The human submandibular gland: immunohistochemical analysis of SNAREs and cytoskeletal proteins.
ABSTRACT Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.
- [show abstract] [hide abstract]
ABSTRACT: It is not known whether the mechanisms involved in amylase release in submandibular and parotid glands are similar. Here, the participation of different signalling pathways in amylase release by the parotid and submandibular glands of the male rat was compared by studying the secretory response after beta-adrenergic stimulation. The beta-adrenergic agonist isoproterenol induced an increase of cAMP in both salivary glands, but while in the parotid it triggered amylase release, in the submandibular it was unable to increase amylase secretion. Parotid amylase release was dependent on adenylate cyclase activation, as SQ-22536 inhibited the secretory effect. In contrast, submandibular amylase secretion did not depend on the intracellular concentration of cAMP, as SQ-22536 did not modify its secretory response. Moreover, other activators of adenylate cyclase, such as forskolin and prostaglandin E2, also failed to modify amylase release by the submandibular gland. Neither ionophores nor calcium-blocking agents, as well as calcium-calmodulin and nitric oxide synthase inhibitors, were effective in modifying basal amylase release by the submandibular gland. However, the disruption of microfilaments with cytochalasin B, but not the disruption of microtubules with colchicine, prevented amylase release in that gland. It is concluded that amylase exocytosis in the submandibular gland is a constitutive non-regulated phenomenon, as it is independent of extracellular or intracellular signals. It depends only on the integrity of the microfilaments, probably used by the vesicles to travel from the Golgi apparatus to the plasma membrane.Archives of Oral Biology 11/2002; 47(10):717-22. · 1.55 Impact Factor
- Journal of Electron Microscopy 02/1966; 15(1):1-14. · 1.44 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: SNAP-23 is the ubiquitously expressed homologue of the neuronal SNAP-25, which functions in synaptic vesicle fusion. We have investigated the subcellular localization of SNAP-23 in polarized epithelial cells. In hepatocyte-derived HepG2 cells and in Madin-Darby canine kidney (MDCK) cells, the majority of SNAP-23 was present at both the basolateral and apical plasma membrane domains with little intracellular localization. This suggests that SNAP-23 does not function in intracellular fusion events but rather as a general plasma membrane t-SNARE. Canine SNAP-23 is efficiently cleaved by the botulinum neurotoxin E, suggesting that it is the toxin-sensitive factor previously found to be involved in plasma membrane fusion in MDCK cells. The localization of SNAP-25 in transfected MDCK cells was studied for comparison and was found to be identical to SNAP-23 with the exception that SNAP-25 was transported to the primary cilia protruding from the apical plasma membrane, which suggests that subtle differences in the targeting signals of both proteins exist. In contrast to its behavior in neurons, the distribution of SNAP-25 in MDCK cells remained unaltered by treatment with dibutyryl cAMP or forskolin, which, however, caused an increased growth of the primary cilia. Finally, we found that SNAP-23/25 and syntaxin 1A, when co-expressed in MDCK cells, do not stably interact with each other but are independently targeted to the plasma membrane and lysosomes, respectively.Journal of Biological Chemistry 02/1998; 273(6):3422-3430. · 4.65 Impact Factor
Journal of Histochemistry & Cytochemistry 60(2) 110 –120
© The Author(s) 2012
Reprints and permission:
The submandibular glands are located, on either side,
between the mandible and the muscles that form the floor of
the mouth. As a mixed gland, the serous acini are more
numerous than the mucous acini. The intercalated ducts are
relatively short; the striated ducts are longer. In humans,
70% of the salivary gland accounts for the submandibular
glands (Bloom and Fawcett 1994). Salivary proteins are
secreted by exocytosis (i.e., the fusion between secretory
granule membranes and the apical plasma membrane of
salivary acinar cells).
