Epidermal growth factor and insulin-like growth factor I are localized in different compartments of salivary gland duct cells. Immunohistochemical evidence

Immunohistochemical methods were used to map EGF (epidermal growth factor) and IGF-I (insulin-like growth factor I; somatomedin C) immunoreactivities in salivary glands of adult rodents. Epidermal growth factor is, as is NGF (nerve growth factor), limited in distribution to the granules in granular duct cells in the submandibular gland. Insulin-like growth factor I is, in contrast, cytoplasmic and has a much more widespread distribution. It is seen in intercalated, striated and granulated duct cells as well as in apical parts of excretory duct cells. The parotid and the palatine salivary glands, lacking EGF immunoreactivity, have their IGF-I immunoreactivity similarly distributed as the submandibular gland. Isoproterenol treatment of adult male rats results in rapid and extensive growth of the submandibular and the parotid glands, which double their weights in just a few days. Isoproterenol causes release of granules from the submandibular granular duct cells and decrease in frequency of EGF immunoreactive cells. However, there is no or only minor concomitant changes in the distribution and intensity of the IGF-I immunoreactivity in these duct cells. Our results indicate that the trophic peptides EGF (and NGF) and IGF-I are localized in different compartments in salivary gland duct cells and that divergent pathways control their release.     

Cellular and subcellular colocalization of nerve growth factor and epidermal growth factor in mouse submandibular glands

Immunocytochemical methods have been used to compare the cellular and subcellular distribution of nerve growth factor (NGF) and epidermal growth factor (EGF) in mouse submandibular glands. Rabbit antisera raised against purified proteins were characterized by immunoblot methods and were used to stain sections of salivary glands embedded in plastic. For light microscopy, antibodies were visualized by indirect immunofluorescence. For electron microscopy, thin sections were treated simultaneously with IgG against NGF and EGF coupled to colloidal gold particles of different size. Data indicate that NGF and EGF are present in all granular convoluted tubule cells and in no other cell type within the salivary gland. Ultrastructural analyses indicate that NGF and EGF are evenly distributed together within mature secretory granules, although a population of small granules was identified that is not immunoreactive for either protein. Taken together, the data suggest that granular convoluted tubule cells are homogeneous in the production and storage of NGF and EGF.     

Expression and localization of cell growth factors in the salivary gland: a review

The salivary gland secretes not only digestive enzymes but also various cell growth factors. Especially in rodents, granular ducts which develop between striated and intercalated ducts, are known to secrete epidermal growth factor (EGF) and nerve growth factor (NGF). Out of newly discovered growth factors, we have examined hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor I (IGF-I), and basic fibroblast growth factor (FGF-2) and have demonstrated their expression and localization in the rat submandibular gland. HGF and TGF-beta show a very similar distribution pattern to EGF, i.e., exclusive localization in the secretory granules of granular duct cells. These factors are suggested to function as i) exocrine salivary factors, or ii) endocrine factors after reabsorption. In contrast, FGF-2 shows a different pattern of cellular and subcellular localization from EGF and others. FGF-2 is localized in cytoplasm of striated and excretory duct cells and pillar cells in granular ducts, but not in secretory granules of granular duct cells. Since the receptor for FGF also present in the same cells as its ligand, FGF is suggested to function in the salivary gland as an autocrine/paracrine factor. The possible physiological roles of salivary growth factors in the digestive organs are discussed based on our own data and an extensive literature.     

Epidermal growth factor, renin, and protease in hormonally responsive duct cells of the mouse sublingual gland

In the striated ducts of the sublingual glands of normal adult male, but not female, Swiss-Webster mice a few scattered cells have apical secretion granules. These sublingual duct cells resemble the granular convoluted tubule (GCT) cells of the submandibular glands of adult female mice, in that they are smaller than submandibular GCT cells of adult males, and contain fewer apical granules, and prominent basal striations. These cells stain immunocytochemically for epidermal growth factor (EGF), renin, and protease A. Such granular striated duct cells could be induced in the sublingual glands of adult female mice by treatment with either testosterone propionate or thyroxine; the two hormones given simultaneously acted synergistically in this induction.     

