J Oral Pathol Med
doi: 10.1111/jop.12409
Melatonin localization in human salivary glands
Michela Isola, Maria Alberta Lilliu
Department of Biomedical Sciences, University of Cagliari, Monserrato, Italy
BACKGROUND: Circulating melatonin is believed to
reach body fluids by crossing passively the cell mem-
branes, but alternative ways for melatonin transport also
are hypothesized. This investigation was carried out to
furnish ultrastructural evidences for melatonin transport
by salivary gland cells in order to indicate plausible routes
by which circulating melatonin can reach saliva.
METHODS: Bioptic samples of parotid, submandibular
and labial glands were processed for the electron
microscopy and treated to demonstrate melatonin reac-
tivity by the immunogold staining method.
RESULTS AND CONCLUSIONS: The preferential sites
of melatonin reactivity were the granules and vesicles of
serous cells. Our results suggested that the acinar cells
are able to store melatonin and that the hormone can be
released into saliva through granule and vesicle exocyto-
sis. The quantitative evaluation of labelling showed that
the parotid gland is the most involved in the release of
melatonin in saliva.
J Oral Pathol Med (2015)
Keywords: immunogold method; melatonin; salivary gland
Introduction
Melatonin (N-acetyl-5-methoxytryptamine) is principally
produced by pinealocytes, but its synthesis occurs in other
tissues also (13). According to the classical theory,
melatonin is released by pineal gland into the blood
circulation and, due to its lipophilic nature, crosses the cell
membranes, enters the cells, and becomes a natural
component of several body uids, such as milk, urine and
saliva (37). However, there is an increasing evidence that
passive diffusion is not the only possible way for melatonin
transport and that some tissues have a still unknown
intracellular machinery that regulates the melatonin move-
ment (810).
Correspondence: Michela Isola PhD, Department of Biomedical Sciences,
University of Cagliari, Cittadella Universitaria, 09042 Monserrato, CA,
Italy. Tel: +39 070 6754029, Fax: +39 070 6754003, E-mail: misola@
unica.it
Accepted for publication November 23, 2015
2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
wileyonlinelibrary.com/journal/jop
Recently, salivary melatonin has received considerable
attention because of its antioxidant, anti-inammatory, and
antimicrobial properties which exert benecial effects on the
oral mucosa (1114). In coherence with the theory that
melatonin moves by passive diffusion, its concentration in
saliva undergoes circadian oscillations that reect those in
blood, so that its measurement is currently accepted as a
reliable tool to monitor blood melatonin changes (5, 15, 16).
However, the discrepancies between blood and salivary
levels (17) and the capability of salivary glands to produce
melatonin (18) conict with the general belief that salivary
melatonin comes exclusively from systemic circulation by
passive diffusion. Thus, many questions about the source
and transport of salivary melatonin still remain unanswered.
In a recent paper (19), we have proposed that salivary
glands, like other tissues (810), might have an intracellular
machinery that facilitates melatonin uptake from blood, its
transport through the cells and its storage. To conrm this
hypothesis, it should be important to identify the specic
cell compartments involved in the transport of melatonin as
well as in its storage.
The present investigation was carried out to show the ne
distribution of melatonin in salivary gland cells with the aim
to identify the intracellular storage sites and to propose a
plausible mechanism by which it is discharged into saliva.
Materials and methods
Fragments of salivary glands were obtained from 15 male
patients, aged around 65 years, undergoing surgery for the
removal of tumours of the mouth and neck regions at the
Otorhinolaryngology Clinic, University of Cagliari. Patients
were not habitual smokers, alcohol consumers or obese.
Moreover, they were not affected by severe oral disorders,
by autoimmune diseases, and were not subjected to
chemotherapy or radiotherapy before surgery. Samples of
5 parotid, 5 submandibular and 5 labial glands were
collected at about 11 am. Written informed consent was
obtained from each patient. All the procedures were
approved by the local Institutional Committee for human
experimentation at the ASL 8 (Azienda Sanitaria Locale 8),
Cagliari.
Transmission electron microscopy
The samples were cut into small pieces and xed for 2 h
with a mixture of 3% paraformaldehyde and 1% glutaralde-
 Melatonin in salivary glands
M. Isola and M. A. Lilliu