Soluble NSF attachment protein receptor (SNARE) pro-
teins were first studied in neuronal cells and are involved in
the exocytosis system, that is, in neurotransmission (Pfeffer
1996; Goda 1997). The SNARE hypothesis indicates that
v-SNAREs are localized in vesicles and t-SNAREs at the
target membrane (Söllner, Bennett, et al. 1993). A transport
vesicle chooses its target for fusion when a soluble NSF-
attachment protein (SNAP) receptor on the vesicle
(v-SNARE) pairs with its cognate t-SNARE at the target
membrane. SNARE proteins not only are found in synapto-
somes but also have various homologues dealing with the
common form of vesicular transport in many cells (Bennett
2012© The Author(s) 2012
elhuber et al.Exocytosis in the Human Submandibular Gland
Reprints and permission:
Received for publication July 7, 2011; accepted November 11, 2011.
Mechthild Stoeckelhuber, Department of Oral and Maxillofacial Surgery,
Technische Universität München, Ismaninger Strasse 22, 81675 Munich,
The Human Submandibular Gland: Immunohistochemical
Analysis of SNAREs and Cytoskeletal Proteins
Mechthild Stoeckelhuber, Elias Q. Scherer, Klaus-Peter Janssen, Julia Slotta-Huspenina,
Denys J. Loeffelbein, Nils H. Rohleder, Markus Nieberler, Rafael Hasler, and Marco R.
Department of Oral and Maxillofacial Surgery (MS,DJL,NHL,MN,RH,MRK), Department of Otorhinolaryngology (EQS), Department of Surgery (K-PJ),
and Institute of Pathology (JS-H), Technische Universität München, Munich, Germany.
Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the
exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)–
mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant
data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated
the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry
and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2,
syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles
in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and
α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2
in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at
the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the
complex exocytosis mechanisms. (J Histochem Cytochem 60:110–120, 2012)
salivary glands, secretory pathway, exocytosis, cytoskeleton, immunohistochemistry
Exocytosis in the Human Submandibular Gland
et al. 1993; McMahon et al. 1993). Mammalian SNAREs
comprise three conserved families: synaptobrevin/vesicle-
associated membrane proteins (VAMPs), syntaxins, and
SNAP-25 homologues. VAMPs are categorized as v-SNAREs,
syntaxins, and SNAP-25 homologues as t-SNAREs. In neu-
ronal cells, VAMP-2/synaptobrevin binds specifically to a
heterodimeric complex of syntaxin-1 and SNAP-25 in the
plasma membrane (Söllner, Whiteheart, et al. 1993).
Syntaxin-3 and VAMP-2 are known to form an apical
SNARE complex in stimulated lacrimal acini (Sou et al.
2005) and in gastric parietal cells (Ammar et al. 2002). In
acinar cells from the pancreas gland, VAMP-2 was expressed
at the apical region as a concentrated border around the aci-
nar lumen (Braun et al. 1994). Oishi et al. (2006) demon-
strated VAMP-2 in small cytoplasmic vesicles of a human
parotid epithelial cell line using a VAMP2-GFP construct.
Syntaxin-4, SNAP-23, and VAMP-8 regulate the exocytosis
in mast cells (Paumet et al. 2000). In parotid and pancreatic
acinar cells, several proteins that are expressed ubiquitously
in non-neuronal cells have been detected—for example, NSF,
α-SNAP, VAMP-2, syntaxin-4, and SNAP-23 (Braun et al.
1994; Ravichandran et al. 1996). However, when VAMP-2
was immunoprecipitated from lysates of parotid acinar cells,
syntaxin-4 and SNAP-23 were not coprecipitated with
VAMP-2 (Takuma et al. 2000). Imai et al. (2003) studied the
intracellular localization of SNARE proteins by Western blot-
ting and immunocytochemistry and found that in rat parotid
acinar cells, syntaxin-2 and -3 were detected in the apical
plasma membrane, and in addition, syntaxin-4 was localized
in the basolateral membrane. Septins, proteins usually identi-
fied in processes of cytokinesis, may be involved in vesicle
targeting or tethering (Kartmann and Roth 2001). Hsu et al.
(1998) isolated a large septin complex, which helps tether
vesicles to specialized regions of the plasma membrane.
Cytoskeletal proteins are essential for regulating cytoskel-
etal dynamics and participate in the process of exocytosis.
