Serotonin-immunoreactive Epithelial Cells in the Main Excretory Ducts of Rat Submandibular Glands

J. Oral Biosci. 460) : 20- 26, 2004

ORIGINAL

Serotonin-immunoreactive Epithelial Cells in the Main Excretory Ducts of Rat Submandibular Glands Kazuyoshi Higashi, Hideko Tsuzuki, Hiroyuki Hayashi, Akira Kawata, Koji Takahashi and Osamu Takahashi Department of Oral Histology, Kanagawa Dental College 82 Inaoka-cho, Yokosuka, Kanagawa 238-8580, Japan [Received on July 22, 2003 ; Accepted on November 4, 2003) Key words: serotonin-immunoreactive cell/ chromogranin and PGP 9.5/epithelium of main excretory duct/ submandibular gland/ rat Abstract: Although several morphological studies of main excretory duct of the submandibular gland have been performed, few reports present immunohistochemical data. Some epithelial cells or basal cells contain dense granules of an array shape by electron microscopic observation. However, the details of granulated cells have not been clarified immunohistochemically. In this study, both protein gene product 9.5 (PGP 9.5) and chromogranin or serotonin immunoreactive cells in the main excretory duct (MED) of the rat submandibular gland were observed. Some nerve endings were also observed among the epithelium of the MED. Therefore, the modifying mechanism of primary saliva in the MED may be regulated by both serotonergic nerve and endocrine cells with serotonin. 1j)~ ~::

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Introduction Many biochemical and morphological studies of glandular bodies in the salivary glands have been performed l - 3). Moreover, striated duct cells contain many physiological substances including growth factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF) 4,5). The main excretory duct of the rat submandibular gland has long been considered to be merely a passive conduit. Recent

histochemical and ultrastructural studies have indicated that primary saliva, which is secreted from acinar cells, is modified in the duct system by secretion of K + and HC0 3- and by absorption of Na+and Cl-3,6,7). However, the details of the modifying mechanism of the saliva are not clarified. Although several morphological studies have been performed and reports accepted 1,2), the morphology of the MED is not clarified immunohistochemically. The epithelium lining the MED of the rat submandibular gland is pseudostratified, consisting of many columnar (light cells,

21 dark cells) and cuboidal cells, brush cells, basal cells and a few granulated cells 8 - 1 1). Some epithelial cells or basal cells contain dense granules of an array shape. Nerve endings are also observed among the epithelium of the MED. The nerve may be related to the regulation of the modifying mechanism of the saliva. The details of the modifying mechanism of the saliva and the function of granulated cells have not been clarified immunohistochemically. In this study, granulated cells in the MED of the rat submandibular glands were immunohistochemically and ultrastructurally investigated. Materials and Methods 1. Experimental animals Male Wi star rats (200-350 g) were used. The rats were maintained under a 12-hr light-dark cycle at 20°C, with food and water ad libitum. 2. Immunohistochemistry Fifteen rats were used. The animals were anesthetized with pentobarbital, 50 mg/kg body weight i.p., and perfused through the left ventricle with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) . The MEDs of the submandibular glands were removed and immediately fixed by immersion in the 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hr, washed overnight in the same buffer, and frozen in liquid nitrogen. Frozen sections were cut at a 15-Jim thickness in a cryostat at - 20°C. Cryosections were treated for double-immunohistochemical demonstration of chromogranin A + B and protein gene product 9.5 (PGP 9.5) or serotonin and PGP 9.5. They were rinsed in PBS for 30 min, and then immersed for 30 min in 0.1 M PBS containing 10% normal goat serum (NGS) /0.75% Triton X -100/ o.05 % NaN 3 • Following rinses in 0.1 M PBS, the sections were incubated overnight at 4°C with either both mouse anti- PGP 9.5 monoclonal antibody (UCL ; Wright, England, 1/200 in 0.1 M PBS containing 10% NGS/O. 75% Triton X-I00/0.05% NaN 3 ) and rabbit anti-serotonin polyclonal antibody (Incstar; Minn, USA, 1/500 in 0.1 M PBS containing 10% NGS/0.75% Triton X-I00/0.05% NaN 3 ) or the anti-

