Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.     

Quantitation and localization of acinar cell-specific mucin in submandibular glands of mice during postnatal development

Summary In this study, antiserum to acinar cell-specific mucin was utilized to determine whether mucin could be detected in the mouse submandibular gland prior to cytodifferentiation of acinar cells. Results from radioimmunoassay indicated that mucin occurs in submandibular glands from newborn mice, i.e., before the appearance of mature acinar cells. Additionally, mucin quantitated in various stages of development was found to be antigenically identical to adult mucin. After sections of glands were treated with immunohistochemical reagents, we observed that the mature acinar cell-specific mucin was present in secretory terminal-tubule cells and in proacinar cells of newborn animals. The present findings suggest that in young animals, the proacinar cells are an immediate precursor of acinar cells and that the secretory terminal-tubule cells may represent an earlier stage in development of acinar cells. In adult female glands, mucin was also detected in the granular intercalating-duct cells. This latter observation is consistent with the hypothesis that these cells are an intermediate in the acinar cell replacement process.     

Morphological changes and EGF expression in the granular convoluted tubule cells of submandibular glands of Trypanosoma cruzi infected rats

We have previously demonstrated in rats that Chagas' disease affects the salivary glands, by promoting an enlargement of the submandibular gland. In order to further investigate possible functional alterations on infected submandibular glands, the objective of the present study was to analyze epidermal growth factor (EGF) expression on rat submandibular glands during Trypanosoma cruzi infection. Results demonstrated that infected rats presented lower levels of testosterone, and morphological changes in the granular convoluted tubule (GCT) cells of the submandibular glands, along with acinar enlargement and delayed ductal maturation at the developing granular ducts. Immunohistochemistry analysis additionally showed that only few cells immunolabelled with anti-EGF on infected rats during the acute phase of Chagas' disease, while after 64 and 90 days (chronic phase) of infection, EGF expression was similar to non-infected rats. The present findings suggest that at the acute phase of Chagas' disease, lower levels of testosterone may lead to a delayed maturation of GCT, which positively correlates with decreased EGF production by submandibular glands cells.     

Redistribution of carbonic anhydrase VI expression from ducts to acini during development of ovine parotid and submandibular glands

 Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands.With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay. Accepted: 19 December 1996     

Monoclonal antibodies against rat saliva and salivary gland antigens

Hybridomas were produced by the fusion of NS1 myeloma cells with spleen cells of a BALB/c mouse immunized with rat submandibular saliva. Growth of hybridomas was evident in 60/96 wells, and colonies secreting antibodies against saliva components were identified in 20 wells by using a solid phase enzyme-linked immunoassay. Cloning of cells from 12 wells yielded originally 43 hybridoma cell lines secreting anti-saliva antibodies. After recloning, one hybridoma (4Cl3) was selected for further studies. The hybridoma (4Cl3) cells were grown as ascites tumors, and the antibodies were purified from the ascitic fluid by diethylaminoethyl Affi-gel Blue chromatography. The purified antibody (MA4), immunoglobulin G1, immunoprecipitated a 39K dalton protein from submandibular saliva, and also reacted with a protein of the same electrophoretic mobility on immunoblots. From extracts of submandibular gland slices, incubated with [3H]leucine, the antibody again immunoprecipitated a 39K protein, indicating that this protein is synthesized in the gland. MA4 was used for immunocytochemical stainings of submandibular glands of rats of different ages. In general, immunostaining was seen only in acinar cells. Thus, there was no staining in the glands of 1-day-old rats that lack differentiated acinar cells. In the glands of 1- to 4-week-old rats the number of immunoreactive cells and the extent of immunostaining paralleled the differentiation of the acinar cells. In the glands of adult rats a uniform staining of the secretory granules of the acinar cells was observed. The immunoreactive 39K protein seemed to be restricted to the acinar cells in the submandibular gland; there was no immunostaining in the parotid, sublingual, or lingual salivary glands, or in the pancreas, colon, and duodenum. Stimulation of saliva secretion by isoproterenol resulted in a virtual depletion of the antigen from the acinar cells. These results indicate the feasibility of producing mouse hybridomas that secrete antibodies against rat saliva components. The monoclonal antibody at hand will be useful in analyzing the differentiation of the acinar cells, and the factors that influence this differentiation process.     