Myosin II plays a role in the secretory processes of a variety
of cells such as pancreatic acinar cells (Bhat and Thorn 2009),
lacrimal acinar epithelial cells (Jerdeva et al. 2005), mast
cells (Ludowyke et al. 2006), natural killer cells (Andzelm
et al. 2007), and neurons (Mochida et al. 1994). Here, myosin II
is necessary for the maintained opening of the fusion
pore (Bhat and Thorn 2009). In pancreatic acinar cells, as in
many other secretory cell types, a breakdown and reorganiza-
tion of the actin cytoskeleton seem crucial for Ca2+-triggered
exocytosis (Valentijn et al. 1999). In human parotid and sub-
mandibular glands, F-actin was localized underneath the
luminal membrane to separate the secretory granules from
the luminal membrane (Segawa et al. 1998). Cofilin, an
actin-depolymerizing protein and one of the key components
that control the turnover and branching of microfilaments,
was supposed to be required in adrenal chromaffin cells to
achieve the rapid reorganization of the cortical actin
cytoskeleton that is necessary to allow the movement of yet
undocked secretory granules to the plasma membrane
(Birkenfeld et al. 2001). Profilin, a G-actin-binding protein,
acts as a regulator of cytoskeletal dynamics and membrane
transport (Birbach 2008). For profilin-2, which has been
highly expressed in brain, a function in the regulation of exo-
cytosis through the interaction with a special member of the
glutamate receptor, the kainate receptor GluK2b (Mondin
et al. 2010), could be demonstrated.
Most of the findings of the regulatory processes in sali-
vary gland exocytosis were made either in animal tissue
such as rat parotid acinar cells (Imai et al. 2003; Nashida
et al. 2004) or with the help of human parotid epithelial cell
lines (HSY cells) (Oishi et al. 2006). There is obviously no
study showing the localization of SNARE proteins in tis-
sues of the human submandibular gland by immunohisto-
chemistry. The aim of our study was to elucidate the
participation of actin, myosin II, cofilin, profilin, SNAP-23,
syntaxin-2, syntaxin-4, VAMP-2, and septin-2 in the exocy-
tosis mechanism in the human submandibular gland.
Materials and Methods
Tissue of human submandibular glands was obtained from
20 persons, 9 males and 11 females, with an age range
between 5 and 83 years. The patients underwent salivary
gland surgery for different indications, and partially healthy
tissue was adjacent to pathological tissue. Four percent
phosphate-buffered formaldehyde was used for the fixation
of the gland tissue. The study was performed according to
the guidelines of the local ethics committee.
For immunohistochemical staining, 5-µm paraffin sections of
formalin-fixed tissue samples were dewaxed and stained with
antibodies to the following proteins: actin (Abcam; Cambridge,
UK), myosin IIa (Sigma-Aldrich; Munich, Germany), cofilin
(Abcam), profilin-1 (Novus Biologicals; Littleton, CO),
SNAP-23 (Abcam), syntaxin-2 (Santa Cruz Biotechnology;
Santa Cruz, CA), syntaxin-4 (Abcam), VAMP-2 (Abcam), and
septin-2 (Santa Cruz Biotechnology). Sections were pretreated
with microwave irradiation in citrate buffer at pH 6.0 for
15 min for all antibodies. The endogenous peroxidase activity
was inhibited with 3% hydrogen peroxide for 10 min.
Blocking of nonspecific binding was achieved by incubating
the sections with 3% normal blood serum: goat serum for
actin, myosin II, cofilin, profilin, SNAP-23, syntaxin-4, and
VAMP-2 and rabbit serum for syntaxin-2 and septin-2.
Sections were incubated with the primary antibodies for 1 hr
at room temperature and overnight at 4˚C. The secondary
antibody (Vector Laboratories, Inc.; Burlingame, CA) was
Stoeckelhuber et al.
applied in a concentration of 1:200 for 45 min at room tem-
perature. The detection of the antibodies was performed with
avidin–biotin–horseradish peroxidase using the Vectastain-Kit
(Vector Laboratories, Inc.). Diaminobenzidine was used as a
chromogen. The sections were counterstained with hematoxy-
lin. Controls, in which the primary antibody was replaced with
buffer, were treated identically. Sections were analyzed with a
Nikon Eclipse 80i microscope, and images were taken by a
digital Nikon camera (Nikon; Duesseldorf, Germany).
