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European Journal of Drug Metabolism and Pharmacokinetics 2007, Vol. 32, No. 3, pp. 123-129
Salivary glands epithelial and myoepithelial cells are
major vitamin D targets
WALTER E. STUMPF1 and NAOHIKO HAYAKAWA2
1
University of North Carolina at Chapel Hill, NC and International Institute of Drug Distribution
Cytopharmacology and Cytotoxicology, 2612 Damascus Church Rd., Chapel Hill, NC 27516, USA, and
2
Chugai Pharmaceutical Company, Ltd., Fuji Gotemba Research Laboratories, I-135, Komakado, Gotemba,
Shizuoka 412-8513, Japan
Received for publication: February 15, 2007
Key words: Autoradiography, receptor microautoradiography, imaging, submandibular gland, parotid,
sublingual gland, digestive system
SUMMARY
Receptor binding with 3H-1,25(OH)2 vitamin D3 (vitamin D) and its oxygen analog 3H-OCT is demonstrated in rat, hamster, and
mice submandibular, sublingual and parotid glands, using receptor microautoradiography high-resolution imaging. Nuclear uptake
and retention of radiolabeled compound exist strongest in epithelial cells of striated ducts, granular convoluted tubules and in
myoepithelial cells throughout, scattered in epithelial cells of intercalated ducts and relatively low in cells of serous and mucous acini.
Deposition and retention of radiolabeled compound is also observed in interstitial spaces. The specific nuclear localization with vitamin D and its analogue OCT, which is absent with 3H-(OH) vitamin D3 and in competition with excess non-radioactive vitamin D,
indicates involvement of vitamin D in the multi-hormonal regulation of salivary gland secretion, excretion, and cell proliferation.
These data - together with previously recognized similar receptor binding in esophagus, gastric glands, entero-endocrine cells, pyloric muscle, and generative and absorptive epithelium of the small intestine and colon, point to the importance of vitamin D for the
digestive system regulation of functions and maintenance with related therapeutic potentials.
.
INTRODUCTION
Discoveries of unexpected target tissues have shifted
the focus and understanding of vitamin D actions by
providing a comprehensive picture beyond the traditional narrow-focus concept of systemic calcium
regulation. Beginning in 1979, during the 1980s and
early1990s, over 50 target tissues have been identified and characterized mainly through discoveries
.
Please send reprint requests to: Walter E. Stumpf, 2612 Damascus Church Rd., Chapel Hill, NC 27516/USA, Tel/Fax:
919 942 8646, E-mail: [email protected],
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with high-resolution receptor microscopic autoradiography (1,2). Accordingly, the main biological
role of vitamin D has been redefined (1,3) as a genomic regulator of seasonal adaption of growth,
reproduction and other vital functions for survival.
Systemic calcium regulation, including bone growth
and metabolism, is an important part of it, but not its
main biological role. Widespread selective regulation
of cell proliferation, differentiation, and secretion are
indicated from the nuclear receptor binding in cell
populations of skin stratum Malpighi, hair and accessory glands, intestinal crypts of Lieberkuehn, atrial
cardiomyocytes, pituitary thyrotropes, gastric gland
isthmus cells, entero-endocrine and neuro-endocrine
cells, female and male reproductive tissues, thymic
reticular cells, and others, most of which are not
primarily involved in systemic calcium regulation.
Receptor microscopic autoradiography with its high
sensitivity and high tissue resolution has made major
contributions to the recognition of target distribution
and function for hormones and drugs that have escaped detection with less sensitive ADME approaches alone. It is especially suited to identify and
monitor target cell populations that are surrounded by
and embedded in non-target tissues which render recognition difficult or impossible with traditional cutand-grind biochemical procedures.
Salivary glands are known to be under multihormonal control (4-70 that includes androgens, adrenal
steroids, and thyroid hormone, to which vitamin D is
added.
time, slides were photographically processed, singlestep stained with methylene blue-basic fuchsin or
methyl green-pyronin, air-dried, coverslipped, and
examined with the microscope. Results from different
exposure times were compared with each other. Short
exposure times are suitable for quantification through
silver grain counting at high magnification, long
exposure times may be selected for display and easy
recognition of labeled compartments at low
magnification, as well as recognition of weak signals
that might be undetectable at short exposure times.
No quantitative silver grain counts were performed.
Receptor microscopic autoradiography was developed in our laboratories for the localization of diffusible compounds and has been described in detail (2).
METHODS
After injection of 3H-1,25(OH)2 vitamin D3 or 3HOCT, in all three major salivary glands a similar nuclear concentration and retention of radioactivity is
seen in select cell types to different degrees in hamsters, rats and mice (Figs. 1 and 2). Strongest nuclear
concentration exits in epithelial cells of striated ducts,
granular convoluted tubules, and in myoepithelial
cells. Cells of intercalated ducts are occasionally labeled, less frequently and less intense compared to
those in striated ducts and granular convoluted tubules. Cells of excretory ducts appear mostly unlabeled, except for myoepithelial cells. Cells of mucous
and serous acini display weak nuclear labeling under
conditions when strong nuclear labeling is visible in
epithelial duct and myoepithelial cells. In some
interstitial spaces increased levels of radioactivity are
noted.
