Why Mouse Airway Submucosal Gland Serous Cells Do Not Secrete Fluid in Response to cAMP Stimulation

Airway submucosal glands are important sites of cystic fibrosis transmembrane conductance regulator (CFTR) chloride (Cl⁻) channel expression and fluid secretion in the airway. Whereas both mouse and human submucosal glands and their serous acinar cells express CFTR, human glands and serous cells secrete much more robustly than mouse cells/glands in response to cAMP-generating agonists such as forskolin and vasoactive intestinal peptide. In this study, we examined mouse and human serous acinar cells to explain this difference and reveal further insights into the mechanisms of serous cell secretion. We found that mouse serous cells possess a robust cAMP-activated CFTR-dependent Cl⁻ permeability, but they lack cAMP-activated calcium (Ca²⁺) signaling observed in human cells. Similar to human cells, basal K⁺ conductance is extremely small in mouse acinar cells. Lack of cAMP-activated Ca²⁺ signaling in mouse cells results in the absence of K⁺ conductances required for secretion. However, cAMP activates CFTR-dependent fluid secretion during low-level cholinergic stimulation that fails to activate secretion on its own. Robust CFTR-dependent fluid secretion was also observed when cAMP stimulation was combined with direct pharmacological activation of epithelial K⁺ channels with 1-ethyl-2-benzimidazolinone (EBIO). Our data suggest that mouse serous cells lack cAMP-mediated Ca²⁺ signaling to activate basolateral membrane K⁺ conductance, resulting in weak cAMP-driven serous cell fluid secretion, providing the likely explanation for reduced cAMP-driven secretion observed in mouse compared with human 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.     

Age-related changes in cellular activity in human submandibular glands as evaluated by argyrophilic nucleolar organizer regions

Objective: To examine the age-related changes in cellular activity of epithelial components of human submandibular glands, evaluated on the basis of argyrophilic nucleolar organizer regions (AgNORs). Design: Epithelial components of human submandibular glands were divided into serous acinar cells, mucous acinar cells, intercalated duct cells, striated duct cells, and interlobular duct cells. The mean AgNOR number of each cell type was compared among six age groups. Setting: The study was conducted at the Department of Oral Pathology, Tohoku University School of Dentistry, Japan. Subjects: Necropsy specimens from 66 males and 57 females 1 to 97 years old. Results: In all cell types except for intercalated duct cells, the mean AgNOR number was lowest in the 0-14 year-old group and highest in the 15-29 year-old group. The value then gradually decreased with advancing age and ultimately reached a similar level to that in the 0-14 year-old group. In intercalated duct cells, the mean AgNOR number did not differ significantly between any age group. There were no significant sex-related differences. Conclusions: The cellular activity of almost all components of human submandibular glands rises in adolescence and young adulthood and then decreases with aging. These results suggest that intercalated duct cells are capable of not only proliferation but also division into other components; these cells may thus compensate for the reduced activity of other components in elderly subjects.     

Binding sites of Ulex europaeus-lectin I in human parotid gland

Summary Twenty non-neoplastic parotid glands (removed during neck dissection for regional tumours) were examined for cellular and subcellular binding sites of Ulex europaeus-lectin I (UEA-I), a lectin reported to be specific for -L-fucose. For light microscopy, an extended peroxidase-antiperoxidase method was applied; for the evaluation of the subcellular localization of bound lectin, three of these glands were examined following immunocryoultramicrotomy and staining by the protein A-gold technique.In addition to the known cytoplasmic affinity of UEA-I for capillary endothelium, acinar cells bound the lectin within the cytoplasmic compartment; the number and distribution of stained acinar cells varied among individuals. Furthermore, cytomembrane-bound labelling that occurred most markedly at the luminar surface was observed in striated-duct epithelium.Using the electron microscope, protein A-gold particles were seen in zymogen granules and in Golgi cisternae of serous acinar cells; primary saliva secreted in the lumina exhibited strong labelling; serous acinar cells had binding sites on their cell membranes, striated-duct epithelium had binding sites on its surface membrane and in the vicinity of apical vesicles. Our results show that UEA-I is a useful tool for the study of the structure and functional states of the parotid gland epithelium and its associated pathological alterations.Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 65th birthday     