Labeling results were categorized as weak, medium, or strong
In total, 4-µm sections of formalin-fixed tissues were
dewaxed. For heat-induced epitope retrieval, sections were
incubated in citrate buffer at pH 6.0 for 10 min. Nonspecific
antibody-binding was blocked by 2% BSA (Sigma-Aldrich).
Antibodies used were anti-septin-2 (Abcam), anti-syntaxin-2
(Santa Cruz Biotechnology), anti-lactoferrin (Abcam), and
anti-α-amylase (Abcam). Sections were incubated for 1 hr
at room temperature. As secondary fluorescence-labeled
antibodies, we used a 1:300 dilution of Cy3-conjugated
(red) or Alexa 488–conjugated (green) antibodies (Jackson
Immuno Research; Suffolk, UK). DAPI was used for
nuclear staining. Coverslips were mounted on glass slides
with 90% glycerol (Sigma-Aldrich). Cells were viewed
using an inverted fluorescence microscope (Zeiss Axiovert
200) or a confocal microscope (LSM510; Zeiss, Goettingen,
Germany). Control sections were processed without treat-
ment with primary antibodies.
In Table 1, the staining procedures for every antibody are
Actin. The luminal membrane of the submandibular aci-
nar cells was stained with a weak to medium intensity.
Myoepithelial cells at the base of the acinar cells stained
strongly. Partially, lateral cell membranes of the acinar cells
stained positively (Fig. 1A).
Myosin II. Myosin II was expressed in the basal part of the
acinar cells with a medium to strong intensity. The staining
decreased in the direction of the lumen. The apical mem-
brane, however, was strongly stained (Fig. 1B). In sections
where the acinus was cut in a direction parallel to the secre-
tory duct, the myosin II–positive contour of the apical
plasma membrane was seen very clearly. When the lumen
of the acinus was cut in a transversal direction, the apical
membrane was strongly stained, indicating a concentrated
border around the acinar lumen.
Cofilin. Cofilin staining was variable; cofilin was par-
tially localized at the apical acinar plasma membrane (Fig.
1C), and to some extent, the apical part of the cells remained
unstained within the same section. In addition, a weak over-
all staining of the whole-cell plasma with a stronger stain-
ing at the basal part of the cell was observed.
Profilin-1. In most cases, the apical membrane of acinar
submandibular cells was stained strongly positive with the
anti-profilin-1 antibody (Fig. 1D). Partially, within the same
section, the luminal acinar membrane was less stained or
unstained. In addition, the cytoplasm and the nucleus
showed a medium intense immunostaining.
Table 1. Primary Antibodies
Staining)HostClone Dilution Blocking Serum 3%
Blocking Serum 2%
Exocytosis in the Human Submandibular Gland
SNAP-23. SNAP-23 immunoreactivity showed a strong
staining at the apical membrane of the acinar cells and partially
at the upper part of the basolateral cell membrane (Fig. 2A).
Syntaxin-2. Syntaxin-2 was strongly expressed at the api-
cal plasma membrane of the acinar cells and in smaller and
larger vesicles in the cytoplasm (Fig. 2B,C). Although the
larger vesicles revealed an unstained matrix, the smaller
were stained as a whole. In the basal part of the cytoplasm,
syntaxin-2 was partially concentrated.
Syntaxin-4. Syntaxin-4 was localized at the apical and
basolateral plasma membrane (Fig. 2D). The staining inten-
sity of the antibody was strong.
VAMP-2. A strong positive staining of the VAMP-2
immunoreactivity could be observed in the apical region of
the acinar cell membrane (Fig. 2E). Positively stained vesi-
cles in the cytoplasm could not be detected.
Septin-2. A large number of positively marked vesicles with
the anti-septin-2 antibody were found in the cytoplasm of aci-
nar cells. Both large and small vesicles with an unstained
matrix as well as smaller, completely stained vesicles showed
a positive reaction with the anti-septin-2 antibody (Fig. 2F).
Immunofluorescence Double Staining
Septin-2 and amylase (Fig. 3A–C) as well as septin-2 and
lactoferrin (Fig. 4A–C) colocalized in vesicles of the serous
acini. Syntaxin-2 and amylase (Fig. 5A–C) as well as
syntaxin-2 and lactoferrin (Fig. 6A–C) showed the same
localization on secretory vesicles. Also, syntaxin-2 and
septin-2 were expressed in the same vesicles (Fig. 7A–C).
