Immunocytochemical study of granular duct cells with a hormonally enhanced granular cell phenotype in the mouse parotid gland

In the parotid glands (PGs) of intact male mice (12 weeks of age, ICR strain), immunofluorescence labels for a true tissue kallikrein, mK1, and for nerve growth factor (NGF) were recognized through the subluminal edges of the striated duct (SD) segments and interlobular duct segments. Because of their small size, secretory granules were not detectable by light microscopy in any of the duct cells. Full-fledged granular cells, containing large secretory granules that were visible by light microscopy, were induced in the SD segments of male mice after the injection of 5α-dehydrotestosterone (DHT) and triiodothyronine (T₃), given either alone or in combination every other day for 2 weeks. A stronger effect was detected in the mice that were concomitantly injected with DHT and T₃, and more abundant, fully developed granular cells appeared in the SD segments of these mice. These full-fledged granular cells were immunoreactive for mK1, NGF, and epidermal growth factor, but not for renin. The present results indicate that some of the SD cells with small granules in the mouse PG can develop a granular cell phenotype, producing more kinds of growth factors, as a result of the actions of androgen and thyroid hormone.     

The effects of ductal obstruction on the acinar cells of the parotid of cat

Twenty-nine parotids ligated for between 1 and 365 days were examined by light and electron microscopy. Major changes in the acini were seen at 4 days and included vacuolation, disintegration, extravasation, apoptosis, phagy and a reduction in number and size of secretory granules. There was a further reduction in secretory granules from 7 to 12 days, but acinar cells persisted even up to 365 days, some contained a luminal concentration of small secretory granules and occasionally acinar cells of a similar appearance to normal were found. These findings contrast with a reported absence of acinar cells from the obstructed parotid of rat and show that parotid acinar cells are able to persist and retain an appearance indicative of secretory activity.     

Repeated androgen and thyroid hormone injection modulates the morphology of hormone-responsive duct cells in the mouse parotid gland

When the parotid glands of normal male and female ICR mice (12 weeks of age) were examined under a light microscope, no granular cells were seen in the duct system. However, transmission electron microscopy revealed that, in both sexes, many striated duct cells contained a few electron-dense secretory granules in their subluminal cytoplasm and had formed so-called granular striated tubules (GSTs) in some of the striated duct segments. These secretory granules were not large enough to be visible with a light microscope. Fully fledged granular cells, containing large secretory granules visible with a light microscope, could be induced in the GST segments of the glands of males by injection with 5α-dihydrotestosterone (DHT), triiodothyronine (T₃), and dexamethasone (Dex), given alone or in combination every other day for 2 weeks. Dex alone showed no effect on the GSTs in this study. Both DHT and T₃, either individually or with Dex, were moderately effective, inducing a few scattered fully fledged granular cells. A stronger effect was detected after concomitant injection of DHT and T₃, with or without Dex, with more abundant fully developed granular cells appearing in the GST segments. Electron microscopy revealed that these granular cells had abundant large secretory granules in their apical two-thirds, a basal nucleus, and modest basal infoldings. By contrast, the effect of the same hormones was very weak in the glands of females, and even the concomitant injection of DHT and T₃, with or without Dex, rarely induced fully fledged granular cells. These results indicate a close similarity between the ductal systems of the major salivary glands of the mouse, in terms of some of the striated duct segments containing secretory granules, being under the same multihormonal regulation, and being sexually dimorphic.     

Radioprotection of the mice parotid gland by isoproterenol: study on morphometry of secretory granules and on autoradiography

Objectives The aim of the present study was to investigate the effect of radiosensitivity on acinar cells in the parotid gland when the secretory granules were released. Methods The parotid glands of mice were exposed to 10 Gy of X-radiation when the acinar cells were degranulated with isoproterenol (IPR). Three days later, morphological images and number and area of secretory granules within the acinar cells in the parotid glands were obtained and light microscope autoradiography (LMARG) was performed using ³H-leucine. Results The light microscopy images showed a disorderly arrangement and pycnosis of acinar cells and cellular atrophy in irradiated groups. The changes were milder in IPR-administered groups than in non-IPR-administered groups. The number of secretory granules in irradiated groups, which included both IPR-administered and non-IPR-administered sets, was significantly less than that in nonirradiated groups. The number of silver grains within acinar cells obtained by LMARG in the non-IPR-administered set of irradiated groups was significantly lower than that in the nonirradiated group or the IPR-administered set after 30 min of radioisotope administration, and it was significantly higher than that of the nonirradiated group after 240 min. Conclusions When the secretory granules of acinar cells in mouse parotid gland were degranulated by isoproterenol, alleviation of the effects of radiation exposure on morphological change as well as the ingestion and egestion of secretory substances were indicated.     

Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, alpha-amylase and B1-immunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

Parotid secretory granules: crossroads of secretory pathways and protein storage

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins-including amylase, proline-rich proteins, and parotid secretory protein (PSP)-to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.     

Immunohistochemical demonstration of acidic mammalian chitinase in the mouse salivary gland and gastric mucosa

Acidic mammalian chitinase (AMCase) is the sole chitinolytic enzyme that has been identified thus far in the gastrointestinal tract of mammals. AMCase mRNA expression has been demonstrated in the salivary gland and stomach of mice and in the stomach of humans, while a bovine homologue of AMCase is produced in the liver and secreted into the blood. The present study using antibody raised against bovine AMCase demonstrates the cellular distribution of AMCase in salivary and gastric secretions at the protein level. Immunostaining using mouse tissues detected intense immunoreactivity for AMCase in serous-type secretory cells of the parotid gland and von Ebner's gland. Gastric chief cells, localized at the bottom of gastric glands, were also immunoreactive for AMCase. Electron-microscopically, the immunoreactivity was localized in granules in the apical cytoplasm of these secretory cells, and not in other structures. Western blot analysis confirmed the existence of AMCase in the parotid gland and stomach, and in their secretions in mice. However, no immunoreactive band was clearly detectable in immunoblots of the human parotid saliva and gastric juice. At least in the mouse, AMCase is secreted into the saliva and gastric juice, and may function as a digestive enzyme or play a defensive role against chitinous pathogens.     

Ultrastructural study of hormonally responsive striated duct cells in the mouse sublingual gland

In semithin sections stained with Heidenhain's iron hematoxylin, a few scattered granular cells were observed in the striated ducts (SDs) of sublingual glands (SLGs) of the mouse; they were seen normally only in the glands of adult males. However, it was shown by electron microscopy that many SD cells, other than these granular cells, had apical secretory granules, thus forming a granular striated tubule (named the GST in this study) in a portion of SD segments in both sexes. Sublingual GST cells had very small dense secretory granules near the apical surface, with the nucleus in the apical one-third to one-half of the cell; small Golgi apparatus; sparse rough endoplasmic reticulum (RER); and well-developed basal infoldings. However, some granular cells in male GSTs had abundant large dense secretory granules in the apical two-thirds of the cell, a basal nucleus, and modest basal infoldings. Such granular SD cells disappeared after castration in males. Granular SD cells could be induced in the GSTs of females by the injection of 5α-dihydrotestosterone (DHT), triiodothyronine (T₃), and/or dexamethasone (Dex); given simultaneously, these hormones acted synergistically in this induction. These results indicate a close similarity between the duct systems of the SLG and those of the submandibulan gland (SMG) of the mouse: granular SD cells of the GST in the SLG resemble GCT cells in the SMG in expressing some of the same biologically active polypeptides, in being sexually dimorphic, and in being under the same multihormonal regulation. Received: October 19, 2000 / Accepted: April 24, 2001     

Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, oamylase and Brimmunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

小型猪与小鼠腮腺、泪腺、垂体超微结构的观察与比较

目的 研究小型猪及小鼠腮腺的超微结构特点,并将腮腺与泪腺、垂体的超微结构对比观察,旨为涎腺基因治疗转基因表达的生物学特性提供腺体形态学的依据.方法选用成年小型猪、昆明种小鼠各5只,分别取腮腺、泪腺、垂体进行超微结构观察.结果小型猪较小鼠的腮腺腺泡细胞内分泌颗粒密度大,质地均匀,细胞器少见,血管内皮细胞内有较多分泌颗粒存在.小型猪与小鼠泪腺超微结构均与各自腮腺相似,小型猪与小鼠垂体结构特点为细胞排列散在,细胞间充满血窦及毛细血管.结论小型猪腮腺腺泡细胞分泌颗粒多,其毛细血管内皮细胞中也可见较多分泌颗粒,提示这些分泌颗粒可能进入血液发挥内分泌功能.     

