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.     

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.     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Expression of CD44 isoforms in normal salivary gland tissue: an immunohistochemical and ultrastructural study

We studied the expression of CD44 isoforms immunoreactivity in normal human salivary gland tissue, aiming at its full characterisation in normal epithelial and myoepithelial cell types. Optical immunohistochemistry techniques using monoclonal antibodies anti-CD44v3, CD44v4/5 and, for CD44v6, together with immunoelectron microscopy, were performed in serous, seromucinous and mucinous glands. Normal human breast and a case of lactating breast adenoma were used for comparative purposes and as controls. CD44v3 was positive in acinar and myoepithelial cells and was absent in mucin-producing cells from the different gland types. CD44v4/5 was consistently negative in all types of salivary tissue. CD44v6 was constantly positive in serous acinar cells, focally positive in basal cells of ducts, and myoepithelial cells consistently expressed it. At the ultrastructural level, CD44v6 was localised to the interdigitating processes of acinar cells, whenever they were not covered by basal lamina and to the cell membrane facing myoepithelial cells. In myoepithelial cells, immunolabelling was found at the membranes facing the acinar cells and in caveolae present at this interface. No labelling was found at cell membranes of both acinar and myoepithelial cells in contact with basal lamina or at the luminal aspect of the former. The finding of CD44v3 and v6 in myoepithelium of normal salivary glands may argue in favour of the role of these molecules in the regulation of growth and renewal of normal tissues and, potentially, on the morphogenesis of salivary gland neoplasms.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Morphology and glycoconjugate histochemistry of the palpebral glands of the adult newt, Notophthalmus viridescens.

The eyelids of the newt were studied in 10 microns serial paraffin and 1-2 microns plastic sections using standard histological stains and special stains for glycoconjugates. The eyelids contain four different glands. Simple acinar serous and simple acinar mucous glands occur in the skin; unicellular mucous glands occur in the conjunctiva; and convoluted tubular seromucous glands are present in connective tissue beneath the conjunctiva. The first two are identical to cutaneous glands found elsewhere on the head and body. The simple acinar serous glands are surrounded by myoepithelial cells and release their secretion, which is composed largely of proteins with minimal glycoconjugate content, by a holocrine mechanism. The secretory product of the simple acinar mucous glands is composed of neutral glycoconjugates with a minor content of acidic glycoconjugates; the mucin exhibits strong PAS and PAPD staining and weak staining by AB and PAPS methods. The unicellular conjunctival mucous glands secrete both neutral and acidic glycoconjugates as shown by positive reactions with PAS, PAPD, PAPS, and AB methods. Convoluted tubular seromucous glands in the ventral eyelid synthesize both proteins and neutral glycoconjugates. The mucous secretions of the conjunctival glands probably provide lubrication and protection for the cornea.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.     

Morphology of the exocrine glands of the frog skin

Frog skin contains three distinct types of exocrine glands: granular (poison), mucous, and seromucous. The granular gland forms a syncytial secretory compartment within the acinus, which is surrounded by smooth muscle cells. The mucous and seromucous glands are easily identifiable as distinct glands. The serous and mucous secretory cells are arranged in a semilunar configuration opposite the ductal end and are filled with granules. Within the acinus, located at the ductal pole of the gland, are distinct groups of cells with few or no granules in the cytoplasm. In both the mucous and seromucous gland there is a cell type with abundant mitochondria; the one in the mucous gland is located in the region adjacent to the secretory cells. The duct of these glands is two-layered, with the individual cells appearing morphologically similar to the layers of the skin epithelium as the duct traverses the skin. The duct appears to be patent throughout its length. The morphological heterogeneity and distinct distribution of the cell types within the gland acinus may be indicative of a functional heterogeneity that allows the production of distinctly different types of secretion from the same gland type, depending on the type of stimulus.     

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.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

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 cephalic glands of Brazilian reptilia and amphibia. IV. Histology of labial, premaxillary, and Duvernoy's glands in the colubrid snake Oxyrhopus trigeminus

A study of the morphology of the salivary glands of the colubrid snake Oxyrhopus trigeminus showed the following: The acini of supralabial, infralabial, and premaxillary glands are formed by mucous and mucoserous cells; the tubules of Duvernoy's gland are formed by seromucous cells; and mucous cells produce neutral and acid mucosubstances, mucoserous cells secrete neutral and acid mucosubstances and protein, and seromucous cells have neutral mucosubstance and protein secretions.     

Cloning and characterization of a novel animal lectin expressed in the rat sublingual gland.

We cloned a rat gene that is expressed primarily in the sublingual gland and named the predicted 503 amino-acid protein SLAMP (sublingual acinar membrane protein). SLAMP has 63% homology with human ERGIC-53-like protein, a member of the family of animal L-type lectins. Using a cDNA probe for SLAMP mRNA and rabbit antisera against SLAMP, we examined the expression and localization of SLAMP in major rat organs and tissues. With both Northern and Western blot analyses, abundant expression of SLAMP was demonstrated predominantly in the sublingual gland, with single sizes of the mRNA and protein 1.8 kb and 50 kDa, respectively, but not in other organs or tissues, including the parotid and submandibular glands. With immunohistochemistry, SLAMP was localized to the mucous acinar cells, but not to the serous demilunes or the duct system. With immunoelectron microscopy, SLAMP was localized predominantly to regions corresponding to the ER-Golgi intermediate compartment. Besides the sublingual gland, SLAMP immunoreactivity was also demonstrated in mucous cells of the minor salivary glands in oral cavity and of Brunner's glands in the duodenum. These results suggested that rat SLAMP plays a specific role in the early secretory pathway of glycoproteins in specific types of mucous cells.     

