshi Ueno1,§
1

Laboratory of Applied Microbiology and Biochemistry, Nara Women’s University,
Nara 630-8506, Japan, 2Department of Anatomy and Cell Biology, Osaka Medical College
Takatsuki 569-8686, Japan, 3Division of Neuroscience, Gunma University Graduate School of
Medicine, Maehashi 371-8511, Japan, 4Brain Science Institute, Riken, Wako 351-0198, Japan
Received August 15, 2007; Accepted August 28, 2007
Expression of glutamate decarboxylase in mouse stomach and intestine was studied by
RT-PCR and immunohistochemical techniques. The RT-PCR study showed GAD mRNA
expressionin fetus and postnatal ages of 4 and 11 weeks old mice. A strong positive staining
against GAD65 was observed at both stomach and intestine of the 4- and 11-week old mice.
GFP positive cells were found in the fetus at both stomach and intestine, suggesting that GAD67
is expressed early in life. In this study, we present preliminary results of our findings on GAD
expressing cells in stomach and intestine and discuss the significance of GAD expression androle(s) of these cells.
Key words: GABA, GAD, stomach, intestine
Introduction
Glutamate decarboxylase (GAD) is an
enzyme [E.C. 4.1.1.15] to catalyze thesynthesis of γ-aminobutyric acid (GABA)[1-3].
GABA functions as an inhibitory
neurotransmitter in the nervous system [4].
In addition, GABA is also found in the
non-neural tissues [5] where the role(s) of
GABA in these tissues has been vigorously
investigated [6]. There are accumulating
evidences that GABA plays several significant
physiological roles, such as lowering bloodpressure, and giving relaxation and diuretic
effects [7,8].
Mammalian species were found to have

two independent genes for GAD and theyexpress two isoforms, GAD65 and GAD67 [9].
Interestingly, both GAD isoforms werereported to be expressed within the same cells,
a rare example that enzymes catalyzing thesame chemical reaction are co-expressed in thesame cells [3]. The GAD isoforms have been
investigated in terms of structural differences,cellular localization, physiological functions,and regulation [1,10].
Studies using
knock-out and/or knock-in mice suggested that
GAD isoforms have different biologicalactivities [11-13]. The knock-in mouse,
GAD67/GAD mouse, developed by Obata andhis colleagues, is a useful tool in studying the___________________________________§
Corresponding author: Phone:+81-742-20-3493, Fax: +81-742-20-3448,
E-mail: hueno@cc.nara-wu.ac.jp†
Present address: Department of Anatomy
and Cell Biology, Osaka Medical College,
Takatsuki, Japan_________________________________
Abbreviations: Ab, antibody; CNS,
central nervous system; GABA, γ-amino
butyric acid; GAD, glutamate
decarboxylase; GFP, green fluorescence
protein; PBS, phosphate buffered saline

56

GABA producing cells in stomach and intestine

exact localization of GAD67 isoform.
Spice extracts have been studies in order
to discriminate the catalytic functions of GADs
[14,15]. Such studies have revealed thatvarious spice extracts could affect enzymatic
activity of GADs. It is suggested that if GAD
is located in the digestive system, GAD
activity could be affected by food components.
Recently, immunohistochemical and RT-PCR
studies made it possible to detect GAD
expression on skins, tongue, submandibular
gland and others [16,17]. In the present study,
we have used GAD67/GFP mice and applied
techniques such as immunohistochemistry and
RT-PCR to study the distribution of GAD
isoforms in order to confirm if GADs are
expressed in the digestive system.
Material and Methods
Animals - ICR mice (Nihon Clea, Osaka,
Japan) was purchased from local breeder.
GAD67/GFP knock-in mice were originallyraised at the National Institute of Physiological
Sciences, Okazaki, and has been kept at Osaka
Medical College [18]. The animals werehoused in cages under the environmental
temperature of 24±2 °C, humidity of 80±5 %,and dark/light schedule of 12/12 hr. Waterand normal food were allowed freely.
RNA isolation and RT-PCR - Male ICR
mice of fetus at 17 days and postnatal at agesof 4 and 11 weeks old were anesthetized andtheir stomach and jejunum were dissected.
The stomach and jejunum were removed andtheir contents were washed with RNase freewater. The total RNA was prepared by using
RNeasy mini kit (Qiagen) and conversion of
RNA to cDNA was carried out by adding
Omniscript reverse transcriptase (Qiagen).
PCR was then performed by using ExTaq DNA
polymerase (Takara, Japan). Primer pairs forthe mice GAD65 and GAD67 were prepared as
described [19] (Table 1). PCR cycle wasprogrammed as 94 °C, 58 °C, and 72 °C for 45
sec, 45 sec, and for 1 min, respectively andcarried out for 38 cycles with a GeneAmp PCRsystem 9700 (Applied Biosystems).
Fluorescent immunohistochemistryFour- -and 11-week-old mice wereanesthetized and their stomach and jejunum
were dissected while 4% phosphate bufferedformalin solution was perfused. The
specimens were cut to 12 μm thickness with a
Leica CM3050 cryomicrotome. The sections
β-actin
M