Ultrastructure of basal cell adenoma in the parotid gland

Basal cell adenoma of the parotid gland was studied with electron microscopy. The cells constituting this tumor were divided into three types of epithelial cells; ductal, myoepithelial, and squamous cells. The ductal cells, which were polygonal and cuboidal in shape, formed a sometimes distinct lumen. Glycogen were recognized in the cytoplasm of these cells. The myoepithelial cells appeared as plasmacytoid cells which contained abundant microfilaments. The squamous cells were characterized by the presence of well-developed tonofilaments and desmosomes. However, no secretory cells could be found, although small, electron dense granules were detected in the cytoplasm of the ductal cells. The granules were unlike secretory granules in their size, number and location. In consideration of the presence of secretory and myoepithelial cells, we reviewed previously reported literature and discussed the identification of secretory granules. From our and other reported results, we tentatively concluded that the electron dense granules described as secretory granules are not intrinsic secretory granules. Further, we suggested that the cell types and the histogenesis of basal cell adenoma are analogous to those of both pleomorphic and clear cell adenomas.     

Amylase and cyclic amp receptor protein expression in human diabetic parotid glands

J Oral Pathol Med (2010) 39 : 715–721 Background: Salivary dysfunction and oral disorders have been described in both type 1 and type 2 diabetes mellitus. However, the cellular and molecular consequences of diabetes on oral tissues remain to be ascertained. The purpose of this investigation was to study, by means of electron microscopy, the morphologic and molecular changes that occur in salivary glands during diabetes. Methods: Biopsy samples of parotid glands were excised from non‐diabetic and diabetic (type 1 and type 2) consenting patients and processed by standard methods for routine morphology and electron microscopic immunogold labeling. Specific antibodies were used to determine and quantify the expression of secretory proteins (alphaamylase and the regulatory subunit of type II protein kinase A). Results: Morphologic changes in the diabetic samples included increased numbers of secretory granules, and alterations in internal granule structure. Quantitative analysis of immunogold labeling showed that labeling densities were variable among the parotid gland samples. In type 1 diabetes amylase expression was greater than in non‐diabetic glands, whereas in type 2 diabetes it was not significantly changed. Expression of type II regulatory subunits was slightly, although not significantly, increased in acinar secretory granules of type 1 diabetic samples and was unchanged in type 2 diabetic samples. Conclusions: Our data show that diabetes elicits specific changes in secretory protein expression in human salivary glands, thus contributing to the altered oral environment and oral disease associated with diabetes.     

Morphological and biochemical changes in the rat parotid gland after compensatory and isoproterenol-induced enlargement

Enlargement of parotid glands can be induced in rats by treatment with isoproterenol (ISP) or by removal of the submandibular and sublingual glands. In this study, morphological changes in the enlarged parotid glands and qualitative changes in secreted proteins were examined in rats that had been treated with ISP for 10 days or that had been partially sialoadenectomized by removal of the submandibular/sublingual glands 2 weeks prior to killing. After ISP treatment or salivary gland ablation, secretory cells were enlarged and contained enlarged secretory granules that stained differently from granules in normal glands. Isoproterenol treatment induced the greatest enlargement of cells and granules. Even though gland structure was altered in both experimental groups, electrophoresis of saliva showed that submandibular/sublingual gland ablation did not lead to significant qualitative changes in secreted proteins, while ISP treatment induced major changes in the pattern of secreted protein. The results suggest that compensatory enlargement of the parotid glands and changes after ISP treatment are induced by stimulation of different regulatory pathways.     