2
hyde in 0.1 M cacodylate buffer. Then, they were rinsed in (a)
cacodylate buffer added with 3.5% sucrose, dehydrated and
embedded in Epon Resin (Glycide Ether 100, Merk,
Darmstadt, Germany). To preserve the antigenicity of the
tissue, treatment with osmium tetroxide was omitted.
Semithin sections (2 lm) stained were examined by light
microscopy to check the histological appearance. Ultrathin
sections (80 nm) were collected on nickel grids and
processed for the immunohistochemical analysis. The grids
were treated with 1% bovine serum albumin (BSA) and 5%
normal goat serum (NGS) in phosphate-buffered saline
(PBS) solution to block non-specic binding. Then, they
were incubated overnight at 4C with a rabbit polyclonal
antibody specic for melatonin (Thermo Fisher Scientic
(b)
Inc., USA) diluted 1:20 in PBS + 1% BSA and 5% NGS.
The grids were then incubated for 1 h at room temperature
with the secondary antiserum, a goat anti-rabbit IgG
conjugated to 15-nm gold particles (GE Healthcare, UK),
diluted 1:30 in PBS + 1% BSA. After washing, they were
stained with uranyl acetate and bismuth subnitrate and
nally observed and photographed with a JEOL 100S
transmission electron microscope. Control sections were
incubated with a non-immune serum or without the primary
antibody.

Quantitative evaluation of labelling and statistical analysis

Electron micrographs of parotid and submandibular glands
were used to quantify the immunoreactivity with the
program Image Pro Plus (Media Cybernetics, Silver Spring,
MD, USA). The morphometric evaluation was not per-
formed in labial glands, as the reactivity was generally
negligible.
A total of 50 randomly chosen images, 5 from each
patient, were considered for calculations. In detail, 25
images were from parotid and 25 from submandibular
samples.
The gold particles were counted inside the secretory
granules of serous cells, and the mean labelling density was
calculated for each gland and expressed as the number of
gold particles per lm2. The percentage of melatonin-
labelled granules was calculated by counting the number of
labelled granules with respect to the total granule number
per surface unit. The differences in labelling density were
statistically evaluated using the MannWhitney U-test. The
values were expressed as mean  standard error (SEM),
and P < 0.05 was considered signicant.
Results
Melatonin labelling in Parotid, Submandibular and Labial
glands
The experiments of the immunogold technique showed
unequivocally that the parotid and the submandibular but
not the labial glands were reactive for melatonin. The
labelling appeared as a prerogative of serous acini and
striated ducts. Serous acini represent the totality of the
secretory elements in parotid, the majority in submandibu-
lar and a minority in labial glands. Melatonin labelling
resulted rather intense in serous cells of parotid, moderate
in those of submandibular and negligible in those of labial
glands (Fig. 1a, b, c). The secretory granules were the
(c)
Figure 1 Serous cells of parotid (a), submandibular (b) and labial (c)
glands treated for melatonin labelling. Gold particles (black dots) were
mainly found in the secretory granules. The strongest reactivity was shown
by parotid cells. Bar = 1 lm.
preferential labelling sites. When a bipartite or tripartite
structure was evident, the staining was restricted to the
pale peripheral portion of the granules (Fig. 1b). More-
over, melatonin labelling was detected in small vesicles
located among the granules and close to the basolateral
membranes (Figs 2 and 3). A few gold particles were also
found on the cell surfaces (Figs 2 and 3). No melatonin
staining was observed in the cells of mucous tubules and
of intercalated ducts, the rst segments of the ductal
system (Fig. 3). The striated duct cells showed melatonin
staining in cell membranes and within small vesicles
located both apically and near the basolateral folds
(Fig. 4). The control samples were always negative
(Fig. 5).
The quantitative evaluation of immunoreactivity
revealed that the labelled granules were 89% and 42%