Immunocytochemical localization of nerve growth factor,epidermal growth factor,renin and protease A in the submandibular glands of Tfm/Y mice

The submandibular glands of mice with testicular feminization (Tfm/Y) and their normal adult male littermates (Ta/Y) were studied by immunocytochemical techniques for the demonstration of epidermal growth factor (EGF), nerve growth factor (NGF), renin and protease A. In the glands of both the affected and normal males, these polypeptides were restricted to cells of the granular convoluted tubules (GCT), with the exception of protease A, which was also found in small amounts in striated duct cells. Compared to those of Ta/Y males, GCTs were narrower in the glands of Tfm/Y mice and contained a markedly reduced number of cells immunoreactive for EGF, NGF and renin. However, the number of GCT cells that stained for protease A in the glands of Tfm/Y males was not as drastically decreased.     

人体正常涎腺组织中成纤维细胞生长因子受体3的表达

目的　探讨成纤维细胞生长因子受体 3 (FGFR3 )在人体正常涎腺组织中的表达情况。方法　正常成人涎腺标本2 4例 ,其中腮腺、颌下腺和舌下腺标本各 4例 ,唇腺、腭腺、磨牙后腺及舌腺各 3例 ,分别用超敏S P免疫组化法检测不同腺体组织中FGFR3的分布。结果　在腮腺的浆液性腺泡的分泌颗粒及其他混合性腺的浆液性腺泡的分泌颗粒中均可见少量的FGFR3的表达 ,在大唾液腺及小唾液腺的小叶内导管和小叶间导管中均可见中等强度的FGFR3的表达 ,其中以颌下腺中表达较强 ,而在舌下腺中表达较弱 ,在间质结缔组织中均无FGFR3的表达。结论　人体正常涎腺的导管系统中及浆液性腺泡中有FGFR3的表达     

Expression of podoplanin and classical cadherins in salivary gland epithelial cells of klotho-deficient mice

We have recently shown that salivary gland myoepithelial cells express podoplanin. Podoplanin indirectly binds the actin filament network which links classical cadherins. The study here is aimed to investigate the expression of podoplanin and cadherins on salivary gland myoepithelial cells and the changes in the aging cells using klotho-deficient (kl/kl) mice. The submandibular glands of kl/kl mouse lack granular ducts which express klotho in wild type mice, suggesting that klotho may be a gene responsible for granular duct development. Although aging resulted in growth suppression of myoepithelial cells because of the sparse distribution of the cells in kl/kl mouse salivary glands, the expression of podoplanin and E-cadherin was shown in aging myoepithelial cells. It is thought that podoplanin participates in the actin-E-cadherin networks which are maintained in aging myoepithelial cells. It was also shown that granular ducts were filled with P-cadherin, and that the P-cadherin amount was larger in the wild type mouse submandibular glands than in the sublingual and parotid glands of wild type mouse, and in the submandibular glands of kl/kl mouse. These findings suggest that the granular duct is an organ secreting soluble P-cadherin into the saliva.     

Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: An immunocytochemical study

Submandibular glands of male mice were stained for nerve growth factor by light microscopic immunocytochemistry. Nerve growth factor (NGF) was present in the granules of granular tubule cells, with the immunoreactive material often concentrated at the periphery of granules. Administration of the α-adrenergic agent, phenylephrine, to animals resulted in a marked depletion of NGF-containing granules from granular tubules. Some release also occurred following administration of the β-adrenergic agent, isoproterenol. Cholinergic stimulation (pilocarpine) did not result in appreciable loss of immunoreactive granules from these cells. In vitro results were not as clear cut, immunocytochemically, as those obtained with intact animals. It is concluded that discharge of NGF from male mouse submandibular glands is mediated predominantly by α-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Secretagogue-mediated discharge of nerve growth factor from granular tubules of male mouse submandibular glands: an immunocytochemical study.

Submandibular glands of male mice were stained for nerve growth factor by light microscopic immunocytochemistry. Nerve growth factor (NGF) was present in the granules of granular tubule cells, with the immunoreactive material often concentrated at the periphery of granules. Administration of the alpha-adrenergic agent, phenylephrine, to animals resulted in a marked depletion of NGF-containing granules from granular tubules. Some release also occurred following administration of the beta-adrenergic agent, isoproterenol. Cholinergic stimulation (pilocarpine) did not result in appreciable loss of immunoreactive granules from these cells. In vitro results were not as clear cut, immunocytochemically, as those obtained with intact animals. It is concluded that discharge of NGF from male mouse submandibular glands is mediated predominantly by alpha-adrenergic activation, and that this phenomenon is readily demonstrated in the intact animal.     