Morphologic changes in the granular convoluted tubule cells of the mouse submandibular gland following hypophysectomy and hormonal replacement

 Morphological changes in the granular convoluted tubule (GCT) cells of the male mouse submandibular gland (SMG) were examined following hypophysectomy and hormonal replacement. Semithin sections stained with Heidenhain's iron hematoxylin showed that hypophysectomy severely regressed the GCT phenotype. Although only a few dispersed cells containing secretory granules were observed in the GCT segments under a light microscope, electron microscopy revealed that many regressed cells continued to constitutively elaborate apical secretory granules (although they were very small) and contained a euchromatic nucleus at the center of the cell, poor rough endoplasmic reticulum (RER) and Golgi apparatus in the perinuclear region, and well-developed basal infoldings. These findings suggest that hypophysectomy resulted in atrophy of GCT cells, but that they retained evidence of being secretory cells. 5α-Dihydrotestosterone (DHT); 3,5,3′-triiodo-l-thyronine (T₃); and dexamethasone (Dex) each enhanced the GCT phenotype of hypophysectomized males to some degree. Combined hormonal replacement with DHT + T₃ in hypophysectomized males restored a nearly normal male GCT phenotype with a full complement of secretory granules and rare basal infoldings, whereas T₃ alone induced a normal female-like GCT phenotype, with considerably abundant secretory granules and the usual short basal infoldings, in hypophysectomized male glands. Furthermore, Dex was found to synergistically enlarge secretory granules when administered with T₃ and/or DHT, although it was only weakly effective in enhancing the GCT phenotype when used alone. Taken together, the above findings confirmed that the GCT phenotype of the mouse SMG is regulated by the synergistic action of pituitary-dependent hormones. Received: October 1, 2001 / Accepted: May 13, 2002     

Expression and localization of calpain 3 in the submandibular gland of mice

ObjectivesCalpains comprise a family of intracellular Ca²⁺-dependent cysteine proteases and are considered to play roles in various physiological phenomena with limited proteolytic activities against specific substrates. We herein revealed the expression and localization of calpain 3, the muscle-type calpain, in the submandibular gland (SMG) of mice.DesignThe expression of the mRNA for conventional, ubiquitous calpains 1 and 2 and skeletal muscle-specific calpain 3 was examined in the major salivary glands of mice using RT-PCR, and the expression and localization of calpain 3 protein was examined in the SMG of mice using immunohistochemistry and Western blotting.ResultsThe large catalytic subunits of calpains 1 and 2 and the small regulatory subunit common to calpains 1 and 2 were weakly expressed in the parotid gland, sublingual gland, and SMG at similar levels in males and females. In contrast, the single large catalytic subunit of calpain 3 was expressed predominantly in the SMG at markedly higher levels in males than in females and in a manner dependent on androgens. Immunoreactivity for calpain 3 was mainly localized in cells of the granular convoluted tubules (GCT) that developed preferentially in the male SMG. In GCT cells, calpain 3 immunoreactivity was localized predominantly in the cytosolic region and was absent in the secretory granules.ConclusionsThese results revealed that the GCT is the primary site of production of calpain 3 in the mouse SMG.     

Presence of BPIFB1 in saliva from non-obese diabetic mice

We previously showed that mRNA expression of BPIFB1 (Bpifb1), an antibacterial protein in the palate, lung, and nasal epithelium clone protein family, was increased in parotid acinar cells in non-obese diabetic (NOD, NOD/ShiJcl) mice, which is an animal model for Sjögren’s syndrome. However, we did not previously assess the protein levels. In this report, we confirmed the expression of BPIFB1 protein in the parotid glands of NOD mice. Immunoblotting of subcellular fractions revealed that BPIBB1 was localised in secretory granules in parotid glands from NOD mice, and was almost not in parotid glands from the control mice. BPIFB1 had N-linked glycan that reacted with Aleuria aurantia lectin, which caused two types of spots with a slightly different pI and molecular weight. The expression of BPIFB1 protein was also demonstrated by immunohistochemistry. BPIFB1 was detected in the saliva from NOD mice but not in the saliva from the control mice, indicating individual constitution. BPIFB1 in saliva may be applied to other research as a diagnostic marker.     