Expression of epidermal growth factor in salivary adenoid cystic carcinoma

This study used an immunohistochemical technique to assess the expression of epidermal growth factor (EGF) in 40 specimens of salivary adenoid cystic carcinoma (ACC), 7 specimens of labial glands adjacent to mucocele, and 5 specimens of normal submandibular glands. In normal submandibular glands, immunohistochemically detectable EGF was demonstrated in all ductal segments, including intercalated, striated, and excretory duct cells. No EGF positive staining was found in acinar compartments. including serous and mucous acinar cells. In degenerated labial glands adjacent to mucocele, no EGF staining was detected in the remaining acinar and ductal cells. In salivary ACCs, positive EGF immunostaining was observed in one of the 5 (20%) ACCs with a solid pattern and in 13 of the 35 (37.1%) ACCs with a tubular-cribriform pattern. The overall EGF expression rate in 40 salivary ACCs was 35%. Positive EGF staining was predominantly found in tubular structures in the tubular ACCs and in duct-like structures in large cribriform patterns or in the stroma of the cribriform ACCs. There was no significant correlation between EGF expression in salivary ACCs and any of the clinicopathological parameters including patient age and sex, cancer location, TNM status, clinical stage, histologic type, perivascular or perineural invasion, focal necrosis of tumor, and cellular atypia. We conclude that the duct segments of the normal submandibular gland are the sites of EGF synthesis and secretion. In degenerated labial glands adjacent to mucocele, EGF synthesis is completely inhibited. Furthermore, EGF is mainly biosynthesized in cells forming tubular or duct-like structures in tubular or cribriform salivary ACCs, and EGF may play a biologic role, particularly as a mitogen in salivary ACC growth.     

Morphometric and fine structural study of experimental autoallergic sialadenitis of rat submandibular glands.

To further our understanding of the immunopathologic mechanisms involved in experimental autoallergic sialadenitis of rat submandibular gland (EAS), histometric and fine structural studies were undertaken. Rats were immunized with allogeneic submandibular glands (SMG) emulsified in complete Freund's adjuvant. Control rats were not treated (C) or adjuvant treated (At). The rats were sacrificed 7, 14, 21 and 28 days after immunization and their SMG were processed for light and electron microscopy. Groups "C" and "at" showed normal acini and ducts. The SMG at 14 days showed significant loss of acini and granular ducts, severe lymphocytic infiltration and the appearance of undifferentiated ducts. The cells of the latter showed abundant free ribosomes, few profiles of rer, no secretory granules and in some cells autophagic vacuoles. Pseudopods of many lymphocytes were found in juxtaposition to degenerating parenchymal cells, mast cells and eosinophils. The extralobular ducts were significantly increased at 7, 14, and 21 days. The immunized glands showed evidence of regeneration at 21 and 28 days. Terminal tubule cells, proacinar cells and acinar cells, at various stages of maturation, were found in the regenerating glands.     

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.     

Immunolocalization patterns of cytokeratins during salivary acinar cell development in mice

Embryonic development of the mouse salivary glands begins with epithelial thickening and continues with sequential changes from the pre-bud to terminal bud stages. After birth, morphogenesis proceeds, and the glands develop into a highly branched epithelial structure that terminates with saliva-producing acinar cells at the adult stage. Acinar cells derived from the epithelium are differentiated into serous, mucous, and seromucous types. During differentiation, cytokeratins, intermediate filaments found in most epithelial cells, play vital roles. Although the localization patterns and developmental roles of cytokeratins in different epithelial organs, including the mammary glands, circumvallate papilla, and sweat glands, have been well studied, their stage-specific localization and morphogenetic roles during salivary gland development have yet to be elucidated. Therefore, the aim of this study was to determine the stage and acinar cell type-specific localization pattern of cytokeratins 4, 5, 7, 8, 13, 14, 18, and 19 in the major salivary glands (submandibular, sublingual, and parotid glands) of the mouse at the E15.5, PN0, PN10, and adult stages. In addition, cell physiology, including cell proliferation, was examined during development via immunostaining for Ki67 to understand the cellular mechanisms that govern acinar cell differentiation during salivary gland morphogenesis. The distinct localization patterns of cytokeratins in conjunction with cell physiology will reveal the roles of epithelial cells in salivary gland formation during the differentiation of serous, mucous or seromucous salivary glands.     

