J. Biol. Macromol., 2(2), 76-77, 2002

Note

Immunohistochemical study of the localizationof glutamate decarboxylase in rodent’s
submandibular gland
Mineko Iwahori, Kanako Akamatsu, Sachie
Kurohara, Kumio Yokoigawa, Hiroshi Ueno* ,Hiroshi Ogawa, ‡ Hitoshi Ozawa‡ and MitsuhiroKawata‡
Laboratory of Applied Microbiology andBiochemistry, Nara Women’s University, Nara
630-8506, Japan and ‡Department of Anatomyand Neurobiology, Kyoto Prefectural Universityof Medicine, Kyoto 602-0841, Japan

Received August 20, 2002; Accepted August 30, 2002

Physiologicalrole(s)ofglutamatedecarboxylase, a GABA-synthesizing enzyme, hasbeen studied by using immunochemical method.
In this study, we report that glutamate
decarboxylase
specificallylocalizes
insubmandibular gland of rat. Our results may
suggest that glutamate decarboxylase plays a rolein salivation.
Keywords:
GABA,
glutamatesalivation, submandibular gland.

decarboxylase,

γ-Amino butyric acid (GABA),1 synthesized byglutamate decarboxylase (GAD) from L-glutamic acid,is an inhibitory neurotransmitter in central nervous
system (CNS) (1). We have shown that GAD
activity is affected by various spice extracts in vitro(2). This earlier results have let us seek GAD
proteins in the digestive system. If GAD proteinsare found in the digestive system, it is highly likelythat GAD is involved in some sort of regulation offood digestion via GABA synthesis.
It is of interests to consider which isozyme of
GAD protein, GAD65 or GAD67, specifically
localized in the digestive system, if there is any.
Ever since the discovery of two isoforms of GAD (3),*To whom correspondence should be addressed:
Tel./Fax: 81-742-20-3493; E-mail: hueno@cc.narawu.ac.jp.
1
The abbreviations used are: BSA, bovine serum
albumin; CNS, central nervous system; DAB,diaminobenzidene; GABA, γ-amino butyric acid;
GAD, glutamate decarboxylase; PBS, phosphatebuffered saline.

distinct roles of two forms have been suggested invarious cellular events, for example, development,metabolism and homeostasis (4, 5).
There was a report that GAD activity wasobserved in rats submandibular gland (6); however,
little information was available for the localizationand physiological functions of GAD in submandibular
gland.
In this study, we have examined rat
submandibular gland by immunohistochemical
staining to reveal the role of GAD in salivation.
Materials and Methods
Rats and Tissue preparation
Wistar rats (9 weeks, Clea, Japan) were used forall of the reported studies. Experimental tissues
were removed, dissected rapidly and fixed in 10%
phosphate buffered formalin, pH7.4 for 24 h. After
fixation, the samples were dehydrated through a
graded ethanol series and embedded in Histosec(Merck Co., Darmastadt, Germany). Sections of 4µm thickness were cut with a microtome, mounted on
glass slides pre-coated with 3-aminopropyl
triethoxysilane (Aldrich Chemical, Milwaukee),deparaffinized with xylene and rehydrated through a
graded ethanol series, then washed for 5 min withrunning water and distilled water.
Immunohistochemistry
The sections were stained by diaminobenzidene(DAB) method using a commercial kit (HistofineSAB-PO(R), Nichirei Co., Japan) to allow
visualization of GAD65 and 67.
Slides wereimmersed in 3% H2 O2 in phosphate buffered saline(PBS) for 7 min at room temperature to inhibit
endogenous peroxidase activity. Additionally, weused a commercial kit (avidin-biotin endogenous
blocking reagent set, Biomeda Co.) to blockendogenous avidin and biotin. After washing withPBS, the sections were pre-incubated with 10%
normal goat serum (Nichirei Co., Japan) for 30 min atroom temperature in wet box, and then rinsed withPBS.
The slides were incubated with primary
antibody for GAD65/67 (Sigma-Aldrich, USA),
GAD65 (Sigma-Aldrich, USA), or GAD67 (Alfa,USA) diluted 750, 2,000, 2500-fold, respectively,with PBS containing 1% bovine serum albumin(BSA) for 1 hr at root temperature in wet box. As
negative controls, sections were incubated with PBS.
After washing with PBS 3 times, the sections wereincubated with biotinylated goat anti-rabbit IgG
antibody (Nichirei Co., Japan) for 30 min at room
temperature in wet box. After 3 washes with PBS,
the sections were incubated peroxidase-conjugatedstrepto-avidin (Nichirei Co., Japan) for 30 min atroom temperature in wet box. The sections werewashed twice for 5 min with PBS, and incubated witha commercial kit of chromagen DAB (DAKO Co.) atroom temperature, then washed twice for 5 min with

