Archives of Oral Biology 113 (2020) 10468 9

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Different expression and subcellular localization of vesicular inhibitory amino acid T

transporter in ducts of major salivary glands: An in situ study in mice
Atthapon Pidsayaa, Anussara Kamnatea, Juthathip Sirisina, Masahiko Watanabeb, Hisatake Kondoa,c,
Wiphawi Hipkaeoa,*
a b
Electron Microscopy Unit, Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Department of Anatomy, Graduate School of Medicine, Hokkaido University, Sapporo, Japan c Department of Anatomy,
Graduate School of Medicine, Tohoku University, Sendai, Japan

ARTI CLEI NF O AB STRACT

Keywords: Objective: The aim of this study was clarify to the mechanism of GABA (□-amino butyric acid)-signaling in the salivary
Vesicular inhibitory amino acid transporter glands by localization of vesicular inhibitory amino acid transporter, a key molecule in GABA-synthesis. Design: Parotid,
Salivary glands sublingual and submandibular glands of mice at various postnatal stages were examined in immuno-light and electron
Mice
microscopy as well as immuno-blotting.
Duct cell
Results: Expression for vesicular inhibitory amino acid transporter was detected in parotid and sublingual glands of both sexes
Subcellular localization
Expression control
and female submandibular gland throughout postnatal development, while it was negligible in male submandibular glands at
and after puberty. The expression in female submandibular glands attenuated after testosterone injection. The
immunoreactivity was localized in striated ductal cells, but not acinar cells, in the salivary glands, and it occurred in
association with intracellular and plasma membranes of the cells. It also occurred in myoepithelial and vascular smooth
muscle cells.
Conclusions: GABA-signaling was suggested to be a significant signaling pathway in salivary ductal cells, which was
suppressed in male submandibular glands at and after puberty. The suppression in the submandibular duct was by
testosterone. In addition, the participation of vesicular inhibitory amino acid transporter in GABA signaling through plasma
membranes of the ductal cells was suggested. The significance of occurrence of the immunoreactivity in myoepithelial and
smooth muscle cells remains to be further elucidated in terms of implication in GABA signaling.

1. Introduction (Bormann, 2000; Chaudhry et al., 1998; Chebib & Johnston,

There have been studies reporting that GABA (gamma-amino
butyric acid), its synthesizing enzyme termed glutamate
decarboxylase and GABA-receptors are contained in three major
salivary glands, parotid, sublingual and submandibular glands, of
rodents, and the GABA levels in the glands have been reported to
significantly decrease by administration of inhibitors to glutamate
decarboxylase. Furthermore, GABA has been shown to inhibit
some cellular functions in the salivary glands (Kosuge et al., 2009;
Sawaki, Ouchi, Sato, & Kawaguchi, 1995; Shida et al., 1995).
Therefore, the localization of molecules involved in the GABA-
signaling is critical to understand its functional significance in the
salivary glands.
⁎
Corresponding author.
E-mail address: wiphawi@kku.ac.th (W. Hipkaeo).
https://doi.org/10.1016/j.archoralbio.2020.104689
Received 21 December 2019; Received in revised form 25 February 2020; Accepted 26 February 2020 0003-9969/ ©
2020 Published by Elsevier Ltd.
Among molecules involved in the metabolism of GABA,
vesicular inhibitory amino acid transporter (VIAAT) for GABA
and glycine has originally been identified in rodent brain
1999; Hsu et al., 1999; McIntire, Reimer, Schuske, Edwards, &
Jorgensen, 1997; Sagné et al., 1997; Watanabe, Maemura,
Kanbara, Tamayama, & Hayasaki, 2002). There have
subsequently been studies indicating that immunoreactivity for
VIAAT is a good marker for GABA-ergic neurons in the brain
and some of peripheral pancreatic islet cells containing GABA
and glutamate decarboxylase (Chessler, Simonson, Sweet, &
Hammerle, 2002; Gammelsaeter et al., 2004; Suckow et al.,
2006). In their studies, VIAAT-immunoreactivity was shown at
ultrastructural levels to be localized in the membranes of
inhibitory neuronal synaptic vesicles in the brain and those of
synaptic-like microvesicles and secretory granules in the
peripheral cells.