Figure 1. (A) Anti-actin immunostaining. Positive staining of the apical membrane of submandibular acinar cells (arrows) and of
myoepithelial cells (arrowheads). (B) Anti–myosin II immunostaining. The luminal membrane showed a strong staining (arrows).
(C) Anti-cofilin immunostaining. Cofilin was strongly expressed at the apical membrane (arrows). (D) Anti-profilin immunostaining. A
strong positive staining of profilin was demonstrated at the apical luminal membrane (arrows) and in the nucleus. The whole cytoplasm
was weaker stained. Scale bars: 25 µm.
Stoeckelhuber et al.
Figure 2. (A) Anti-SNAP-23 immunostaining. SNAP-23 was concentrated at the luminal and the basolateral membrane (arrows).
(B) Anti-syntaxin-2 immunostaining. Syntaxin-2 showed a strong expression at the apical plasma membrane (arrows) and in smaller
and larger vesicles (arrowheads). The basal part of the cell, especially the perinuclear compartment, was also strongly stained. (C) Anti-
syntaxin-2 immunostaining. Larger vesicles with an unstained matrix and smaller completely stained vesicles showed a positive syntaxin-2
immunostaining. A higher magnification of the vesicles is demonstrated in the inset. (D) Anti-syntaxin-4 immunostaining. Syntaxin-4 was
expressed at the apical and basolateral plasma membrane of submandibular acinar cells (arrows). (E) Anti-VAMP-2 immunostaining.
VAMP-2 was localized at the apical region of the plasma membrane (arrows). (F) Anti-septin-2 immunostaining. Positively stained vesicles
with an unstained matrix (arrowheads) and completely stained vesicles (arrows) were visible with the antibody to septin-2. A higher
magnification of the vesicles is demonstrated in the inset. Scale bars: 25 µm; scale bars in insets: 6 µm.
Exocytosis in the Human Submandibular Gland
The staining results do not correlate with age or sex of
The human submandibular gland as a typical exocytotic
gland produces a great amount of proteins that are part of
the oral saliva. Secreted proteins, including mucins, lyso-
zyme, lactoferrin, amylase, statherin, histatin, and many
more, play a role in the maintenance of a healthy oral cavity
and are part of the digestive process (Kouznetsova et al.
2010). There are proteins that are constitutively released
without external stimuli, such as amylase (Busch et al.
2002), kallikrein (Garrett et al. 1996), IgA, and other
Figure 3. (A) Septin-2 colocalizes with amylase in secretory vesicles.
Yellow vesicles indicate the overlay of septin-2 and amylase (arrows).
(B) Septin-2 (red). (C) Amylase (green). Scale bar: 10 µm.
Figure 4. (A) Colocalization of septin-2 and lactoferrin as
a marker for secretory vesicles. Yellow vesicles indicate the
overlay of septin-2 and lactoferrin (arrows). (B) Septin-2 (red).
(C) Lactoferrin (green). Scale bar: 10 µm.
Stoeckelhuber et al.
proteins (Proctor et al. 2003). Regulated exocytosis uses
the SNARE-mediated exocytotic pathway (Wang
et al. 2007). We investigated the secretory molecular machin-
ery of acinar (serous) cells in the human submandibular
gland and looked at proteins that are part of a SNARE-
mediated membrane fusion of vesicles and target membrane,
as well as cytoskeletal components that are necessary for
these processes. This study identifies for the first time the
localization of a considerable number of SNAREs and cyto-
skeletal proteins in the human submandibular gland.
Figure 5. (A) Syntaxin-2 colocalization with amylase. Yellow
vesicles illustrate the overlay of syntaxin-2 and amylase (arrows).
(B) Syntaxin-2 (red). (C) Amylase (green). Scale bar: 10 µm.