mouse monoclonal PGP 9.5 antibody (1/100 in 0.1 M PBS containing 10% NGS/O. 75% Triton X -100/ 0.05% NaN 3 ) and rabbit anti-chromogranin A+B polyclonal antibody (Progen; Heidelberg, Germany, 1/100 in the same solution). The sections were rinsed in PBS for 30 min at room temperature and incubated for 60 min with both biotin-conjugated anti-mouse IgG antiserum (DAKO; Glostrup, Denmark, 1/400 in 0.1 M PBS containing 10% NGS/0.05% NaN 3 ) and FITC-conjugated anti-rabbit IgG antiserum (Wako ; Osaka, lapan, 1/50 in 0.1 M PBS containing 10% NGS/0.05% NaN 3)' After incubation, the sections were rinsed with PBS for 30 min. Then they were immersed in Cy 3-streptavidin (KPL; Guildford, UK, 1/1,000 in 0.1 M PBS containing 10% NGS/0.05% NaN 3) for 60 min in a dark room at room temperature. In each experiment, some sections were picked up at random and they were used as the negative controls. The sections were mounted in PBS/glycerin (3/7), and examined with a confocal laser scanning microscope (Nikon, ECLIPSE. E 800).

3. Ultrastructure Ten rats (200- 350 g) were used for ultrastructural analysis. The animals were anesthetized with pentobarbital, 50 mg/kg body weight i.p., and perfused through the left ventricle with 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The MEDs of the submandibular glands were removed and immediately fixed by immersion in 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 hr, washed overnight in the same buffer at 4°C. They were dehydrated in a graded series of ethanol at - 35°C, and embedded in K 4 M resin. Ultrathin-sections, cut by an ultramicrotome, were incubated in 5% goat serum with 0.1 M phosphate buffer (pH 7.4) for 30 min at room temperature. Then they were incubated in rabbit anti-serotonin antibody (Incstar; Minn, USA, 1/2000) in 5% goat serum with 0.1 M phosphate buffer (pH 7.4) at 4°C overnight. After incubation, they were rinsed with PBS, and immersed in PBS containing 2% 15-nm gold particle-conjugated goat anti-rabbit antibody (Amersham; Buckinghamshire, UK.) for 2 hr. Sections were counterstained with 2% uranyl acetate and examined with a lEOL-200 CX

22

K. Higashi, et al : Immunohistochemistry of MED of the Submandibular Gland

electron microscope. Results 1. Immunohistochemistry 1) MED cells immunoreactive against chromogranin and PG P 9 . 5 PGP 9. 5-immunoreactive cells were observed in the epithelium of the MED of the rat submandibular gland (Fig. 1 a, b). Several immunoreactive areas for PGP 9.5 were seen in the lamina propria of the MED. When the same section was immunostained with the chromogranin A + B antibody, chromogranin immunoreactivity (Fig. 1 c) was observed in the cell which was PGP 9.5 immunoreactive. With a doubleimmunohistochemical demonstration of chromogranin A + Band PGP 9.5, both PGP 9.5 and chromogranin immunoreactivity were observed in the same cell of the epithelium of the MED. Chromogranin immunoreactivity was showed in the central portion of the cell, but only PGP 9.5 immunoreactivity was exhibited in the entirety of the cell (Fig. 1 a). All chromogranin immunoreactive cells in the MED were PGP 9.5 immunoreactive. 2) MED cells immunoreactive against serotonin and PGP 9.5 Both serotonin and PGP 9.5 immunoreactivity were shown in the same cell of the epithelium of the MED. When the specimen was double-labeled with serotonin and PGP 9.5, both immunoreactivities were exhibited in the central portion of the cell except the nucleus using the confocal laser scanning microscope (Figs. 2a, 3). Only PGP 9.5 immunoreactivity was observed in the entirety of the cell (Fig. 2 b). Serotonin immunoreactivity was observed in the central portion of the cell (Fig. 2 c). Both serotonin- and PGP 9. 5-immunoreactive structures and only PGP 9. 5-immunoreactive structures were present in the intercellular space. These structures showed an oval or slender shape. Many nerve fibers which were immunoreactive for PGP 9.5 were observed in the lamina propria (Fig. 3). All serotonin immunoreactive cells in the MED were PGP 9.5 immunoreactive. Sometimes both serotoninand PGP 9. 5-immunoreactive nerve fibers were present in the lamina propria.