Immunocytochemical localization of epidermal growth factor during the postnatal development of the submandibular gland of the mouse

The time of appearance and the pattern of localization of epidermal growth factor (EGF) in submandibular glands of mice was studied during postnatal development immunocytochemically. EGF was first detectable in the granular convoluted tubule (GCT) cells in the glands of males at 20 days of age and of females at 30 days of age. Development of GCT cells containing EGF was rapid in males, approaching adult conditions by 45 days of age. In females EGF-containing GCTs developed more slowly and irregularly, and did not reach adult status by 45 days of age. It is concluded that EGF is restricted during postnatal development to the GCT cells, and that these cells and the distribution of EGF are represented dimorphically from their first appearance in the submandibular glands of both sexes.     

Pituitary adenylate cyclase‐activating polypeptide enhances saliva secretion via direct binding to PACAP receptors of major salivary glands in mice

Xerostomia, or dry mouth, is a common syndrome that is generally treated with artificial saliva; however, no other effective methods have yet been established. Saliva secretion is mainly under the control of the autonomic nervous system. Pituitary adenylate cyclase‐activating polypeptide (PACAP) is recognized as a multifunctional neuropeptide in various organs. In this study, we examined the effect of PACAP on saliva secretion, and detected the distribution of the PACAP type 1 receptor (PAC1R) in major salivary glands, including the parotid, submandibular, and sublingual glands, in 9‐week‐old male C57BL/6 mice. Intranasal administration of PACAP 38 increased the amount of saliva secreted, which was not inhibited by atropine pretreatment. Immunohistochemical analysis showed that PAC1R was distributed in the three major salivary glands. In the parotid and sublingual glands, PAC1R was detected in striated duct cells, whereas in the submandibular gland, a strong PAC1R immunoreaction was detected in tall columnar epithelial cells in the granular ducts (i.e., pillar cells), as well as in some striated duct cells. PACAP significantly increased the concentration of epidermal growth factor in saliva. These results suggest that PACAP directly regulates saliva secretion by controlling the absorption activity in the ducts, and that pillar cells regulate the function of granular epithelial cells in the granular duct, such as the secretion of growth factors into the saliva. Collectively, these results suggest the possibility of PACAP as a new effective treatment of xerostomia. Anat Rec, 299:1293–1299, 2016. © 2016 Wiley Periodicals, Inc.     

Electron microscopic immunostaining of nerve growth factor: secretagogue stimulated submandibular glands

Immunocytochemical localization of nerve growth factor (NGF) was assessed on thin sections of plastic-embedded male mouse submandibular glands by electron microscopy. Both control and secretagogue-stimulated glands were examined. NGF was localized in granules of both granular convoluted tubule (GCT) cells and transition cells. The latter were intermediate in morphology between GCT cells and striated duct cells. Both large and small granules were immunostained in GCT cells; however, considerable variability in immunostaining intensity was observed in both sizes of granules but especially in the small granules of transition cells. Rough endoplasmic reticulum (RER) in both cell types exhibited NGF immunoreactivity. No Golgi-associated immunostaining was observed. Following alpha-adrenergic stimulation with phenylephrine, NGF-containing granules were sharply reduced because of extensive degranulation. Pools of immunostained secretory material suggested intracellular fusion of NGF-containing granules. Immunostaining was also observed on membrane fragments found within large vacuoles in GCT cells. Evidence of NGF secretion after beta-adrenergic or cholinergic stimulation was less dramatic. In isoproterenol-stimulated GCT cells there was evidence of fusion of small, apical, NGF-stained granules. These cells also possessed heavily immunostained apical membrane blebs. Pilocarpine-stimulated cells exhibited pleomorphic immunostained apical granules but less apical membrane immunostaining. Abundant basal lysosomes appearing in GCT cells after pilocarpine stimulation did not stain for NGF.     