Thyroid hormone regulation of granular intercalated duct cells in the submandibular glands of female mice

Intercalated ducts (IDs) in the submandibular glands (SMGs) of mice exhibit a sexual dimorphism, in which a few cells in the IDs of females, but not of males, possess secretory granules. The effects of a hypophysectomy (Hypox) followed by the administration of triiodo-l-thyronine (T₃) on such granular intercalated duct (GID) cells in the female gland were histologically examined. Semithin sections stained with Heidenhain's iron hematoxylin revealed that Hypox resulted in the complete disappearance of the GID cells. In Hypox females, electron-microscopy examination of the ID cells whose localization corresponded to that of the GID cells in normal females showed that these cells had a pale, centric nucleus, poorly developed rough-surfaced endoplasmic reticulum (RER) and Golgi apparatus, and no secretory granules. When T₃ (1mg/kg body weight) was given to Hypox female mice every other day for 2 weeks, the GID cells reappeared in most of the ID segments. Electron microscopy revealed that these cells had abundant secretory granules in their apical cytoplasm, a nucleus located near the base of the cell, and layered cisterna of RER and segments of Golgi apparatus in the perinuclear cytoplasm. The localization and distribution of the GID cells in the T₃-treated Hypox females were almost the same as those in normal females. Taken together, these results suggest that thyroid hormones upregulate the GID phenotype, and that thyroid hormones are essential for the exocrine activities of GID cells.     

Effect of moderate protein-deficiency on ultrastructure in parotid and submandibular acinar cells in the adult rat

Abstract— Powdered diets, of three different levels of protein, were given to groups of adult male rats for 21 days. Two diets with reduced protein content, 5% or 10% casein, were given to experimental rats. A diet with an adequate protein content, 20% casein, was given to controls. A reference group of rats was fed a standard pellet diet for the same period. At the end of the experimental period, the parotid and the submandibular glands were removed and subjected to electron microscopy. The zymogen granules of acinar cells in the parotid glands from the rats receiving the 5% protein diet had lost their normal opaqueness and turned electron lucid whereas parotid glands from the rats fed the 20%, the 10%, and the standard pellet diet exhibited normal acinar cells. The ultrastructure of submandibular acinar cells was not affected in any dietary group.     

Effect of moderate protein-deficiency on ultrastructure in parotid and submandibular acinar cells in the adult rat

Powdered diets, of three different levels of protein, were given to groups of adult male rats for 21 days. Two diets with reduced protein content, 5% or 10% casein, were given to experimental rats. A diet with an adequate protein content, 20% casein, was given to controls. A reference group of rats was fed a standard pellet diet for the same period. At the end of the experimental period, the parotid and the submandibular glands were removed and subjected to electron microscopy. The zymogen granules of acinar cells in the parotid glands from the rats receiving the 5% protein diet had lost their normal opaqueness and turned electron lucid whereas parotid glands from the rats fed the 20%, the 10%, and the standard pellet diet exhibited normal acinar cells. The ultrastructure of submandibular acinar cells was not affected in any dietary group.     

Effects of adriamycin on calcium concentration and morphology of mouse salivary glands

A large single dose (15 mg/kg body wt, ip) of the antitumor agent adriamycin (ADR) caused a marked increase in calcium concentration of submaxillary gland of female mice, and a smaller increase in the parotid gland within 2 days of injection. A small dose (2.5 mg/kg body wt) had no effect. The histological appearance of the glands was also changed and included an increase in size of granules and acinar cells of the submaxillary glands and a decrease in size of acinar cells of the parotid. At the EM level, there was evidence of mitochondrial alteration in the parotid but not in the submaxillary glands. Rough endoplasmic reticulum (RER) was markedly disorganized in the parotid, and abnormal whorls of RER were evident. Submaxillary glands showed no change in RER. Water content of either gland was unchanged from that of controls. Heart ventricles, unexpectedly, showed no change in calcium concentration from that of control tissues, at 3 h, 1, 2 or 4 days after ADR administration. The [Ca] changes induced by ADR in the submaxillary glands are not mediated via beta-adrenoceptor activation since propranolol did not alter the ADR-induced changes. The marked difference in response of the glands (and heart) to ADR, suggests that the mechanisms involved in calcium homeostasis in these organs are very different.