2. Electron microscopy 1) General features of MED epithelium The epithelium lining the MED of the rat submandibular gland was pseudostratified, consisting of many columnar cells and some cuboidal cells, brush cells and basal cells, with a few granulated cells. The epithelial cells contained a round nucleus with small invaginations, many mitochondria and short roughsurfaced endoplasmic reticulum. Numerous short microvilli in the apical portion of the epithelial cell extended into the lumen. Basal cells, which contain large and dense nuclei, were present in the basal layer of the epithelium. The brush cells had long and thick microvilli, and numerous microfilaments extending from the cytoplasm to the core of the microvilli. Many processes of the epithelial cells were present in the intercellular space (Fig. 4 a). Sometimes nerve endings which contained many clear vesicles and some mitochondria were present in the intercellular space (Fig. 4 a, b). A few of the epithelial and basal cells contained many dense granules. The granulated cells had a round nucleus with invagination and dense cytoplasm (Fig. 5). Several granules were scattered in the cytoplasm. The granules showed an oval shape. Nerve fibers, which contained many filaments and mitochondria, and blood capillaries were located at the basal portion of the epithelium (Fig. 5) . 2) Immunoelectron microscopy for serotonin Some epithelial cells or basal cells showed serotonin immunoreactivity. Several gold particles were concentrated on some of the granules of the epithelial or basal cells. However, concentrated particles were never observed in the other non-granulated epithelial cells and basal cells (Fig. 6) . Discussion

The immunohistochemical and ultrastructural studies have indicated that the primary saliva, which is secreted from acinar cells, is modified in the duct system by secretion of K+ and HC0 3- and absorption of Na+ and CI-3,6). Moreover, the striated duct and the granulated duct of the rat submandibular gland contain many physiological substances including growth factors such as FGF and EGF4,5). Morphological

23

Fig. I n-c Double- label immunohistochemical confocal images of PGP 9.5 (red) and chromogranin (green) in the MED of the rat submandibular gland. With the double- immunohistochemical demonstration of chromogranin A + Band PGP9.5. both PGP 9.5 and chromognmin immunoreactivity were observed in the same cell (yell ow) of the epithelium of the MED (Fig. I n). Fig. 1 bshows the immunoreactivity of PGP 9.5 (red). and Fig. I c shows the immunoreactivity of ch romogranin (green). Fig. 2 a-c Double- label immunohistochemical confOCal images of PCP 9.5 (red) and serotonin (green) in the . MED. Cells in the MED indicated the immunoreactivity of both I'G P9.5 (red) ;md serotonin. Fig. 2 b shows the immunoreactivi ty of pcp 9.5 (red). and Fig. 2 c shows the immunoreactivity of serotonin (green). Fig.3 Double- label immunohh;lochemical confocal images of PGP 9.5 (red) and serotonin (green) in the 1'\'IED. Several cells (.6. ) with double immunoreactivity of PGP 9.5 and serotonin were observed in the IviED epithelium. Nerve fibers (arrows) in the lamina propria indicated the immunoreactiv ity of PGP 9. 5 and serotonin. NG : submandibular ganglion

24

K. Higashi, et a l : Immunohistochemistry of MED of the Submandibular Gland

Fig. 4 a, b By electron microscopic observation, the epithe lium lining the MED of the fat submandibular gland was shown as pseuc\ostratified, consisting of many columnar cells and some cuboidal cells, brush cells (br) and basal cells. Nerve endings which contained many clear vesicl es and some mitochondria were present in the in tercellular space (arrow) . Fig. 4 b shows a high magnification of the nerve end ings in Fig. 4 a. Fig. 4 a : X 5,000 Fig. 4 b : X 22,000

Fig.5 The granulated cells (Ga) have a round nucleus with invagination and dense cytoplasm. Several granules were scattered in the cytoplasm (arrows). Nerve fibers (N) and blood ca pilla ri es (C) were located at the basal portion of the epithe lium. X7,000 Fig.6 Serotonin immunoe lectron microscopy of the MED epithelium. Severa l immuno- go ld particles were concentrated on the granules of the basal zone in the ep ithelia! cells. X 22, 000