小鼠下颌下腺发育中转化生长因子β受体的表达

背景：小鼠的下颌下腺是研究唾液腺的发育的良好模型,转化生长因子β是器官发育和疾病中重要的生长因子,但是在下颌下腺中转化生长因子β受体的表达以及作用机制至今并不明确。 目的：观察胚胎小鼠下颌下腺发育过程中转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2的表达,揭示转化生长因子β在小鼠涎腺发育中的作用。 方法：取C57BL/6J小鼠胚胎期第14.5天的标本,使用转化生长因子βⅠ型受体和Ⅱ型受体以及p-ERK1/2抗体,对小鼠的下颌下腺进行免疫组化染色。取新生小鼠标本,大体观察下颌下腺,并且使用苏木精-伊红染色观察其形态。 结果与结论：①小鼠出生时,下颌下腺位于下颌骨下方;苏木精-伊红染色发现小鼠下颌下腺的腺泡、导管和闰管细胞也已经分化完成。②在胚胎期第14.5天,转化生长因子βⅠ型和Ⅱ型受体在腺泡上皮和导管上皮内高表达,而腺体上皮细胞外的间充质没有表达。③p-ERK1/2主要也是表达在下颌下腺的上皮细胞中,与转化生长因子βⅠ型受体和Ⅱ型受体在下颌下腺中的表达基本一致。说明在小鼠下颌下腺的发育过程中,转化生长因子β蛋白可能通过与上皮细胞表面的受体结合,激活ERK信号通路来调节涎腺腺泡和导管的发育。     

Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland

The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected. The question of renewing or expanding populations is resolved by quantitating and integrating the rates of autologous cell division, differentiation, and apoptosis for each cell type. The integrated data shows that both acinar and granular duct cell populations exhibit a substantial positive growth index, whereas the growth index for the intercalated duct cells is moderately negative. On balance, it suggests that the submandibular gland of the young adult female mouse is still growing. Comparison of young female mice with older females suggests that, although overall parenchymal growth slows with age, there is no longer a net loss of intercalated duct cells. Comparison with young adult male submandibular glands indicates that gender differences exist in the rates and mechanisms used for maintaining the different cell populations. The acinar and granular duct cell populations in young adult female mouse submandibular glands are expanding at the expense of the intercalated duct cell population, which appears to be contracting.     

Rat cytomegalovirus replication in the salivary glands is exclusively confined to striated duct cells

The salivary gland is the preferred organ for cytomegalovirus (CMV) replication and viral persistence. In order to identify the nature of infected cells and to study viral replication in more detail, several experiments were conducted. Using the rat CMV (RCMV) model, acutely infected young adult rats (6 weeks of age) and new-born rats (3 days of age) were infected, and submandibular, parotid and sublingual glands were harvested at different time points after infection. For identification of the nature of infected cells, immunohistochemistry, in situ hybridisation and electron microscopic techniques were used. In young adult animals, the submandibular gland was the preferred organ for RCMV replication. The parotid and sublingual glands contained fewer viruses than the submandibular gland. In contrast, in new-born rats, the main site of RCMV replication was the sublingual gland, while the submandibular and parotid glands contained low amounts of virus. No virus could be detected in the parotid glands. In all glands of RCMV-infected animals, the infection was exclusively confined to striated duct cells. Infection resulted in a cellular inflammatory response which was mostly located in the interlobular duct region, whereas only few inflammatory cells were found in the neighbourhood of infected striated duct cells. This phenomenon may contribute to the long persistence of the virus in this organ. Received: 25 May 2000 / Accepted: 26 May 2000     

Effect of advanced age on the induction by androgen or thyroid hormone of epidermal growth factor and epidermal growth factor mRNA in the submandibular glands of C57BL/6 male mice

We have compared the responsiveness of the submandibular glands of mature (12 month old) and senescent (26-28 month old) male C57BL/6 mice to dihydrotestosterone (DHT) or triiodothyronine (T3) in terms of steady state levels of epidermal growth factor (EGF) protein and EGF mRNA. Northern blot analyses did not disclose any differences with age in the apparent sizes of EGF mRNA species. In untreated animals, submandibular glands of 26-28-month-old mice contained approximately 50% less EGF, and 75% less EGF mRNA than those of 12-month-old males. With advanced age, there was a 20% reduction in the absolute volume of the granular convoluted tubule (GCT) compartment, which is the exclusive site of EGF and EGF mRNA in the gland. In general, GCTs of old mice were composed of smaller cells with fewer secretion granules, but there was considerable cell-to-cell variation. In addition, there was greater variation in the intensity of immunocytochemical staining for EGF in senescent GCT cells, which also gave a lower and more variable in situ hybridization signal for EGF mRNA. After hormonal stimulation for 1 week with either tri-iodothyronine (T3) or dihydrotestosterone (DHT), EGF protein concentration in the glands was induced to the same level at both ages. However, EGF mRNA was 50% less abundant in old hormonally stimulated glands, compared to similarly treated young ones. Although many GCT cells in treated glands of senescent males respond to hormonal stimulation by increases in size and in content of secretion granules, there was cell-to-cell variation in responsiveness, especially after treatment with T3. These findings indicate that the decreases seen in the entire gland in EGF and EGF mRNA are caused by a wide-spread deterioration of the GCT cells themselves, which apparently can be reversed in many but not all GCT cells by stimulation with supraphysiologic doses of either T3 or DHT.     