Considering this information described above, the present
authors have previously reported the occurrence of VIAAT-like
immunoreactivity in the submandibular glands of postnatal mice
using immuno-light microscopy (Toomsan et al., 2015). In that
study, the immunoreactivity was localized in ductal epithelial cells
with the striated duct more intensely immunopositive at young
postnatal stages, and it was progressively attenuated throughout the
ducts at and after puberty, while no significant immunoreactivity
was seen in the acinar cells throughout the postnatal development.
A. Pidsaya, et al.
As a result, the study strongly suggested the possible synthesis and
secretion of GABA mainly in ductal epithelial cells of the glands at
early postnatal mice. The study leads us to examine the expression
and localization of VIAAT in the other two major salivary glands,
parotid and sublingual glands, and the subcellular localization of
VIAAT in ductal cells of the glands. The results of that
investigation, based on immuno-blotting and immunoelectron
microscopy, are presented here.
2. Materials and methods
2.1. Animals
A total of thirty three ICR mice at stages of the postnatal 2
week were purchased from Northeast Laboratory Animal Center,
Khon Kaen University, Khon Kaen, Thailand and kept under
standard laboratory conditions with foods and water ad libitum.
Three pairs of male and female mice were prepared at each age
stage of the postnatal 2, 4 and 8 weeks (total 9 males and 9
females) and used to examine the expression and localization of
VIAAT in the major three salivary glands during postnatal
development. For analysis of testosterone effects, fifteen female
mice at postnatal 6 week were prepared and divided into five
groups of three each: four groups of them were subjected to a daily
subcutaneous injection of testosterone (Panreac Applichem,
Ottoweg, Germany) at 25 mg/kg dissolved in 0.1 ml of olive oil.
Each of the testosterone-injected groups were sacrificed at 1st, 3rd,
7th, and 14th days after start of the injection. The remaining one
group of three female mice at the postnata 6 week were subjected
to a subcutaneous injection of the 0.1 ml olive oil alone as the
vehicle for 14 consecutive days until their age reached the postnatal
8 week. All procedures were performed in accordance with
Guidelines for the Care and Use of Laboratory Animals at Khon
Kaen University. This study was reviewed and approved by the
Institutional Animal Care and Use Committee of Khon Kaen
University, following the Ethics of Animal Experimentation of the
National Research Council of Thailand (Reference No.
IACUCKKU-103/62).
2.2. Immunoblotting analysis
Mice at the various postnatal stages were sacrificed by deep
anesthesia with sodium pentobarbital (60 mg/body weight) and
subsequently the three major salivary glands at the right hand side
were extirpated, after ligation of blood vessels relating to them
under stereo microscope, and frozen at −80 °C until use. The frozen
glands were homogenized in a lysis buffer containing the proteinase
inhibitor cocktail (Roche; Mannheim, Germany). The lysates were
centrifuged at 12,000 rpm. The supernatants were measured for the
protein concentration, and proteins of 40 μg in total from each
lysate were boiled for 10 min in 2x SDS (sodium dodecyl sulfate)
sample buffer and subjected to SDS/10 % PAGE (polyacrylamide
gel electrophoresis). They were then blotted to a PVDF
(polyvinylidene difluoride) membranes (GE Healthcare;
Buckinghamshire, UK). After blocking non-specific binding sites
with 5% skim milk (wt/vol)/TBS (Tris-buffered saline)/ 0.3 %
tween-20, the membranes were incubated overnight at 4 °C with an
antibody against VIAAT (0.1 μg/ml) in 5% skim milk
(wt/vol)/TBS/ 0.1 % tween-20. The antibody, the same as used in
our previous study (Toomsan et al., 2015), was a rabbit IgG against
mouse vesicular inhibitory amino acid transporter (VIAAT) (RRID,
AB_2571622; Fukudome et al., 2004). The membranes were then
treated with mouse peroxidase-conjugated anti-rabbit IgG (dilution
1: 2500) for 1 h at room temperature. Goat polyclonal antibody
against β-actin was used as the internal control. Student’s t-test of
Archives of Oral Biology 113 (2020) 104689
statistical analysis was used to analyze the difference between two
mean values. The post hoc tests in the ANOVA was used to analyze
the difference among multiple mean values, and resulting P-value
less than 0.05 was regarded as significant difference.