B

S

GAD67

GAD65
J

B

S

J

B

S

J

A
B
C
Fig. 1. RT-PCR analysis of GAD65 and
GAD67 mRNAs from ICR mouse stomach,jejunum and brain. A:fetus at 17 days
mRNA, B: 4 weeks, C:11 weeks. M :Maker, B : Brain, S : Stomach, J : Jejunum

K. Akamatsu, et al.

were incubated at 4 °C for overnight withrabbit polyclonal GAD65 antibody (No.
G5038, Sigma) at 1000-fold dilution or
GAD67 antiserum (Alpha Diagnostic) at
1000-fold dilution with PBS. Then, sections
were incubated with second antibody, Alexa
Fluor 488 goat anti-rabbit IgG (Molecular
Probes) at 300-fold dilution with PBS.
Propidium iodide (Molecular Probes) was used
for staining nuclei. Fluorescent images wereacquired with a Nikon ECLIPSE E600
fluorescent microscope.
Observation of GFP-positive cells Stomach and jejunum of GAD67/GFP
knock-in mouse of fetus at 17days old, andthose of 1, 4, and 11weeks old were dissected
as described above, except the thickness was
30 μm. The sections were washed with PBS
3 times, and then GFP-positive cells wereobserved on a fluorescent microscope.
Results
RT-PCR analysis of GAD65 and GAD67- RT-PCR analyses were carried out toexamine the expression of mRNA for GAD65
and GAD67. When mRNA was isolated from
ICR mouse stomach, jejunum and brain, we
have found all animals express GAD65 and
GAD67 mRNAs (Fig.1). Although all
samples gave positive results, the band
intensity of GAD67 from jejunum wassignificantly weaker than those from the others.
GAD65 appears to be a predominant form inboth stomach and jejunum as the mRNA
expression levels indicated.
Immunohistochemical analysis Anti-GAD65 Ab staining was carried out on
stomach and jejunum specimens from the 4and 11- week-old ICR mice. As shown in
Figs. 2 and 3, both stomach and jejunum
showed GAD65 immunoreactivity. The
immunohistochemical results agreed well withthe results of RT-PCR. Thus, it is likely that
GAD65 is expressed in both stomach andjejunum. Another immunohistochemical
study was carried out with anti-GAD67 Ab.
However, we were unable to detect any stains
in both stomach and jejunum specimens. It is57

also noteworthy that immunohistochemical
study on fetus was unsuccessful due todifficulties in handling the specimen. The
location of GAD65 stains was carefully
examined. It is probable to assume that the
principal cells (Figs. 2a and 3a) and surface of
mucosa (Figs. 2b and 3b) of stomach are likely
to be stained. Also, the endocrine cells atepithelium (Figs. 2c and 3c) and crypts (Figs.
2d and 3d) of jejunum are assumed to be the
GAD65 positive cells.
Expression of GFP-positive cells GAD67/GFP knock-in mice were used in orderto locate the cells expressing GAD67.
Because these knock-in mouse express GFP inplace of GAD67, it has been demonstrated thatthe direct visualization of GAD67 can be
possible by following green fluorescent[12,18]. The knock-in mice of fetus at
17days and postnatal ages of 1, 4, and 11weeks
were used. When the fetus stomach
specimens were examined, we found
GFP-positive cells at the surface of mucous
that appeared as a line (Fig. 4a). Also foundin stomach was a dense fluorescent spot at theserous membrane that appears to dote at
submucosa (Fig. 4b ). When the fetus
jejunum was examined, there were no
GFP-positive cells in the epithelium. We
found a few GFP-positive cells in the muscularlayer (Fig. 4c) where those fluorescent cell
distributions were scattered. While
GFP-positive cells were found in the fetus, wewere unable to detect any GFP-positive cells
for the mice after the birth.
Discussion
Glutamate decarboxylase (GAD)
catalyzes decarboxylation reaction of
L-glutamate to produce GABA. In the CNSof higher mammals, GABA plays as an
inhibitory neurotransmitter. Those neuronalcells have been shown to express two
GABA-producing enzymes, GAD65 and
GAD67. With the advancement in detecting
mRNA and protein by RT-PCR and fluorescentlabeled Ab, respectively, it has become
possible to detect protein expression in various

58

GABA producing cells in stomach and intestine

a

b

c

d

Fig. 2. Immunohistochemistry for GAD65 on 4 weeks old mice. GAD65
immunostaining is shown in green, whereas propidium iodide (PI) staining isshown red. Photos cover general region to contain principal cells in
stomach (a), surface of mucosa in stomach (b), endocrine cells in jejunum(c), and crypts in jejunum (d). Bar is indicated 50μm.
a

b

c

d

Fig. 3. Immunohistochemistry for GAD65 on 11 weeks old mice. GAD65
immunostaining is shown in green, whereas PI staining is shown red.
Photos cover general region to contain chief cells in stomach (a), surface of
mucosa in stomach (b), endocrine cells in jejunum (c), and crypts in jejunum(d). Bar is indicated 50μm.