25 studies of the MED have been reported by many authors l,2,9,lO). Epithelial cells in the MED have been classified into two groups, light cells and dark cells, based on their cytoplasmic electron densityI,9). Brush cells with long and thick microvilli including numerous microfilaments have been seen scattered between the epithelial cells 9,lO). Many basal cells existed on the basal zone of the epithelium. Several basal cells have primary cilia. The primary cilium might be a sensory . or chemoreceptive receptorS). Nerve endings have also been observed in intercellular space of the epithelium 9). Although there have been many morphological studies, the details of the modifying mechanism of the saliva in the MED have not been clarified. Granulated cells with dense oval-shaped granules were scattered in the epithelium of the MED. On the other hand, the endocrine cells including oval-shaped granules have been seen in the epithelium of the gallbladder 12 ). The structures of the epithelial cells of gallbladder seem to be similar to those of the epithelial cells of the MED. Therefore, it is possible that the same cells exist in the MED. When MED epithelium was treated with the Fa1ck-Hillarp method 13 ), some cells in the epithelium of the MED showed fluorescence of catecholamine 14 ). In general, endocrine cells with catecholamine in the digestive tract were examined by the Falck-Hillarp method. Therefore catecholamine-reactive cells may be localized in the MED. However, the details of the granulated cells of the MED have not been clarified. In addition to morphological studies of the MED, several immunohistochemical studies of the MED have been performed. C-type natriuretic peptide which modifies the composition of induced-salivary secretion 15 )and P-glycoprotein 16 ) which is related on secretion of primary saliva. PGP 9.5 immunoreactivity has been observed in the cells derived from the neural crest, and in nervous structures 17). In this study, PGP 9. 5-immunoreactive cells in the MED were observed. The existence of PGP 9.5 in neuroendocrine cells together with amines in the gastrointestinal tract was already known. Neuroendocrine cells in the digestive tract show both chromogranin and PGP 9.5 immunoreactivities IS). Both immunoreactivities for chromogranin and

PGP 9.5 were located in the central portion of the cells of the MED, except the nucleus, using the confocal laser scanning microscope. Moreover, only PGP 9.5 immunoreactivity was observed in the peripheral area of the cells. It seemed that immunoreactivity of chromogranin was shown in the cytoplasm, but PGP 9.5 immunoreactivity was shown on the cell membrane. The cells, which exhibited both chromogranin and PGP 9.5 immunoreactivity, seemed to resemble neuroendocrine cells in the digestive tract. Several types of neuroendocrine cells in the digestive tract show chromogranin and PGP 9.5 immunoreactivity IS). Therefore, we also attempted to clarify what type of neuroendocrine cells exist in the MED by the serotonin- PGP 9.5 immunohistochemical method. Serotonin-immunoreactive cells were scattered in the MED. All serotonin-immunoreactive cells were immunoreactive for PGP 9.5. These findings indicate that some epithelial cells and some basal cells in the MED are endocrine cells. Furthermore, other ovalshaped structures in the MED showed PGP 9.5 immunoreactivity. These structures may have been nerve endings. Nerve endings in the MED epithelium of the rat was already observed by Sato and Miyoshi. 9). Therefore, the PGP 9. 5-immunoreactive structures in the MED may be nerve endings. Serotonin in the endocrine cells of the digestive tract has a function as a regulator of motor nerves in blood vessels. The MED of the hamster and rat are surrounded by a network of blood capillaries I.2,19}. It was reported that the primary saliva was modified by the blood capillaries around the MED. The granulated cells containing serotonin in the MED are related to the regulation of the modifying mechanism of primary saliva in the MED through the regulation of blood vessel movement by serotonin 20 }. The presence of nerve endings in the MED was reported by Sato and Miyoshi 9}. However, these studies have not yet characterized the composition of nerve endings. In this study, serotonin-immunoreactive nerve endings were observed in the intercellular space between the epithelial cells of the MED. Serotonergic nerves have a function of inhibition of glandular secretion 21 ,22}.

26

K Higashi, et al : Immunohistochemistry of MED of the Submandibular Gland

Therefore, the modifying mechanism of primary saliva in the MED may be regulated by both serotonergic nerves and endocrine cells with serotonin.

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