2.3. Immuno-light microscopy
The mice at the various postnatal stages, soon after the right
glands were extirpated, were perfused through the heart with 10 ml
physiological saline, followed by 10 ml of 4% paraformaldehyde in
0.1 M phosphate buffer. Three major salivary glands at the left
hand side were removed, then postfixed with the same fixative for
overnight. Specimens were dipped into 30 % sucrose/PB for cryo-
protection. Sections of 20 μm thickness were made on a cryostat
and mounted on glass slides. After incubation for 10 min with 0.3
% H2O2/methanol to inhibit intrinsic peroxidase activity, the
sections on glass slides were further incubated for 30 min with 10
% normal goat serum in phosphate buffer saline to prevent non-
specific binding of the antibody. They were then incubated at room
temperature overnight with the rabbit IgG against VIAAT (1
μg/ml), and subsequently with biotinylated goat anti-rabbit IgG (H
+ L) (Abcam; Cambridge, MA USA) for 1 h at room temperature.
The antigen-antibody reaction sites were visualized using
diaminobenzidine reaction with an ABC kit (Vector Laboratories;
Burlingame, CA, USA).
2.4. Immuno-gold electron microscopy
Cryostat-sections were mounted on 0.1 % poly-L-lysine coated
plastic slides. After blocking nonspecific reactions by 5% bovine
serum albumin and overnight incubation with the primary antibody
(5 μg/ ml), the sections were reacted with goat anti-rabbit IgG
covalently conjugated with ultrasmall gold particles (1:100 in
dilution) (Nanoprobes; Yaphank, NY, USA). Following silver
enhancement using a silver enhancement kit (Aurion; Hatfield, PA,
USA), the sections were then postfixed with 1% osmium tetroxide,
dehydrated, embedded in Epon, and polymerized in 60 °C oven for
48 h. Ultrathin sections were prepared and mounted on single-hole
grids covered with formvar-films. The sections were then stained
with uranyl acetate and lead citrate for observation and analysis in
electron microscopy.
In the antigen pre-absorption tests for control of the
immunohistochemistry, the primary antibody was first incubated
with synthetic VIAAT antigen (100 □g/ml). Sections were then
incubated with the pre-absorption antibody for 1 h at room
temperature, and subsequently treated in the same procedure for
immunohistochemistry as described above.
3. Results
3.1. Protein expression levels of vesicular inhibitory amino acid
transporter (VIAAT) in major salivary glands of adult male mice
In immunoblot analysis, a band with a size of approximately 57
kDa was seen in parotid and sublingual glands of young adult male
mice at the postnatal 8 week without statistical significances of the
band density difference between the two glands. In contrast, no
such an immunoblot band was clearly discerned in male
submandibular glands at the stage. A band with the same size and a
much higher density was seen in the brain, used as the positive
control, from mice at the postnatal 8 week (Fig. 1).
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A. Pidsaya, et al.
3.2. Localization of VIAAT in adult parotid and sublingual glands
in immuno-light microscopy
Based on the expression pattern described above, the
localization of VIAAT-immunoreactivity in male parotid and
sublingual glands at the postnatal 8 week was examined in
immuno-light microscopy. The immunoreactivity was seen
throughout the cytoplasm, but not in the nuclei, of almost all
striated and excretory ductal cells. In numerous ductal cells, the
immunoreactivity was higher in their apical/supra-nuclear cell
domain than the basal/infra-nuclear cell domain, resulting in an
apical-to-basal polarity in terms of the immuno-intensity. Because
cells with such apically polarized immunoreactivity are often
aligned consecutively, their apical domain tended to appear as a
densely immunoreactive zone adjacent to the lumen along the
trajectory of the duct. In a relatively small number of cells, the
immunoreactivity in the basal domain including the basal striations
was as dense as in the apical domain, leading to indistinct polarity.
In addition, a few cells showed weaker immunoreactivity for
VIAAT without distinct polarity. The immunoreactivity was weak
in intercalated ductal cells and numerous myoepithelial cells
surrounding acini, but no significant immunoreactivity was
discerned in almost all acinar cells (Fig. 2a–c). In addition,
immunoreactivity for VIAAT was found in vascular medial smooth
muscle cells of interlobular arterioles (Fig. 2a–c), in accord with
the finding of our previous study on the submandibular gland
(Toomsan et al., 2015).