J Oral Pathol Med


(a)
(b)
Figure 2 Acinar cells of parotid gland. (a): in the apical cytoplasm,
labelled small vesicles (arrows) were seen among the secretory granules;
(b): in the basolateral cytoplasm, melatonin labelling appeared in both cell
membranes and nearby vesicles (arrows). Bar = 1 lm.
Figure 3 Acinus-intercalated duct junction of submandibular gland.
Acinar cells (ac) showed a moderate melatonin staining in secretory
granules and vesicles (arrows) scattered among the granules. Intercalated
duct cells (id) usually were unstained. Bar = 1 lm.
Melatonin in salivary glands
M. Isola and M. A. Lilliu
3
(a)
(b)
Figure 4 Striated duct cells of submandibular gland. Apical (a) and basal
(b) portions showed melatonin labelling within small vesicles (arrows)
throughout the cytoplasm and near the basolateral membranes, also stained
(asterisks). Bar = 1 lm.
Figure 5 Serous cell of parotid gland used as a control. In all the controls,
the melatonin staining was completely absent. Bar = 1 lm.
of total granules in parotid in submandibular gland,
respectively (Fig. 6a). The labelling density was
28.12  1.036 gold particles/lm2 in parotid granules and
12.64  0.427 gold particles/lm2 in submandibular gran-
ules (Fig. 6b).
J Oral Pathol Med
 Melatonin in salivary glands
M. Isola and M. A. Lilliu
4
Figure 6 Quantitative evaluation of melatonin labelling in parotid and
submandibular glands. (a): percentage of melatonin-labelled granules with
respect to the total granule number per surface unit; (b): labelling density
(number of gold particles/lm2) in secretory granules. ***P < 0.001.
Discussion
This investigation shows the subcellular localization of
melatonin in the human salivary gland parenchyma and
reveals several new information about melatonin trafc. For
the rst time, our study highlights that the major glands are
the principal supplier of salivary melatonin. This statement
was already acclaimed by other authors (20), but, to the best
of our knowledge, no morphological evidence has been
recorded until now. The quantitative analysis pointed out
that parotid gland has a twofolds higher labelling density
and a twofolds higher number of labelled granules than
submandibular gland. This nding indicates that, among the
major salivary glands, parotid is the most involved in
melatonin release. It is noteworthy that the degree of
participation to melatonin supply represents a further feature
distinguishing the salivary glands from one another, in
addition to many anatomical, embryonic, histochemical and
metabolic diversities (2129).
J Oral Pathol Med
The distribution pattern of labelling demonstrates that the
serous cells are able to accumulate melatonin in secretory
granules, vesicles and membranes. As the serous cells have
not the enzymes needed to obtain melatonin from serotonin
(18), the only source of the stored hormone should be the
blood. The specic staining in secretory granules clearly
shows that melatonin is discharged by serous cells with
other secretory products when the granules open into the
lumen. The granule exocytosis takes place following a
multitude of stimulus when enhanced salivation is required
and represents the so-called major regulated secretion (30).
By this way, the granule emptying would cause a rapid
increase in salivary melatonin during activities such as
chewing or speaking, in addition to the circadian variations.
The small reactive vesicles located apically among the
granules could be representative of the constitutive-like or
of the minor regulated secretion pathways. They would
originate from granule budding and open into the lumen
independently from the large granules following low or no
stimulation (31, 32). By this way, small melatonin amounts
would be withdrawn from granules and continually dis-
charged into resting saliva.
Therefore, our results on one hand suggest that passive
diffusion is not the only way by which melatonin reaches
saliva and on the other hand address the problem of the
mechanism by which the cells receive and concentrate the
hormone. Through the years, evidence has grown that
several cell types actively acquire and store melatonin from
blood. For example, Venegas et al. (10) reported that
different cell compartments accumulate melatonin at vari-
able extent according to the specic functions characterizing
the cell types. The mechanism by which melatonin is
accumulated in cell organelles should require the involve-
ment of specic carriers, until now only hypothesized,
which capture the hormone and allow its concentration in
storage compartments (810). In our recent paper, melatonin
receptors have been proposed for this role in salivary gland
cells (19). Melatonin receptors (MT1 and MT2) are
transmembrane G protein-coupled receptors whose activa-
tion triggers several intracellular signal cascades mediating
cell responses (20, 33) such as salivary secretion (34).
Although dened as membrane receptors, their presence in
the cytoplasm also was recorded in human salivary gland
cells, leading to propose that, beyond starting the signal
cascades, they may be involved in melatonin transport
within the cells (19, 35, 36). The melatonin localization
shown here, quite identical to that recently described for
receptors (19), strengthens the assumption that melatonin
and receptors travel together into and throughout the cells.
In particular, the staining in basolateral cell membranes and
nearby vesicles might indicate that circulating melatonin is
captured by the membrane receptors and rapidly internal-
ized. Then, the hormonereceptor complex might be carried
by vesicles either towards the storage organelles such as the
secretory granules, or directly towards the glandular lumen.
As suggested above, the capability of both granules and
vesicles to retain melatonin could be ensured just by the
presence of the melatonin receptors.
The striated duct cells also showed melatonin staining in
membranes and vesicles. As in acinar cells, the exact
correspondence between the localization of melatonin and

that of its receptors (19) leads us to think that the melatonin
receptor complex is carried throughout the cytoplasm. The
large amounts of vesicles characterizing these cells are
mostly considered as absorptive, in that related to the
electrolyte transport, but some of them could move in the
opposite direction from the base to the apex (transcytosis) or
could carry some secretory products towards the lumen (37).
Therefore, reactive vesicles possibly carry secretory mela-
tonin towards the glandular lumen. Interestingly, it cannot
be ruled out that melatonin within these cells can be of
endogenous origin, given the presence of enzymes needed
to the synthesis (18) so that the striated duct cells can be
even considered an extrapineal source of this indoleamine.
Taken together, our data suggest that (i) the passive
diffusion is not the only way by which melatonin reaches
saliva; (ii) the capability of both granules and vesicles to
retain melatonin could be ensured by the melatonin
receptors; (iii) the parotid gland is the most involved in
melatonin trafc.
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thetised rat. J Pineal Res 2008; 45: 41321. M Isola and MA Lilliu acknowledge Sardinia Regional Government for the
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The authors declare no conict of interest.
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