The nuclear labeling pattern varies depending on
experimental conditions. At short exposure times,
nuclear radioactivity may be visible only in
myoepithelial cells and certain duct cells, while cells
of acini appear unlabeled or weakly labeled.
In the competition experiment with excess unlabeled 1,25(OH)2 vitamin D3, nuclear uptake and
retention of radiolabeled compound is not apparent.
No nuclear concentration of silver grains is visible at
0-day exposure time.
After injection of 3H-(OH) vitamin D3 concentration of radioactivity is absent in cell nuclei and visible only in regions of blood vessels.
Quantification of nuclear uptake has not been done,
and differences among animals and experimental
conditions cannot be excluded.
Young adult male rats (Sprague-Dawley), hamsters,
and C57 BL/6J mice were injected with 3H1,25(OH)2 cholecalciferol (Dupont,Boston, Mass,
USA) or 3H-1,25(OH)2-22-oxa-vitamin D3 (OCT),
spec. act. 160 Ci/mM, dissolved in 20% ethanol-isotonic saline. The animals (n-2 each condition)) were
injected i.v. or subcutaneously with 0.2-0.4 μg/100g
bw and sacrificed 1 to 3-hr afterwards (for details of
the hamster experiment see (8). One additional rat
was injected i.v. with 0.4 μg/100g bw of 3H-(OH)
cholecalciferol, specific activity 160 Ci/mM, and tissues processed similar to the experiments with 3H1,25-(OH)2 cholecalciferol.
Competition control was conducted with 1000x excess of unlabeled 1,25-(OH)2 cholecalciferol, injected
prior to 3H-1,25(OH)2 cholecalciferol and processed
like the experiments with radiolabeled compound.
Further controls against artifacts include 0-day exposure autoradiograms of experimental animals.
The experiments were designed primarily for the
study of other vitamin D target tissues, the results of
which have been reviewed (1). Salivary glands were
included in these studies for pilot exploration.
Individual salivary glands were excised, placed on
tissue holders and freeze-mounted by immersion in
isopentane cooled with liquid nitrogen, then stored in
liquid nitrogen until sectioning. Four micrometer sections were cut in a cryostat and thaw-mounted on nuclear emulsion-coated slides. The slides were placed
in a light-proof desiccator box and kept in a freezer
or refrigerator for exposure. After latent image
formation through exposure for different lengths of
RESULTS
Figure 1. Autoradiograms of hamster submandibular (A and B) and sublingual (C and D) glands after injection of 3H-1,25
dihydroxycholecalciferol showing strong nuclear concentration and retention of radiolabeled hormone in epithelial cells of striated
ducts (SD) and granular convoluted tubules (GCT; B and D). Epithelium of mucous and serous acini shows comparatively low
nuclear uptake. Variable deposition of radioactive compound exists in interstitial ground substance. Radiolabeled epithelium in
intercalated duct (D, insert), and myoepithelial cell (M). Exposure time 24 months. Bar 10 µm
Figure 2. Autoradiograms after injection of the vitamin D analogue 3H-OCT (A and D), 3H-(OH) vitamin D3 (C), or 3H-1,25(OH)2
vitamin D3 (B, E-G), showing nuclear concentration of radiolabeled hormone in rat parotid striated duct cells (A, center) when serous
acinar cells are still unlabeled two hours after iv injection of 0.4 ug/100g bw and 2-month exposure time. Nuclear concentration in rat
(A, B, E) and mouse (D, F, G) submandibular (B, E, F, G) and sublingual (D) granular convoluted tubule epithelium (GCT in D and
E), and intercalated duct (ICD in B). Granular convoluted tubules (GCT) stand out through strong nuclear labeling when acinar cells
appear weakly labeled or unlabeled at exposure times of more than six months. Myoepithelial cells (M) and epithelial cells of
granular convoluted tubule (D and E) display strong nuclear concentration after injection of 3H-1,25 dihydroxyvitamin D3 that can be
recognized at short exposure times when acinar cells appear only weakly labeled or unlabeled. Myoepithelial cells (M), apparent by
their marginal position, frequently stand out among other cells with their strong nuclear concentration of radiolabeled compound,
demonstrated after short (F) and long exposure time (G). After injection of 3H-25(OH) vitamin D3 concentration of radioactivity is
seen in rat parotid in regions of blood vessels only (C), but not in nuclei of epithelial and duct cells, even after long exposure time of
more than one year. Bar 10 µm.