3.3. Subcellular localization of VIAAT in adult parotid and
sublingual gland
In immuno-electron microscopy, gold particles representing the
immunoreaction were often seen more conspicuously in the apical/
supranuclear domains than in the basal/infranuclear domains in
most ductal cells, while relatively reflecting the immuno-light
microscopic finding (Fig. 3a–c). They were often in association
with apical plasma membranes, especially those of microvilli (Fig.
3a). Intracellularly, gold particles were associated with round and
oblong vesicles of small sizes which were randomly found in apical
cell domains. No particles were found in association with Golgi
apparatus or rough endoplasmic reticulum (Fig. 3a, b). On the other
hand, gold particles were relatively sparse in the basal cell domain
of most ductal cells and they were associated with the plasma
membranes of basal infoldings (Fig. 3c).
Reflecting the immuno-light microscopic finding, gold particles
were scarcely seen in almost all acinar cells containing typical
secretory granules (Fig. 4a). On the other hand, a considerable
number of gold particles were deposited in myoepithelial cells in
close apposition to the acinar cells, and also in vascular medial
smooth muscle cells, and they were associated with myofilament
bundles, but not with the plasma membranes including caveolar
membranes of the smooth muscle cells (Fig. 4a, b).
Archives of Oral Biology 113 (2020) 104689
Fig. 1. Immunoblotting bands with bar charts
representing their densities as the mean± SEM for
vesicular inhibitory amino acid transporter (VIAAT)
in parotid (PG), sublingual (SLG) and
submandibular (SMG) glands as well as whole brain
of male mice at the postnatal 8 week (P8W) (young
adult). Note the absence of expression of VIAAT in
SMG in contrast to its positive expression in PG and
SLG at P8W. Simultaneous blot-bands for β-actin
were utilized as controls for evaluation of statistical
differences in the expression levels between two of
the three glands. According to Student’s
t-test, expression levels of VIAAT in the
PG and the SLG were significantly
greater (P < 0.05) than in the SMG, as
signified by horizontal lines in the bar
charts.
3.4. Sexual dimorphism in VIAAT-expression in the submandibular
gland
Because of a lack of distinct expression for VIAAT in
immunoblot of young adult male submandibular glands, in order to
see whether or not the lack was the case only in adult male
submandibular glands, the expression was examined first in
immunoblots of submandibular glands of both sexes throughout the
course of postnatal development, in comparison with parotid and
sublingual glands. First, in immunoblots of parotid and sublingual
glands, such a blot-band for VIAAT as seen in the two male glands
at the postnatal 8 week was constantly seen throughout the stage
from the postnatal 2 week to the postnatal 8 week without
statistical differences in the density between male and female at
each stage (Fig. 5a). In submandibular glands, on the other hand, a
blot band for VIAAT was discerned at the postnatal 2 week without
statistical difference in the density between male and female. The
immunoblot band was attenuated at the postnatal 4 week, and
almost negligible at the postnatal 8 week in the male glands, while
it remained detectable in the female glands at postnatal 4 and 8
weeks (Fig. 5b).
Based on the immunoblot findings, immunohistochemical
analysis was applied to female submandibular glands at the
postnatal 8 week. Distinct immunoreactivity for VIAAT was seen
mainly in striated duct cells, but not in granular convoluted tubule
cells which often appeared in mixture with striated cells along the
duct trajectory (Fig. 6a). In contrast, in accord with the findings in
the present immunoblot analysis described above and in our
previous immunohistochemical study (Toomsan et al., 2015), well-
developed granular convoluted tubule cells comprising a majority
of intralobular ducts in male submandibular glands showed actual
absence of the immunoreactivity although a few intermingled
striated cells showed the immunoreactivity weakly (Fig. 6b). Weak
immunoreactivity was also seen in myoepithelial and vascular
smooth muscle cells similar to those in the parotid and sublingual
glands at the postnatal 8 week described above.
Because of the sexual difference in the expression intensity in
immunoblots of submandibular glands at the postnatal 4 and 8
weeks, the daily injection of testosterone in female mice was
performed starting at the postnatal 6 week and lasting for two
weeks, and the expression levels were examined in
immunoblotting. The immunoblot band in female submandibular
glands weakened in density gradually with postinjection days, and
it was at a considerably low level at 14 th post-injection days, with a
significant difference from that at the postnatal 8 week in normal
postnatal development of female submandibular glands (Fig. 5c).