Different localizations of 21 and 27 kDa gap-junction proteins in rat salivary glands

Antibodies against 21 and 27 kDa gap-junction proteins from rat liver were used to examine the identification and localization of gap-junction proteins in rat salivary glands. Acinar cells of the submandibular glands and parotid glands stained well for the 27 kDa gap junction protein and less intensely for the 21 kDa protein. Acinar cells of the sublingual glands were stained heavily for the 27 kDa gap junction protein and stained well for 21 kDa gap junction protein. No 27 kDa protein was observed in the ducts of the salivary glands. The 21 kDa gap-junction protein was distributed in some of the intercalated ducts in the parotid and submandibular glands. Immunoblotting of an extract of parotid glands with antibodies against 21 and 27 kDa gap-junction proteins revealed the presence of 21 and 27 kDa proteins in the parotid glands. It is concluded that the 27 kDa gap-junction protein in tistributed as a major component of the gap junctions in the acinar cells of all the salivary glands; the 21 kDa protein is localized as a minor component in the acinar cells and some portions of the intercalated ducts in the salivary glands. It is possible that these gap-junction proteins might contribute to the regulation of function of the salivary glands.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

Quantitative histochemical investigation of rat salivary gland at different age stages of involution of the endocrine system

A complex quantitative histochemical investigation of the submandibular salivary glands in female albino rats at different age periods (4-5 month, 12 to 14 month- and 20 to 25 month-old) revealed some structural and functional changes during the oestrus cycle. The animals were grouped according to the age changes of the endocrine system. The salivary glands were sensitive to hormonal balance changes at all age periods but their metabolic interrelations varied. The functional changes in the salivary glands of young rats were accompanied by synchronous changes in the indices of energy, synthesis and transport metabolism. The gradual increase of disintegration of the endocrine system resulted in the uncoupling between the indices of the parenchyma and the microcirculation, as well as between nucleo-cytoplasmic relationships and intracellular transport processes in the salivary glands. That was a condition under which the impairment of cellularly excretory processes occurred (secretory stasis). The intercalated ducts and the striated tubules were especially sensitive to hormonal balance fluctuations which is consistent with the hypothesis of the endocrine nature of their function.     

Immunohistochemical and ultrastructural investigation of acinar cells in submandibular and sublingual glands of rats fed a liquid diet

In atrophic parotid glands induced by liquid diet, acinar cell apoptosis is increased while proliferative activity is reduced. This study aimed to clarify how liquid diet affects submandibular and sublingual glands, including acinar cell apoptosis and proliferation. Seven-week-old male Wistar rats were fed either a liquid (experimental group) or pellet diet (control group) from 3 to 21 days, respectively. Submandibular and sublingual glands were weighed and examined histologically, ultrastructurally, and immunohistochemically using antibodies to cleaved caspase-3 (Casp-3) and 5-bromo-2′-deoxyuridine (BrdU). Weights of submandibular and sublingual gland from the experimental group were not significantly different from controls at any time point. Histological and ultrastructural characteristics of experimental acinar cells in both glands were normal. Acinar cells in control and experimental submandibular glands were positively stained with periodic acid Schiff (PAS) and weakly stained by alcian blue (AB). In control and experimental sublingual glands, mucous acinar cells were PAS-positive and strongly AB-positive. Although Casp-3- and BrdU-positive acinar cells were identified in both glands in the experimental group, their labeling indices were not significantly different from controls. In conclusion, liquid diet in rats does not induce atrophic alterations to acinar cells, including apoptosis and proliferative activity in submandibular and sublingual glands.     

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.     

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.     

Immunofluorescence-microscopic demonstration of myosin and actin in salivary glands and exocrine pancreas of the rat

Summary Actin and myosin were localized in various salivary glands (parotid, submandibular, sublingual, lingual and Harderian gland) and the exocrine pancreas of rats by indirect immunofluorescence microscopy using specific rabbit antibodies against chicken gizzard myosin and actin. A bright immunofluorescent staining with both antibodies was observed at three main sites: (1) In myoepithelial cells of all salivary glands, (2) in secretory gland cells underneath the cell membrane bordering the acinar lumen (except Harderian and mucous lingual gland), and (3) in epithelial cells of the various secretory ducts (of all glands) in similar distribution as in acinar cells. The present immunohistochemical findings in acinar cells could lend further support to a concept suggesting that myosin and actin are involved in the process of transport and exocytosis of secretory granules.Supported by grants form Deutsche Forschungsgemeinschaft (Dr. 91/1, Ste. 105/19 and U. 34/4). We thank Mrs. Ursula König, Mrs. Christine Mahlmeister and Miss Renate Steffens for excellent technical assistance.