Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Immunolocalization of PTHrP in the sublingual glands of three mammals

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.     

Immunolocalization of PTHrP in the submandibular glands of three rodent species.

The present study deals with immunohistochemical localization of PTHrP in bank vole, pine vole and white mouse submandibular glands. PTHrP immunoreactivity was observed in epithelial cells of all ductal segments (intercalated, striated, interlobular and main excretory ducts) of the salivary glands in all the three animal species tested. We also found PTHrP expression in myoepithelial cells surrounding the mucous alveoli of submandibular glands in those animals. The reaction was less intense than that found in the epithelial cells of excretory ducts. We occasionally observed a very slight positive reaction for PTHrP in smooth muscle cells of small blood vessels. We also found PTHrP expression in the neurons of ganglion in the submandibular gland.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.     

The expression of water channel proteins during human salivary gland development: a topographic study of aquaporins 1, 3 and 5

Some members of aquaporin family (AQP) plays crucial functions in salivary synthesis and secretion. These proteins expression has already been reported during salivary gland formation, however no previous studies in human developing glands have been performed. We evaluated AQP1, 3 and 5 expression through the stages of human salivary gland morphogenesis and discuss the possible role of AQP for glandular maturation. Human salivary glands derived from foetuses aged between 14 and 25 weeks were submitted to immunohistochemistry. At the bud stage, membrane expression of AQP1, 3 and 5 were observed within the epithelial bud cells presenting a similar apicolateral pattern, also found at the pseudoglandular stage, present within the terminal portions of future acini, while AQP5 was also particularly strong at the apical membrane of pre-acinar and pre-ductal cells. AQP5 was co-localised with Cytokeratin 7. Similar AQP1, 3 and 5 expression were observed at the following canalicular stage, where distinct and strongly luminal and acinar AQP5 expression is present. During the final terminal bud stage, AQP1 was only identified in serous acini, myoepithelial and endothelial cells, while differentiated mucous acinar cells and ducts were negative. AQP3 was detected at apicolateral membranes of both mucous and serous acini. AQP5 also showed a diffuse expression in mucous and serous acini, in addition to strong apical membrane expression within lumen of intercalated ductal cells. This topographic analysis of AQP1, 3 and 5 revealed differences in the expression pattern throughout salivary gland developmental stages, suggesting different roles for each protein in human glandular maturation.     

The distribution and origin of nerve fibers immunoreactive for substance P and neurokinin A in the anterior buccal gland of the rat

The distribution and origin of substance P (SP) and neurokinin A (NKA) were studied in rat in the anterior buccal glands, which are minor mucous salivary glands. Indirect immunofluorescence staining showed moderate SP and NKA innervation of salivary acini and interlobular ducts, whereas blood vessels were more sparsely innervated, and there were few nerve fibers in the stroma and around the intralobular ducts. About 10%–20% of the trigeminal ganglion cells showed equally strong immunoreactivity to both SP and NKA. Unilateral denervation of the branches of the trigeminal nerve caused complete disappearance of the stromal fibers and greatly reduced the number of all other SP-immunoreactive and NKA-immunoreactive nerve fibers. In the superior cervical ganglia, SP and NKA immunoreactivity was restricted to small intensely fluorescent cells; SP and NKA immunoreactivity was absent from principal ganglionic cells, and thus sympathectomy had no any effect on the number or distribution of fibers immunoreactive for SP and NKA in the anterior buccal glands. The fibers remaining after sensory denervation could have been of parasympathetic origin, indicating a dual origin of nerves immunoreactive for SP and NKA in these glands. The present data demonstrate that the major part of the glandular SP and NKA innervation in the minor salivary glands derives from the trigeminal ganglia. The distribution of the peripheral nerve fibers indicates that they may play a role in the delivery of potent neuropeptides involved in the vascular, secretory, and motor (myoepithelial cells) functions of salivary glands.     

The structure of the salivary glands of the human soft palate

The glandular layer constitutes the greatest bulk of the human soft palate and is composed of individual compound tubulo-acinar salivary glands. Connective tissue partitions of the submucosa divide the glandular layer into lobules of irregular shapes and sizes. The glands are interwoven and bound firmly together by a connective tissue stroma rich in elastic fibers. The secretory units consist of elongated, branched, and sometimes convoluted tubules lined by a single layer of pyramidal mucous cells. Mucous secretion by acini is supplemented to some degree by mucous acinar cells, which were found as epithelial components of all ducts except the main excretory ducts, suggesting a diffuse distribution of progenitor cells. Some mucous acini communicate with highly convoluted intercalated ducts which occupy partially isolated positions within inter- and intralobular connective tissue septa. These ducts follow the connective tissue septa and eventually join the main duct system. The significance of this system of intercalated ducts is not known. A supplemental functional role is hypothesized.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands.

BACKGROUND INFORMATION: TSPO (translocator protein), previously known as PBR (peripheral-type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High-affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. RESULTS: Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam-binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, (3)H-labelled PK 11195, as shown by B(max) and K(d) values of 10.0+/-0.5 pmol/mg and 4.0+/-1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and alpha-adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K(+), Na(+), Cl(-) and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. CONCLUSIONS: High-affinity ligand binding to mitochondrial TSPO modulates neurotransmitter-induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

SD大鼠和Beagle犬大唾液腺的形态学观察

目的-研究及观察SD大鼠和Beagle犬大唾液腺正常比较组织学.方法-SD大鼠和Beagle犬三对大唾液腺剖取后进行石蜡切片、HE染色和PAS染色,光学显微镜观察.结果-SD大鼠腮腺是纯浆液腺,Beagle犬腮腺属混合腺,以浆液性腺泡为主,偶见小的粘液细胞群.SD大鼠的下颌下腺属于以浆液腺泡为主的混合腺,Beagle犬的下颌下腺属于以粘液腺泡为主的混合腺.SD大鼠与Beagle犬的舌下腺均为粘液性腺泡为主的混合腺.Beagle犬的眶腺亦是以纯粘液性腺泡为主的混合腺结构.     