No significant differences in the density of the blot bands were
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A. Pidsaya, et al.
discerned between submandibular glands from female mice with
the vehicle-injection for 14 days and those without injection at the
postnatal 8 week (data not shown).
In the control with antigen pre-absorption test, no significant
immunoreaction was found in any portions of sections from all the
major salivary glands (Fig. 7).
4. Discussion
We believe that the VIAAT (vesicular inhibitory amino acid
transporter)-immunopositive sites we detected in the salivary
glands of mice represent GABA-synthesizing sites. The evidence
to support this statement includes the following: 1) we obtained an
immunoblot band with a size equivalent to the brain VIAAT of
mouse VIAAT (Bedet, Isambert, Henry, & Gasnier, 2002;
Dumoulin et al., 1999) in adult parotid and sublingual glands as
well as the brain used as a positive control; 2) the presence of
enzyme molecules involved in the synthesis of GABA in rat
salivary glands has already been shown by others (Kosuge et al.,
2009; Sawaki et al., 1995) as cited in Introduction, 3) We discerned
no significant immunoreaction in any portions of the sections
treated as controls for immunohistochemistry by antigen-
absorption of the primary antibody. The final confirmation requires
examination of the glands from gene-knockout mice for VIAAT.
Two major findings should be noted in the present study. The
first is the constant expression without sexual differences and
localization of VIAAT in ductal cells, but not acinar cells,
throughout the postnatal development of parotid and sublingual
glands. The second is the sexual dimorphism in its expression in
submandibular glands at and after puberty.
The lack of significant immunoreactivity for VIAAT in most
acinar cells, the major producers of saliva, in contrast to its
Archives of Oral Biology 113 (2020) 104689
immunoreactivity in ductal cells, the main modifiers of saliva, was
a finding that we had not expected. In fact, previous
pharmacological studies have shown that benzodiazepines, whose
binding site is contained in GABA receptor molecules (Barnard et
al., 1998), decrease the secretion/release of fluids and amylase
elicited by secretagogues in acinar cells of the adult rat parotid
Fig. 2. a-c Immuno-light micrographs for VIAAT of
the parotid gland (PG) (2a, and 2b at higher
magnification) and the sublingual gland (SLG) (2c)
of male mice at P8W. Note distinct
immunoreactivity for VIAAT in epithelial cells of
striated (S) and excretory (E) ducts, in contrast to
actual absence of immunoreactivity in acinar cells
(a) and faint immunoreactivity in intercalated (I)
ductal cells. Also note occurrence of higher
immunoreactivity in the apical cellular domain than
in the basal domain in numerous ductal cells,
resulting in a densely immunoreactive zone adjacent
to the lumen and an apical-to-basal polarity along
the ducts (white arrowheads). Broken line (2b)
indicates dense immunoreactivity in the basal
domains as well as the apical domains of some cells,
resulting in indistinct polarity. Black arrows indicate
weakly immunoreactive myoepithelial cells among
immunonegative acinar cells. Further note distinct
immunoreactivity in vascular walls (*) of intra-
glandular arterioles (V).
Scale bars represent 25 μm.
gland in vivo and in vitro (Okubo & Kawaguchi, 1998).
Assuming that non-detection of distinct immunoreaction in
immunohistochemistry does not necessarily represent the real
absence of the molecule, it can be at least concluded that the
activity of VIAAT is lower in most acinar cells than in ductal cells
in situ in the two glands, at least in ICR mice.
Next, the subcellular localization of VIAAT-immunoreactivity
in the salivary glandular cells of young adult mice requires
comments. Immunoreactivity for VIAAT was localized to certain
small vesicles randomly distributed largely in the
apical/supranuclear cell domains of ductal cells of the parotid and
sublingual glands. This is compatible with the known findings:
VIAAT was originally identified as a protein in synaptic vesicle
membranes of central nerve terminals (Chaudhry et al., 1998), and
immunoreactivity for VIAAT has subsequently been localized in
synaptic vesicle membranes of central neurons and in the
membranes of synaptic-like microvesicles and endocrine secretory
granules of peripheral non-neuronal cells, that is, its localization on
intracellular membranes (Gammelsaeter et al., 2004; Kudo et al.,
2012; Miyazaki, Fukaya, Shimizu, & Watanabe, 2003). In contrast,
the present frequent association of immunoreactivity for VIAAT
with the plasma membranes in ductal cells is rather difficult to be
reconciled with the original information of this molecule structure.