tissues. In this study, we have focused on an
expression of GAD isoforms in stomach andjejunum.
We have found that GAD65 and GAD67
mRNAs are expressed in both stomach andjejunum at all age groups we have examined.
However, GAD65 appears to be expressedabstronger than GAD67. We have assumed thatthe difficulty in detecting GAD67 with
antibody was attributed to the quality of theantibody we have used; therefore we have used
the GAD/GFP knock-in mice. The knock-in
mice showed positive fluorescence only for the
fetus specimens. The absence of fluorescencecFig. 4. GFP positive cells in 17days fetus mice.
GFP
positive cells are shown in green. Photos cover theregion containing the surface of mucosa in stomach (a),serous membrane and submucosa in stomach (b), andmuscular layer in jejunum (c). Bar is indicated 50μm.

K. Akamatsu, et al.

for 1, 4, 11 weeks old mice suggests that thesemice express little or no GAD67. It is ofinterest to note that our current observation
indicates the age-dependent expression of
GAD isoforms; GAD67 expressed
predominantly during the prenatal stage while
GAD65 in postnatal stage.
Distinctly different biological roles for
GAD isoforms have been demonstrated
previously. GAD65-deficient mice are shown
to be highly susceptible to seizure and exhibit
emotional behaviors such as anxiety andaggression [11,20,21]. GAD67-deficient
mice are born with cleft palate and die shortly
after the birth [22] which suggests GAD67
may produce GABA acting at the basal level inbrain. Our results are consistent with an ideathat GAD67 protein may be involved in the
fetal development, probably in growth of thedigestive system, while GAD65 protein may
be involved in probably functional roles ofdigestive system, i.e., excreting acid and/or
hormones.
GAD65 protein was detected on thesurface of stomach mucous of postnatal miceand GAD67 protein was at the similar region
in the fetus stomach. Recently, the presenceof G-protein coupled receptor (mGluR1), a
candidate for umami receptor in taste bud, wasreported at the stomach glandular [23]. It wasalso reported that GABA-immunoreactive cells
were found at the stomach pylorus and upper
part of the small intestine [24]. Theseobservations and our current results havesuggested that GABA at the gastrointestinal
tract may act as a gut hormone, as well as
another role as an enteric neurotransmitter. A
subpopulation of mucosal endocrine cells may
play this role.
Since GAD proteins are found at thesurface of stomach and at the epithelium andcrypts of jejunum, GAD may participate in thesynthesis of GABA at these sites by utilizing
dietary L-glutamate as a substrate. In
addition to previously identified location of
GAD-immunoreactive cells in stomach by
Gilon et al.[23], we found that GAD is locatedat the pyloric and oxyntic mucosa, endocrine

59

cells in mouse and rat stomach in this study.
It has been suggested that GAD should belocated at a part of endocrine and exocrine
glands that participate in the secretion ofgastric acid. We have also observed GAD on
pancreas where various hormones and proteinsare excreted. Pancreas is an organ where
GAD is thought to be active and a depletion of
GAD from β-cells was reported to cause type I
diabetes and GAD65 is identified as an
autoantigenic protein [25]. Further studies
are need to explore the possibility of GAD
being involved in the excretion mechanism.
In the above described Gilon’s study, isoformsof GAD were not discriminated. Since thepresence of isoforms had been identified in the
beginning of 1990, it was not clearly distinct
which GAD isoforms were dealt with at thetime of the Gilon’s study and even the RT-PCR
study was not carried out then. Our present
study may suggest that GAD65 is thefunctional isoform since it is the major isoform
expressed after the birth.
The endocrine cells found in digestivesystem are the likely cell types that are
immunoreactive to GAD65 in epithelium andthey are the ones typically found in theepithelium of absorbed cells and the goblet
cells. Those endocrine cells form
neuroendocrine complex in the intestine may
have a close connection with nerve system. If
GAD in the neuroendocrine complex
synthesizes GABA, it is probable that GABA
may participate in digestion of foods by acting
as a hormone in the intestine in secreting
digestive enzymes [26,27]. A part ofendocrine cells at the crypt of intestine was
known to move as these cells become a part ofvilli during the growth. Our current findingof the location of GAD in intestine isconsistent with GAD being expressed in theendocrine cells. A significant amount of
GABA was found in the duodenum where
GABA may participate in the development of
duodenum [28]. Along major curvature of
the antrum pyloricum, a high density of GABA
immunoreactive cells was exhibited [29].
Our present finding was consistent with the

60

GABA producing cells in stomach and intestineprevious description of GABA distributions.
It is likely that those cells exhibiting GABA
immunoreactivity contain GABA synthetic
enzyme, GAD. Since it has been suggested
that GABA plays a role in intestinal endocrine
regulation, GAD should have some extensiveroles in the digestive system. Moreover, thedifference in expression of GAD isoforms in
different developmental stages suggests that
GAD may also be involved in the growth anddevelopment of stomach and intestine.
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Contributed by Ishijima Sumio