Peroxidase activity in the submandibular gland of the house musk shrew, Suncus murinus (Soricidae, Insectivora)

Endogenous peroxidase activity in the submandibular gland of the house musk shrew, Suncus murinus was cytochemically investigated by light and electron microscopy using 3,3'-diaminobenzidine-tetrahydrochloride salt (DAB). The submandibular glands of male Suncus murinus at 8-month-olds were excised and diced into small pieces. In general, salivary glands are structurally divided into a terminal portion comprising a secretory portion and duct system. The submandibular gland of the Suncus murinus, the terminal portions consisted of proximal and distal acinar cells. On the other hand, a granular duct cell of the duct system contained a number of characteristic myelin-like bodies. In the present study, the peroxidase reaction products were localized in the secretory granules of the proximal acinar cells and in the endoplasmic reticulum, Golgi apparatus and myelin-like bodies of the granular duct cells. These reaction products were reduced when 5 mM 3-amino-1,2,4-triazole was added to the reaction medium. Additionally, release of peroxidase into the lumen was observed. In conclusion, the proximal acinar and granular duct cells formed peroxidase and may have performed excretory secretions. Moreover, the peroxidase positive myelin-like body consisted of lamellated membrane and its outer surface membrane continued to the endoplasmic reticulum.     

Phenotypic expression of human epidermal growth factor in foetal submandibular gland and pleomorphic adenoma of salivary gland

Summary The phenotypic expression of the human epidermal growth factor (EGF) was investigated immunohistochemically in human foetal submandibular glands from the 5th to 10th month of gestation, adult normal submandibular glands and 48 cases of pleomorphic adenomas. In foetal submandibular glands, both the terminal buds and primary ducts at the intermediate stage of gestation were positive for EGF, and in particular, the outer layer cells of primary ducts showed strong EGF-immunoreactivity. EGF-positive cells decreased as the gestational stage advanced and only ductal cells were weakly positive for EGF at the terminal stage of gestation. In the adult normal submandibular gland, weak immunoreactivity for EGF was restricted to ductal cells. However, 41 (86%) of the 48 pleomorphic adenomas had EGF-positive cells which were distributed among the ductal, chondroid and myxoid portion. No EGF-immunoreactivity was detected in the solid portion of pleomorphic adenomas. These results suggest that EGF may play an important role in the growth and differentiation of foetal cells as well as the proliferation of tumour cells in pleomorphic adenomas.     

On the ultrastructure of the parotid and submandibular gland of the mongoose Herpestes edwardsi (Viverridae) (author's transl)

The parotid and submandibular glands of the mongoose are described. Essential differences between the 2 glands were recognized in the acini; however, the intra- and interlobular ducts are built up similarly. The parotid gland is acinar. Its secretory cells are filled with distinct types of granula, which show a considerable variation of size and structure of their secretory material. Organelles are found sparsely. The submandibular gland, however, is tubuloacinar. Its tubuli are capped with cells which belong to the demilunes of v. EBNER, but because of their pale granules they occupy an exceptional position. As the acinar cells of the parotid gland, they form intercellular canaliculi by their plasmalemmata. In the secretory cells of the tubules an intimate contact between the rER and the granules is observed. The intralobular duct surface is built up by an onelayered epithelial cell formation. The cytoplasm of the intercalated duct cells is rich in bundles of filaments, and these cells contain mitochondria with a particular dense matrix. Some microvilli cover the apical surface. In the cells of the striated ducts several populations of granules differing in size and electron density are found. The material of the dense granules shows a marginal plate-like condensation, sometimes it cristallizes. It is supposed that they were released by an apocrine extrusion mechanism. Terminal axons innervate the acini, the duct cells, and also the myoepithelial cells. The findings are compared with the well-known morphology of the salivary glands of the cat.