VIAAT-immunoreactivity in association with plasma
membranes has recently been reported for the first time in a
previous study by the present author group (Sakaew et al., 2019). In
4
A. Pidsaya, et al.
that study, VIAAT-immunoreactivity was mainly localized on
basal, but not ‘apical’ as seen in the present study, plasma
membranes of the basal infoldings of renal distal tubular cells.
Several possible interpretations, as already discussed in that study,
are also applicable to the present finding: For example,
immunoreactivity for VIAAT on the plasma membranes is ascribed
to an incorporation of VIAAT originally located on the vesicle
Fig. 3. a-c Immuno-gold electron micrographs for VIAAT in parotid ductal
cells at P8W. Note gold particles associated with apical plasma membranes
including apical microvilli (white asterisks in Fig. 3a) as well as with small
elliptical and flattened vesicles/vacuoles in the supranuclear cellular domain
(white arrowheads). Insets show areas marked by rectangles at higher
magnification. Also note some gold particles associated with basal infolding
membranes (white arrows in Fig. 3c). B: basement membrane; G: Golgi
apparatus; L: lumen; m: mitochondria; Nu: nucleus; rER: rough endoplasmic
reticulum. Scale bars represent 600 nm.
Archives of Oral Biology 113 (2020) 104689
membranes into such selected domains of the plasma membranes
as sites of active exocytosis, as already suggested for choline
transporter in nerve endings by Ferguson et al. (2003). Another
interpretation is that an as yet-unidentified plasma membranous
GABA transporter (GAT) cross-reacts with the VIAAT antibody we
used. This is, however, less likely because none of the four already
known GAT isoforms show any similarity to VIAAT in their
molecular structure (Zhou & Danbolt, 2013). On the other hand,
inositol triphosphate (IP3)-receptor type 2 may be localized in the
plasma membrane (IP3 is generally found in intracellular
membranes of endoplasmic reticulum (Dingsdale, Voronina,
Haynes, Tepikin, & Lur, 2012; Vermassen, Parys, & Mauger,
2004). If the first possible interpretation is correct, GABA could be
released more frequently into the ductal luminal space, and
somehow also into the interstitial spaces surrounding the ducts,
resulting in its autocrine and paracrine effects on the ductal cells
which have been reported to be immunoreactive for GABA
receptors (Shida et al., 1995).
The second major finding is the detection of VIAAT-expression
only in female submandibular glands, but not in male glands, at and
after puberty (approximately postnatal week 4), in contrast to the
expression in the submandibular glands of both sexes before
puberty. This occurs despite the similar levels of expression for
VIAAT in the parotid and sublingual glands of both sexes
throughout the postnatal 8 week period studied. The significant
attenuation of its expression in response to the injection of
testosterone in the female submandibular glands after puberty
suggests a role of testosterone. Female dominant expression in
Fig. 4. a, b Immuno-gold electron micrographs for VIAAT of an
immunoreactive myoepithelial cell (ME) (4a) and a medial smooth muscle
cell (SM) (4b) of interlobular arterioles in a parotid gland at P8W. Note gold
particles associated with filament bundles in both cells. Also note absence of
gold particles in an acinar cell (A) characterized by specific secretory
granules (g) and direct contacts, without intervening basal lamina, with the
myoepithelial cell (ME) (4a). Further note absence of gold particles in
association with plasma membranes including caveolae (white ellipses) in
the smooth muscle cell (SM) (4b). E: endothelial cell; IS: interstitial space;
L: vascular lumen; Nu: nucleus. Scale bars represents 800 nm (4a) and 1 μm
(4b).
the submandibular glands of adult mice has been reported for
several molecules, such as members of Jun and Fos families in
ductal cells of the intercalated and adjacent striated ducts, and
submandibular androgen-repressed protein in acinar cells (Hipkaeo,
Wakayama, Yamamoto, & Iseki, 2004; Sakulsak, Wakayama,
Hipkaeo, & Iseki, 2007), phospholipase D1 in granular convoluted
tubule cells and diacylglycerol kinase δ in terminal tubule cells
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A. Pidsaya, et al.