Histochemical localization of prostaglandin-synthetase in the salivary glands of Praomys (Mastomys) natalensis

The cellular distribution of prostaglandin-synthetase was studied in the salivary glands of Praomys (Mastomys) natalensis. Only the epithelial cells of the ducts showed discrete prostaglandin-synthetase activity, whereas the acini and the stroma gave completely negative results in all three salivary glands of both sexes. The presence of exogenous substrates was not required for the reaction, since identical results are observed following incubation with and without arachidonic acid. The localization of prostaglandin synthetase in the ductal epithelia of Praomys salivary glands is related to a possible regulatory role of the prostaglandins on the reabsorptive activity of the ductal cells.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.     

The sympathetic and parasympathetic nature of neuropeptide Y-immunoreactive nerve fibres in the major salivary glands of the rat

Summary The distribution and origin of neuropeptide Y in the major salivary glands of the rat was studied by indirect immunofluorescence technique. Numerous nerve fibres immunoreactive for the peptide were seen in the parotid and sublingual glands. Most of the fibres were located around blood vessels and salivary acini. In the submandibular gland the number of immunoreactive nerve fibres around the acini was lower in comparison with that in the parotid and sublingual glands. Some immunoreactive nerve fibres were also found around or along intra- and interlobular ducts in all major salivary glands.A large number of the neuropeptide-containing neuronal cell bodies and nerve fibres were detected in the sympathetic superior cervical ganglion. Sympathetic postganglionic nerve trunks of this ganglion contained numerous immunoreactive nerve fibres as well. A subpopulation of the neuronal cell bodies in the submandibular ganglion were immunoreactive to neuropeptide Y.Both uni- and bilateral superior cervical ganglionectomies caused a significant decrease in the number of immunoreactive nerve fibres around the blood vessels in all the major salivary glands. However, these denervations did not affect the density of nerve fibres around the acini and ducts. On the contrary, unilateral parasympathetic denervation by sectioning the auriculotemporal nerve reduced the fibres around the secretory acini in the parotid gland remarkably, while only a minor reduction in the density of immunoreactive fibres associated with the blood vessels of the gland was detected. Unilateral electrocoagulation of the trigeminal nerve branches caused no detectable change in the density of immunoreactive nerve fibres in any of the major salivary glands.On the basis of the present findings it is concluded that neuropeptide Y-reactive nerve fibres present in all major salivary glands around the blood vessels seem to be mainly sympathetic, whereas those around the acini and ducts seems to be of parasympathetic origin.     

Localization of neonatal secretory proteins in different cell types of the rat submandibular gland from embryogenesis to adulthood

In the neonatal rat submandibular gland, Type III cells contain a group of related proteins that we call the B1-immunoreactive proteins (B1-IP; 23.5, 26, and 27.5 kDa). Type I cells lack these, but synthesize a different protein, Protein C (89 kDa). With maturation of the gland, these neonatal cell types are no longer seen in the seromucous acini, which are no longer reactive for the B1-IP. Here, we report the ultrastructural immunocytochemical localization of the B1-IP and Protein C over the course of development. From their first appearance in the embryo, the B1-IP and Protein C are present in different cells which become morphologically typical Type I and III cells prior to birth. At all stages, Type I cells have strong Protein C labeling and no B1 labeling. By 3 days postpartum, ultrastructurally atypical Type III cells are seen (Type IIIP); these label for the B1-IP, but also show labeling with antibody to Protein C. In the next week, as mucous cells appear in the acini, these show both B1-IP and C labeling; the B1 marker is lost by 30 days postpartum, but adult mucous acinar cells continue to show Protein C reactivity. In view of the appearance of Protein C reactivity in neonatal Type IIIP and then in mucous cells, and the presence of B1 reactivity in early but not mature mucous cells, we suggest that Type III cells differentiate into mucous cells and that Type IIIP cells are intermediates in this transformation. We see no evidence for the differentiation of either Type III or mucous cells from Type I cells, although our data cannot rule out this possibility. In adult glands, cells with B1 labeling are seen in intercalated ducts. Cells that appear to be Type I cells are also present in these ducts and label for Protein C. Double labeling for B1-IP and Protein C demonstrated that the two markers were exclusively present in different cells within intercalated ducts. This is of considerable interest, as intercalated ducts have been reported to be the stem cell population for normal and trauma-induced cellular replacement.     

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.     

Immunohistochemical distribution of calcitonin gene-related peptide in nerve fibres of the anterior buccal gland of the rat

Summary Calcitonin gene-related peptide immunoreactive (CGRP-IR) nerves in rat were studied as to their distribution and origin in anterior buccal glands, which are unique minor mucous salivary glands in the rat. A moderate number of CGRP-IR nerve fibres were located, mostly perivascularly and around intralobular ducts, but they were also found around acini. The latter fibres were mainly of sensory origin, as suggested by their disappearance after denervation of the ophthalmic and maxillary branches of the trigeminal nerve. On the contrary, CGRP-IR nerves around interlobular ducts and some of those in a perivascular location remained both after sensory denervation and after extirpation of the sympathetic superior cervical ganglion. Whether these fibres originate in dorsal root ganglia C₃−C₄ or represent parasympathetic fibres is not known. Based on the present data and the previous findings showing a regulatory role of CGRP both on blood and salivary flow, it is possible that CGRP in sensory, and possibly also in parasympathetic nerves, participates in the regulation of reflex blood flow and salivary secretion in the anterior buccal gland of the rat.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.