Figure 6. (A) Colocalization of syntaxin-2 and lactoferrin. Yellow
vesicles demonstrate the overlay of syntaxin-2 and lactoferrin
(arrows). (B) Syntaxin-2 (red). (C) Lactoferrin (green). Scale bar:
Exocytosis in the Human Submandibular Gland
Actin and Actin-Binding Proteins in the
Process of Exocytosis
Studies indicate that the actin cytoskeleton, which is local-
ized under the plasma membrane, prevents secretory gran-
ules from reaching their exocytotic destination (Burgoyne
and Cheek 1987; Aunis and Bader 1988). In most models,
the actin cytoskeleton is disassembled upon stimulation and
rearranged, thereby allowing secretory granules to reach
the site of exocytosis (Cheek and Burgoyne 1986; Perrin
et al. 1992). In contrast, investigations by Williams (1977),
Muallem et al. (1995), and others have shown that disrup-
tion of the actin cytoskeleton in exocrine glands inhibits
stimulated secretion. Moreover, the actin cytoskeleton may
play a positive role in secretion from exocrine glands—for
example, by regulating an early step in the formation of
retrieval vesicles and/or movement of membrane back into
the cell (Valentijn et al. 1999). The variable staining inten-
sity of the luminal border with the actin antibody in our
studies reflects this phenomenon, too. The localization of
myosin II in the acinar cells of the submandibular gland is
the same as in pancreatic acinar cells, predominantly but
not exclusively the apical cell domain, especially a narrow
band along the lumen (Bhat and Thorn 2009). Cofilin is an
actin-depolymerizing protein that has been dephosphory-
lated by various secretory stimuli in parotid acini (Takuma
et al. 1996). Cofilin is involved in many membrane-modulat-
ing activities, such as cell growth, motility, and secretion; for
instance, cofilin is activated upon Ca2+-regulated noradrena-
lin secretion from adrenal chromaffin cells (Birkenfeld et al.
2001). In our investigation, cofilin was found concentrated at
the apical part of the membrane, indicating its role as a part
of the actin cortical network where a breakdown and reorga-
nization of the actin cytoskeleton are necessary for exocyto-
sis. The fact that cofilin could not be demonstrated at the
apical membrane in every acinus might indicate the regional
dynamic processes in secretory cell types reflected by the
breakdown and reorganization of the actin cytoskeleton. In
recent studies, profilin has been attributed with a role in syn-
aptic vesicle exocytosis in the brain (Pilo Boyl et al. 2007).
Mondin et al. (2010) found out that the specific interaction of
profilin-2 (PfnIIa) to a diproline motif in a kainate receptor
(GluK2b), a member of glutamate receptors, leads to the
control of exocytosis of this receptor. The ubiquitously
expressed G-actin-binding protein profilin-1, a key molecule
for regulating actin dynamics in all cell types, was strongly
expressed at the apical membrane of submandibular glands
in our study. We speculate that profilin might be involved in
exocytotic events similar to presynaptic exocytotic processes
in synapses of the brain.
SNARE Proteins Play a Central Role in
SNAP-23. It has been reported that SNAP-23 is necessary
for both apical and basolateral transport in polarized cells
such as hepatocytes, kidney cells (Low et al. 1998), and rat
parotid acinar cells (Takuma et al. 2000). We demonstrated
the expression of SNAP-23 at exactly these positions in the
human submandibular acinar cells.
Figure 7. (A) Syntaxin-2 colocalizes with septin-2. Yellow
vesicles indicate the overlay of syntaxin-2 and septin-2 (arrows).
(B) Syntaxin-2 (red). (C) Septin-2 (green). Scale bar: 10 µm.
Stoeckelhuber et al.
VAMP-2. VAMP-2 was located at the apical membrane of
the submandibular acinar cells, but we could not detect the
protein in vesicles in the cytoplasm, although it is called a
vesicle-associated protein. This is in accordance with other
investigations in acinar cells of the rat exocrine pancreas.
Braun et al. (1994) demonstrated the localization of VAMP
protein as a border around the acinar lumen by immunoflu-
orescence microscopy in frozen pancreas sections. How-
ever, the identification of a VAMP-like protein in the
zymogen granule membrane fraction was proved biochemi-
cally. It was speculated that the detrimental effects of alde-
hyde fixation make it difficult to detect VAMP within other
membrane compartments. Takuma et al. (2000) investigated
the lysate of parotid acinar cells and found VAMP-2 in the
crude secretory-granule fraction by immunoprecipitation.