(Hipkaeo et al., 2015; Khrongyut, Polsan et al., 2019). Other
examples for the female dominant expression in the submandibular
glands are phosphodiesterase 2A in acinar cells and striated ductal
cells (Adthapanyawanich, Nakata, & Iseki, 2018), and
submandibular gland protein C (SMGC) in terminal tubule
/granular intercalated duct cells (Hecht, Connelly, Marchetti,
Ball, & Hand, 2000; Yamamoto, Nakata,
Kumchantuek,
Adhapanyawanich, & Iseki, 2018). In addition, we have recently
demonstrated no or weak expression of endogenous
phosphatidylinositol 4-phosphate 5-kinase (PIP5K) and
phospholipase Cβ3 (PLCβ3) in adult male submandibular glands,
despite the significant expression of these molecules in adult male
parotid and sublingual glands (Khrongyut, Rawangwong et al.,
2019; Rawangwong et al., 2019). This situation for PIPK and
PLCβ3 in adult male submandibular glands may also represent
female dominance in expression after puberty. The molecular
Archives of Oral Biology 113 (2020) 104689
mechanism controlling the expression of VIAAT as well as the
other molecules listed above remains to be elucidated.
The suppression control of VIAAT-expression in the
submandibular gland by testosterone suggests that this hormone
might influence the level of GABA itself and its signaling in the
submandibular gland, separately from the parotid and/or sublingual
glands. No studies on salivary glands have so far supported this
hypothesis. However, a sexual dimorphism exists in the cell
population density of GABA-ergic neurons marked by VIAAT-
immunoreactivity in the brain of rodents (Marshall, Desroziers,
McLennan, & Campbell, 2017; Ottem, Godwin, Krishnan, &
Petersen, 2004). There have also been studies on quantitative
changes in GABA amounts in the brain of fishes (Bosma et al.,
2001) and changes in expression of mRNA for GAD in the brain of
rats by extrinsic administration of testosterone (Davis, Grattan,
Selmanoff, & Mccarthy, 1996). Whether or not the sexual
dimorphism in GABA signaling occurs in mouse submandibular
glands remains to be elucidated.
Fig. 5. a, b. Immunoblotting bands for
VIAAT with bar charts representing their
densities as the mean ± SEM in parotid
(PG) and sublingual glands (SLG) (5a)
and submandibular glands (SMG) (5b) of
mice of both sexes at the postnatal 2
week (P2W) (before puberty), P4W
(puberty) and P8W (after puberty/young
adults), and in female SMGs after
testosterone administration (5c). Note the
constant expression of VIAAT without
statistical differences in expression
intensities between male and female in
PGs and SLGs at P2W-P8W, and its
expression without sexual difference in
intensity in SMGs at P2W. Also note the
attenuation of VIAAT expression in male
SMGs at P4W and its expression only in
female SMGs at P8W. Further note the
progressive and significant suppression
of expression of VIAAT by testosterone
(TT) injection in female SMG at post-
injection 1 day (1D TT), 3 days (3D TT),
7 days (7D TT) and 14 days (14D TT).
Simultaneous blot-bands for □-actin were
utilized as controls for evaluation of
statistical differences between VIAAT-
expression levels at individual normal
and experimental stages. Where
significant differences (P < 0.05) are
detected, according to Student’s t-test, in
band densities, these are indicated by
horizontal lines bridging columns in the
bar charts.
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Fig. 7. Immuno-light micrograph of a PG of a male mouse at postnatal 8
week (P8W) treated with antigen-pre-absorption for vesicular inhibitory
amino acid transporter (VIAAT) as the control. No significant
immunoreaction is seen anywhere in the section. a: acini, E: excretory duct,
I: intercalated duct, S: striated duct, V: blood vessel. Scale bar represents 50
μm.
On the other hand, a previous study has shown that the
concentration of GABA and activities of glutamate decarboxylase
and GABA-transaminase are higher in the submandibular gland
than in the parotid gland of adult male rats (Sawaki et al., 1995).