Antigenic profile of human bronchial gland

Bronchial glands have been regarded as modified salivary glands. It is well known that there no previous reviews concerning the antigenic profile of the bronchial wall. The aim of this study is a systematic survey of the antigenic profile and to describe the histology of normal human bronchial glands. Six formalin-fixed, paraffin-embedded surgical specimens were studied using a panel of 22 polyclonal and monoclonal antibodies by the avidin-biotin-peroxidase method. Bronchial glands disclosed a tubuloacinar structure. The smallest ducts intercalated originated from a cluster of secretory acini and converge to form an excretory duct. No striated duct was observed. Acinar united is composed by mucous, serous and mixed units. Myoepithelial cells are found in relation to the intercalated ducts and secretory acinis. Secretory cells of bronchial glands reacted strongly with cytokeratin AE1 and moderately for CK7, CK18. Additionally, serous acinar cells reacted with AE3, CK19, CK5/6/8/18, CK8/18/19, and Leu7. Myoepithelial cells reacted strongly with a-smooth muscle actin, CD10 and CK34betaE12. Ductal system cells differed from acinar secretory cells in expressing CK34betaE12 and HSP27. In conclusion, the detailed knowledge of the immunohistochemical reactivities of normal cell types of normal human bronchial glands will prove useful in studies of bronchial pathology, especially of neoplastic processes.     

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.     

Postnatal growth of the rat palatine gland

To elucidate how the palatine glands grow postnatally, the palatine glands of rats from 0 to 8 weeks of age were investigated histologically and immunohistochemically. Under light microscope, three dimensions of the right part of the palatine glands were measured and the total number of excretory ducts of the glands was counted from the parasagittal serial sections. Immunohistochemistry with anti-5-bromo-2'-deoxyuridine (BrdU) monoclonal antibody was also employed to detect the cellular proliferative activity. At birth (0 weeks), the palatine glands consisted of ducts and immature acini. The ducts in the glands were connected with excretory ducts. After 2 weeks, there was no duct in the glands. Most acinar cells became mature as mucous cells and took the form of tubulo-acini connected directly with excretory ducts. In the posterior region of the glands, serous acinar cells forming demilunes were occasionally seen. All three dimensions of the palatine glands became longer, and the number of excretory ducts tended to increase. Immunohistochemistry showed acinar and duct cells were highly proliferative in early stage of postnatal life and their proliferative activity decreased thereafter. This study demonstrated that immature rat palatine glands of newborn rats grow three-dimensionally during maturation, and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from the excretory ducts formed postnatally.     

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.     

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.     

Distribution of lactoferrin in human salivary glands

Summary An immunoperoxidase technique was used to study the distribution of lactoferrin (LF) in human salivary glands from autopsy tissues fixed in Carnoy's fluid for the optimal preservation of LF antigenicity. Specific LF staining was seen in the intralobular ducts of all salivary glands but never in the interlobular ducts; acinar LF was detected in most serous demilunes of the mixed glands and in some but not all acinar cells of the pure serous glands. No LF was detected in the acinar cells of the pure mucous glands. In the oral tissues studied the only additional cells containing LF were polymorphonuclear leucocytes. Whether LF in the salivary glands is synthesized locally and/or ultrafiltrated from the blood is at present unclear. Present evidence indicates that the biological role of salivary LF is antibacterial.Studies supported by grants from the Sigrid Jusélius Foundation and Finska Läkaresällskapet     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

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.     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.     

Secretion of carbonic anhydrase isoenzyme VI (CA VI) from human and rat lingual serous von Ebner's glands.

Salivary carbonic anhydrase VI (CA VI) appears to contribute to taste function by protecting taste receptor cells (TRCs) from apoptosis. The serous von Ebner's glands locating in the posterior tongue deliver their saliva into the bottom of the trenches surrounding the TRC-rich circumvallate and foliate papillae. Because these glands deliver their saliva directly into the immediate vicinity of TRCs, we investigated whether CA VI is secreted by the von Ebner's glands, using immunochemical techniques. The immunohistochemical results showed that CA VI is present in the serous acinar cells, ductal cells, and ductal content of von Ebner's glands and in the demilune and ductal cells plus ductal content of rat lingual mucous glands. More importantly, CA VI was also detected in taste buds and in the taste pores. Western blotting of saliva collected from the orifices of human von Ebner's glands and CAs purified from rat von Ebner's glands confirmed that CA VI is expressed in these glands and secreted to the bottom of the trenches surrounding the circumvallate and foliate papillae. These findings are consistent with the hypothesis that locally secreted CA VI is implicated in the paracrine modulation of taste function and TRC apoptosis. (J Histochem Cytochem 49:657-662, 2001)     

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.     

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.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.