Fujita-Yoshigaki et al. (1999) reported that an unknown
protein x makes it difficult to detect VAMP-2 on the secre-
tory granules of the parotid acinar cells. Moreover, syn-
taxin-3 was detected on the granule membrane fraction of
rat parotid acinar cells by Western blotting but was not
detectable by immunocytochemistry. Imai et al. (2003)
speculated that the reason for that phenomenon is similar to
that described by Fujita-Yoshigaki et al. (1999).
Syntaxins. Syntaxin-2 and -4 have been classified as
t-SNARE proteins (Braun et al. 1994; Ravichandran et al.
1996); syntaxin-2 also has been found in intracellular vesicular
structures in normal rat kidney (NRK) cells (Band and Kuis-
manen 2005). We corroborate this finding with our results. We
could find syntaxin-2 in the apical plasma membrane of the
submandibular acinar cells but also in granules in the cyto-
plasm. Syntaxin-2 was expressed in the limiting membrane of
larger granules with an unstained matrix and in smaller gran-
ules with an overall staining. In electron microscope images,
typical serous granules of human salivary glands display a
complex internal substructure as a result of the presence of
components of various electron densities, reflecting a different
chemical composition (Tandler and Erlandson 1972; Tandler
and Phillips 1993). On the electron microscope level, secretory
granules of the submandibular gland are generally surrounded
by a limiting membrane, showing an oval corpuscle of medium
density in the matrix. In addition, small secretory granules
exist in the Golgi area (Sato et al. 1966). Presumably, the dif-
ferent sizes of the granules represent different maturation
stages. In our observations, smaller granules might be granules
released by the Golgi apparatus, and the larger granules are
obviously mature granules. Both express syntaxin-2 and are
therefore destined for the exocytotic pathway. Antibodies to
septin-2 react only with the limiting membrane of the larger
vesicles and not with the smaller granules, pointing to a role in
the latter process of exocytosis.
Septin-2. Septins participate in diverse aspects of cell biol-
ogy. Originally, septins were characterized as proteins involved
in cytokinesis in yeast (Haarer and Pringle 1987). In the mean-
time, the identification of numerous septin family members
with functions, for example, in cell cycle control (Barral et al.
2000) and septin homologues in postmitotic cells (Longtine et
al. 1996) has suggested additional roles for these proteins. In
vesicle transport and exocytosis, septins interact with compo-
nents of the so-called exocyst complex (Hsu et al. 1998) and
with syntaxin (Beites et al. 1999). We could determine the
localization of septin-2 to vesicles in the cytoplasm near the
luminal outline, which suggests a clear role in exocytosis. Fur-
thermore, the localization of syntaxin-2 in vesicles also con-
cludes an interactive role between septin and syntaxin. Whether
the interaction is the same as for the septin CDCrek-1, which
binds syntaxin and inhibits exocytosis, has to be elucidated in
further studies. The syntaxin localized at the plasma membrane
might also be a target for septin in the regulation of exocytosis.
Localization of SNARE Proteins on Secretory
Vesicles by Double Staining
We used α-amylase as a marker for secretory vesicles that was
localized in serous acini. Lactoferrin is another well-investi-
gated marker, especially for the acini in submandibular glands
(Korsud and Brandtzaeg 1982). In contrast, lysozyme has been
present predominantly in mucous acini (Kouznetsova et al.
2010) and in intercalated ducts of the serous duct system
(Korsud and Brandtzaeg 1982). In electron microscope studies
by Marchetti et al. (2000), lysozyme and amylase were found
in pale granules in acinar cells of the mouse submandibular
gland. The colocalization in the immunofluorescence of
syntaxin-2 and lactoferrin as well as of syntaxin-2 and
α-amylase showed that syntaxin-2 is localized on secretory
vesicles. Furthermore, we showed an overlay of the fluores-
cence signal in a double staining of septin-2 and lactoferrin as
well as of septin-2 and α-amylase, pointing to a localization of
septin-2 on exocytotic vesicles. Both septin-2 and syntaxin-2
are present on the same secretory vesicles and involved in the
exocrine secretory mechanisms.
We are grateful to Kaori Ochi for excellent technical assistance.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to
the authorship and/or publication of this article.
The authors received no financial support for the research and/or
authorship of this article.
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