Distinct immunohistochemical expression of osteopontin in the adult rat major salivary glands

Osteopontin is a multifunctional protein secreted by epithelial cells of various tissues. Its expression in the adult rat major salivary glands has not yet been studied. We examined osteopontin expression by immunohistochemistry using a well characterized monoclonal antibody. Submandibular glands of young adult male rats (70–100 days old) showed specific expression in secretion granules of granular duct cells but also in cells of the striated ducts and excretory duct. In the major sublingual as well as the parotid gland expression was found solely in the duct system. In addition, a few interstitial-like cells exhibiting very strong immunostaining for osteopontin could be found in either organ. Expression could neither be seen in acinar cells nor in cells of the intercalated ducts. Moreover, in submandibular glands of more aged rats (6- to 7-month old) which show well developed granular convoluted tubules, there was almost exclusive expression of osteopontin in granular duct cells as well as in some interstitial-like cells, but barely in the striated/excretory duct system. Western blot analysis of the submandibular gland showed a specific band migrating at approximately 74 kDa, detectable at both age stages. Osteopontin secreted fom granular duct cells may influence the compostion of the saliva, e.g. thereby modulating pathways affecting sialolithiasis. Its expression in striated duct cells may also hint to roles such as cell–cell attachment or cell differentiation. The cell-specific expression detected in the rat major salivary glands differs in part from that reported in mice, human and monkey.Nicholas Obermüller and Nikolaus Gassler contributed equally to this work.     

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.     

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.     

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.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

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.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

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.     

Expression of the Na+-HCO3- cotransporter and its role in pHi regulation in guinea pig salivary glands

Patterns of salivary HCO(3)(-) secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO(3)(-) transport and the underlying mechanism of intracellular pH (pH(i)) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na(+)-HCO(3)(-) cotransporter (NBC) and its role in pH(i) regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO(3)(-) transport in human salivary glands. RT-PCR, immunohistochemistry, and pH(i) measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na(+)/H(+) exchanger (NHE) played a putative role in pH(i) regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pH(i) regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO(3)(-) and is involved in pH(i) regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pH(i) regulation. We conclude that NBC1 is functionally expressed and plays a role in pH(i) regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.     

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.     

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     

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.     

Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.     

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.     

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.