77M. Iwahori et al.

distilledwater.
The sections weresubsequently
counterstainedwith methylgreen,
followed
bydehydration andmounted with axylene-based
mounting medium(entellan)
andthenobserved
under an optical
microscope(BX50, Olympus,Tokyo, Japan).

ResultsandDiscussion
Wehaveinvestigatedimmunolocalization of GADs in rat
submandibulargland.
WhenFig. 1 Immunohistochemistry for GAD65 and/or GAD67 in rat submandibular gland.
anti-GAD65/67
a: Stained with anti-GAD65/67 Ab. Locations of ducts (D) are marked inantibody (Ab) wasarrowheads. Nuclei (N) are stained with methylgreen. b: Stained with anti-GAD65
employed,
Ab. c: Stained with anti-GAD67 Ab. d: No staining with Ab. Photos were taken
epidermal cells ofthrough video camera attached to the microscope having an objective lens of 40X.
ducts were stained,
whereasacinartechnical assistance of M. Yamaguchi-Yamada and S.
cells were not stained (Fig. 1a). To determine which
Imanishi are also acknowledged.
isoform, GAD65 or GAD67, exists, we stained tissues
with anti-GAD65 Ab and anti-GAD67 Ab (Fig. 1b
Referencesand 1c).
In the case using anti-GAD65 Ab,
epidermal cells of ducts were strongly stained.
1) Roberts, E. and Frankel, S., J. Biol. Chem. 188,
While in the case using anti-GAD67 Ab, epidermal
789-795 (1951).
cells of ducts were also stained but only weakly.
2)
Kurohara, S., Asai, M., Hayashi, M., Yokoigawa,I n general, GAD65 is considered to localize inK., and Ueno, H., J. Biol. Macromol. 1, 45-48
neurons and GAD67 is more widely distributed.
(2001).
However, our present results indicate both GAD3)
Erlander, M.G., Tillakaratne, N.J.K., Feldblum, S.,
isoforms being localized in ducts of submandibularPatel, N., and Tobin, A.J., Neuron 7, 91-100
gland; but, GAD65 appears to exist more than(1991).
GAD67. It is of interests to investigate the role(s) of4)
Dupuy, S.T. and Houser, C.R., J. Neurosci. 16,
GAD65 in submandibular gland.
6919-6932. (1996).
In rat’s submandibular gland, GABAA receptor
5)Ueno, H., J. Mol. Catalysis B: Enzymatic 10,
was found in acinar cells and striated ducts(7). It67-79 (2000).
is also reported that GABA inhibits salivation by6)
Sawaki, K., Ouchi, K., Sato, T., and Kawaguchi,nerve stimulation.
Therefore, it is probable toM., Jpn J Pharmacol 67, 359-363. (1995).
assume that salivation in rat is controlled by the action
7)
Shida, T., Kondo, E., Ueda, Y., Takai, N., Yoshida,of GAD locating at the ducts of submandibular gland.
Y., Araki, T., Kiyama, H., and Tohyama, M.,
Brain Res Mol Brain Res 33, 261-268. (1995).
Acknowledgements
We would like to express sincere thanks to Drs. H.
Miyamoto and N. Manabe of Kyoto University
Graduate School of Agriculture for the contribution of
their time and advice on this study. The skilful

Communicated by Sumio Ishijima