Par-1b is required for morphogenesis and differentiation of myoepithelial cells during salivary gland development

The salivary epithelium initiates as a solid mass of epithelial cells that are organized into a primary bud that undergoes morphogenesis and differentiation to yield bilayered acini consisting of interior secretory acinar cells that are surrounded by contractile myoepithelial cells in mature salivary glands. How the primary bud transitions into acini has not been previously documented. We document here that the outer epithelial cells subsequently undergo a vertical compression as they express smooth muscle α-actin and differentiate into myoepithelial cells. The outermost layer of polarized epithelial cells assemble and organize the basal deposition of basement membrane, which requires basal positioning of the polarity protein, Par-1b. Whether Par-1b is required for the vertical compression and differentiation of the myoepithelial cells is unknown. Following manipulation of Par-1b in salivary gland organ explants, Par-1b-inhibited explants showed both a reduced vertical compression of differentiating myoepithelial cells and reduced levels of smooth muscle α-actin. Rac1 knockdown and inhibition of Rac GTPase function also inhibited branching morphogenesis. Since Rac regulates cellular morphology, we investigated a contribution for Rac in myoepithelial cell differentiation. Inhibition of Rac GTPase activity showed a similar reduction in vertical compression and smooth muscle α-actin levels while decreasing the levels of Par-1b protein and altering its basal localization in the outer cells. Inhibition of ROCK, which is required for basal positioning of Par-1b, resulted in mislocalization of Par-1b and loss of vertical cellular compression, but did not significantly alter levels of smooth muscle α-actin in these cells. Overexpression of Par-1b in the presence of Rac inhibition restored basement membrane protein levels and localization. Our results indicate that the basal localization of Par-1b in the outer epithelial cells is required for myoepithelial cell compression, and Par-1b is required for myoepithelial differentiation, regardless of its localization.     

The role of capsulin in the morphogenesis and differentiation of fetal rat gastric mucosa.

The signals that guide the morphogenesis and differentiation of rat fetal gastric mucosa remain largely unknown. We have investigated the role of capsulin in pit/gland formation and epithelial cell differentiation in cultured stomach tissue. Embryonic day 16.5 (E 16.5) stomach tissue cultured for three days in the presence of 1 microM hydrocortisone underwent dramatic transformation, from undifferentiated, stratified cells to differentiated epithelia composed of polarised columnar cells with mucous cells and pit/glands. In the presence of capsulin antisense oligonucleotides directed against capsulin mRNA, tissues do not undergo further development. Significantly, both mucous granules and pit/gland formation were inhibited compared to capsulin sense/scrambled oligonucleotide treated controls. However, in tissues treated with specific anti-rat HGF-antiserum to neutralise secreted HGF, pit/gland formation was inhibited, but the number of mucous granules remained unchanged compared to controls treated with non-specific antiserum (mouse monoclonal cytokeratin 8 antiserum). This data suggests that capsulin may have a role in the morphogenesis of pit/glands and mucin granule formation in the developing rat gastric mucosa. We discuss the possibility that this role of capsulin may be partly mediated through the actions of HGF.     

A high resolution sem study of human minor salivary glands

By SEM we have investigated the human minor salivary glands using the NaOH method for the visualization of endpieces and myoepithelial cells, and the osmium maceration technique that reveals membranous intracellular structures. With the former method all minor glands, including the posterior deep (Ebner's) lingual glands, consist of tubules sometimes dilated into alveoli, while true acini of the kind observed in human major salivary glands, are absent. Tubules of the posterior deep lingual gland exhibit stellate myoepitelial cells that leave a substantial part of the secretory cells uncovered. The latter cells, at variance with serous cells of major glands, do not show basal folds. In contrast, tubules of the other minor glands, like the mucous ones of major glands, are covered almost completely by band-like myoepithelial cells. The osmium maceration method clearly demonstrates that posterior deep lingual glands are serous in character and that all the other minor glands, together with the predominant mucous cells, possess a variable number of seromucous cells that, despite variations among individuals, increase in order from palatine and posterior superficial lingual (Weber's), to minor sublingual, labial, anterior lingual (Blandin and Nuhn's), and buccal glands.     

A neonatal secretory protein associated with secretion granule membranes in developing rat salivary glands

In the perinatal submandibular gland, the secretion granules of Type I cells contain protein C (89 KD) and those of Type III cells have Bl-immunoreactive proteins (Bl-IP, 23.5-27.5 KD). In this report we used immunocytochemistry at the light and electron microscopic levels to describe the developmental distribution and localization of protein D (175 KD), which is secreted by both Type I and Type III cells. At its first appearance in Type I cells at 18 days and in Type III cells at 19 days post conception, protein D immunoreactivity (D-IR) is associated with secretion granule membranes; this is more pronounced in Type I than in Type III cells. In early postnatal life the label remains membrane associated, but as Type III cells differentiate into seromucous acinar cells, the lower level of label present in these cells is found in the granule content. Label is found associated with the membrane in secretion granules of Type I cells as long as these cells are identifiable in acini, and subsequent to this similarly labeled cells are seen in intercalated ducts. In the sublingual gland (SLG), D-IR is membrane associated in secretion granules of serous demilune cells, and is present in the secretion granule content in mucous acinar cells. D-IR is also found in the lingual serous (von Ebner's) glands, lacrimal gland, and tracheal glands, primarily in the ducts, where it is localized in the content of secretion granules.