Their finding is discrepant from the present lack of VIAAT-
immunoreactivity in the submandibular gland despite its positive
reaction in the parotid gland of adult male mice. In order to clarify
this discrepancy, the species difference should be first taken into
consideration, because the development of the granular convoluted
tubules, to which the sexual dimorphism is largely ascribed, is
evident in mice, but not in rats (Gresik, 1994). Therefore, the
biochemical analysis of the GABA-related molecules is first
necessary in the salivary glands of mice. Another issue to be
deliberated is the occurrence of discrepancy between findings on
bioactivities (in biochemistry/pharmacology) of a given molecule
and those on its fixed immunoreactive products (in microscopy).
This is exemplified by the rather unexpected lack of VIAAT-
immunoreactivity in acinar cells as already discussed in the
beginning of this Discussion. Needless to say, any methodologies
have merits and demerits, and it is difficult to clarify how much the
immunoreactive intensity of a given molecule is correlative to its
bio-activity level, and how accurate the
biochemical/pharmacological methodology is able to differentiate
acini from ducts. Therefore, further careful comparison between
Archives of Oral Biology 113 (2020) 104689
Fig. 6. a, b. Immuno-light micrographs of
a submandibular gland of a female
mouse (6a) and a male mouse (6b) at
P8W. Note distinct immunoreactivity in
striated duct cells (S), and lack of
significant immunoreactivity in granular
convoluted tubule cells (GCT) which
often appear in mixture with striated
cells along the duct trajectory in the
female submandibular gland. In contrast,
well-developed GCT cells comprising a
majority of ducts in the male
submandibular gland show actual
absence of the immunoreactivity with a
few intermingled striated cells (S)
showing the immunoreactivity. Also note
weak immunoreactivity in forms of thin
lines along the bases of acinar cells
(arrows). E: excretory duct. Scale bars
represent 50 μm.
findings by the two methodologies would eventually give us some
clarification on this discrepancy.
Lastly, the deposits of immunoreactive gold particles in
myoepithelial cells and vascular smooth muscle cells in parotid and
sublingual glands are noted. The subcellular localization of VIAAT-
immunoreactivity in these two cell types is another enigma based
on the original information about this molecule structure as
discussed above, because we mainly saw the immunoreactivity in
association with myofilaments, but not with intracellular vesicle
membranes or even with plasma membranes including caveolae.
On the other hand, one study showed the expression of glutamate
decarboxylase isoforms and GABA transporter-2 and -4 in native
and cultured airway smooth muscles of humans but failure to detect
neuronal GABA transporter (Zaidi et al., 2011). According to that
study, functional inhibition of 3HGABA uptake occurred in the
smooth muscles of humans, and a mechanism was proposed in
which locally synthesized GABA can be released from the muscle
cells into the airway to activate GABA receptors, with subsequent
autocrine and/or paracrine signaling effects on the smooth muscle
cells. A similar mode of GABA-signaling may occur in
myoepithelial cells and intraglandular vascular smooth muscle
cells. Although the preabsorption control was negative as shown in
Fig. 7, and other controls using non-immune IgG and several
antibodies unrelated to VIAAT also were negative (data not
shown), further analyses remain to be elucidated concerning the
functional significance of VIAAT-immunoreactivity associated with
myofilaments in these two cells of the glands, including
localization of GABA-signaling molecules other than VIAAT and
careful confirmation of the authenticity of VIAAT- immunoreaction
by using its mutant mice.
Author statements
AP, AK and JS performed acquisition of data and analysis and
interpretation of data, while.
MW, HK and WH performed conception and design of the
study. AP, AK, JS, WH and HK performed drafting the article,
while.
MW, WH and HK performed revising it critically for important
intellectual content.
All (AP, AK, JS, MW, HK and WH) performed final approval
of the version to be submitted. Declaration of Competing Interest
None.
7
A. Pidsaya, et al.
Acknowledgments
This work was supported by the Invitation Grants, Faculty of
Medicine, Khon Kaen University, Thailand (grant number IN63110
to W.H.). Sincerely thanks go to Mr. D. Hipkaeo and Ms Y. Polsan
for their technical supports. We would like to acknowledge Prof.
David Blair for editing the manuscript via Publication Clinic KKU,
Thailand.
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