DISCUSSION
In the present study, in all major salivary glands nuclear concentration and retention of radiolabeled
compound is observed after injection of 3H1,25(OH)2 vitamin D3 or 3H-OCT which is abolished
or diminished in competition studies with excess
unlabeled 1,25(OH)2 vitamin D3, similar to other target tissues in the same experiment. This suggests that
the nuclear concentration of radioactivity represents
specific and limited capacity binding of the original
compound. After administration of 3H-25(OH) vitamin D3 no nuclear concentration of radioactivity is
observed which further supports the specificity of the
nuclear labeling with 3H-1,25(OH)2 vitamin D3. In
previous biochemical experiments with homogenized
parotid all of the metabolites of the cholecalciferol
were present which normally occur in known target
tissues of vitamin D (9), also vitamin D receptors in
isolated rat parotid gland acinar cells were present,
but absent in the submandibular gland (10), while in
both parotid and submandibular gland the salivary
flow was stimulated after 1,25(OH)2 vitamin D3 treatment in vitamin D-deficient rats (11).
In epithelial cells of striated ducts and granular
convoluted tubules, the strong concentration of vitamin D is of functional significance. Granular convoluted tubule cells are under complex multihormonal
regulation for the synthesis of various growth factors
(12). Striated duct cells of rat salivary glands readily
incorporate 3H-fucose into newly-synthesized glycoproteins, packaged into apical granules and incorporated into plasma membranes (13). In the secretory
duct system of the rat submandibular gland, principal,
dark (pillar), and tuft cells with different secretory
granules and basal infoldings have been identified
(14). Striated duct cells and cells of the granular
convoluted tubules in the submandibular and sublingual gland resemble each other ultrastructurally, being under the same hormonal regulation (15). Agerelated regressive changes have been noted in rat
granular ducts with decreased hight and content of
mature secretory granules (16,17), involving NGF,
EGF, and protease (6). Changes in the expression of
transcription factor JunD in the duct system, dependent on testosterone, have been reported (18). After
injection of 3H-testosterone or 3H-dihydrotestosterone strong nuclear labeling has been demonstrated in
rat parotid serous acinar cells, weak or absent in ductal epithelium (19) and present in different cell types
of the submandibular gland, including acinar, granular convoluted duct and striated duct cells (20). Hypophysectomy affects various salivary gland functions (4,12) which further indicates endocrine links
and hormonal dependency of salivary gland functions.
Intercalated duct cells occasionally show nuclear
labeling in the present autoradiograms. The intercalated duct harbors proliferating stem cells that give
rise to both acinar and duct cells (17,21). This suggests involvement of vitamin D in cell renewal,
apparently similar to vitamin D targets in the gastric
gland isthmus region, intestinal crypts, skin stratum
Malpighi, hair sheaths and bulbs, and dental pulp
odontoblast precursor cells (reviewed 1).
Myoepithelial cells have been recognized as vitamin D target also in other glands, such as sweat
glands (22). Myoepithelial cells function as contractile element in the promotion of secretion and expulsion of saliva. They may participate in neoplasms
(23). Myoepitheliomas, benign and malignant, have
been reported to originate from major and minor salivary glands (24-26). Possible relationships to vitamin
D effects, causative and therapeutic, deserve to be
investigated because of their conspicuous nuclear
concentration of vitamin D as demonstrated in the
present study.
Conclusions
The present results demonstrate concentration and
retention of radiolabeled vitamin D in specific cell
populations of salivary glands in a fashion similar to
the many other target tissues identified through
receptor microscopic autoradiography since its first
application in 1979 (1,27). In salivary glands, while
striated duct cells, granular convoluted tubule cells
and myoepithelial cells clearly can be recognized and
stand out as concentrating and retaining radiolabeled
hormone, a low and variable concentration can be
seen in intercalated duct cells and acinar cells. Acini
and excretory ducts remain to be further studied. Follow-up experiments with the high resolution approach are required to clarify the hierarchy of receptor expression and hormone (drug) binding in the
different cell types and locations and their variations
related to age and endocrine conditions. Comparison
of these data with those of sex, adrenal, and other
hormones will elucidate salivary gland regulations
and multiple functions. While specific functions of
vitamin D in salivary glands remain to be explored,
the significance of the present findings is apparent
from previous data about functions of striated ducts
and granular convoluted tubules (28).
Therapeutical potential
The importance of vitamin D for the digestive system
can be gleaned from the presence of multiple target
tissues - identified and characterized through microautoradiography (1) - that include in addition to salivary glands, epithelium of the oral mucosa and
esophagus, gastric gland isthmus cells, antrum Gcells, pyloric muscle cells, epithelium of villi and
crypts in the duodenum, jejunum, and ileum, epithelium of the colon, and pancreatic B-cells.
Impairment of digestion that involves non-neoplastic salivary gland diseases (29) and the finding that
the total amount of salivary components and not only
salivary flow rates are reduced in the elderly, is of
great clinical importance (30). All of that points to a
high therapeutic potential for vitamin D and its
congeners for the maintenance and repair of
gastrointestinal functions, independent of or in
conjunction with systemic calcium regulation and
other salubrious actions of this polyfunctional hormone (3,31).
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ACKNOWLEDGEMENT
17.
Some of the experiments with vitamin D were conducted at the Department of Cell Biology and Anatomy, University of North Carolina, Chapel Hill,
involving graduate students and coworkers; related
results have been reviewed (Stumpf, 1995).
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19.
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