Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained. These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated and treated acinar cells.     

Reduction capacity of untreated and of repeatedly isoproterenol treated parotid gland of the mouse

Summary Osmium impregnation was used to show possible differences of reduction capacity of perinuclear space, rough endoplasmic reticulum and the Golgi apparatus of unstimulated mouse parotid gland and in the gland after repeated pharmacological doses of isoproterenol. There were some significant differences between the staining of acinar and duct cells. In all intercalated and striated duct cells the staining is dense in the perinuclear space and in the rough endoplasmic reticulum. Osmiophility was not detected in the Golgi complex of intercalated duct cells. The staining was also lacking in the perinuclear space and endoplasmic reticulum of the acinar cells. The cis face of the Golgi complex and numerous transitional vesicles in the acinar cells showed variability of the reduction capacity of their membrane segments. In chronically treated acinar cells Os black was lacking in the Golgi cisternae, except that the numerous transitional vesicles were heavily stained.These results reveal characteristic differences of reduction capacity of endomembrane compartments in different parotid glandular cells, as well as between untreated und treated acinar cells.     

Localization of cellular regulatory proteins using postembedding immunogold labeling

Cyclic AMP-dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP-binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RII) of differing molecular weight, primary structure, and cyclic AMP-binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK subunits in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP-binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK subunits in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory cell function.     

Ultrastructural localization of epidermal growth factor receptor in human parotid gland

The epidermal growth factor receptor (EGFR) is widely distributed in several organs in which, following interaction with its ligand, it can affect development and differentiation. The aim of this study was to define the distribution of EGFR in human parotid gland by means of a post-embedding immunogold staining method. Normal human parotid glands obtained at surgery were routinely prepared for electron microscopy. Semithin and ultrathin sections were treated for immunocytochemistry using a mouse monoclonal antibody specific for EGFR and a goat anti-mouse gold conjugated secondary antiserum. At the light microscope level, EGFR reactivity was revealed by a specific dark staining in both acinar and ductal cells. At the electron microscope level, EGFR was strongly stained in the cytoplasmic compartments and occasionally labeled on cell surfaces. In acinar cells, it appeared to be associated with small vesicles of uncertain nature that were scattered among the secretory granules. EGFR-positive vesicles were also observed in the ductal cells, with the most intense labeling being localized in striated ducts. Since cytoplasmic vesicles were previously found to be EGF-positive, these results may be due to the presence of the EGF-EGFR complex that is internalized after binding of EGF to the surface EGFR.     

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.     

Subcellular localization of epidermal growth factor in human parotid gland

The intracellular distribution of epidermal growth factor was investigated in human parotid gland by immunogold cytochemistry at the electron-microscopy level. Epidermal growth factor immunoreactivity was demonstrated in both acini and ducts. In acinar cells, secretory granules appeared moderately stained, clearly indicating that parotid gland contributes to salivary epidermal growth factor through granule exocytosis. In ductal cells, gold particles were found to decorate numerous cytoplasmic vesicles, particularly abundant in striated duct cells. Since epidermal growth factor reactive vesicles were seen not only at the cellular apex, but nearby lateral plasma membranes as well, it leads to the hypothesis that epidermal growth factor may be discharged both apically into the saliva, and basally into the interstitium.     

Fine structure of the parotid gland in the pika and the volcano rabbit

The parotid glands of the pika and the volcano rabbit were examined by light and transmission electron microscopy. The acinar cells of the pika consisted of light cells containing basophilic granules of low density, while in the volcano rabbit the acinar cells consisted of light and dark cells containing acidophilic granules of moderate density. Intercalated duct cells were composed of light cells containing a few granules of moderate density. These segments of the two animals were similar in morphology. The striated duct cells in both species were composed of light and dark cells. Most of those in the pika contained a few moderately dense granules. In both animals, no myoepithelial cells were detected around the acini, intercalated ducts or striated ducts, while nerve terminals were observed among the adjacent acinar cells.     

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.     

Cytochemical localization of carbohydrates in intercalated duct and acinar cells of mouse parotid gland

Summary The glycoconjugate composition of mouse intercalated duct and acinar cells of parotid gland has been compared. Mucins containing 1,2-glycols were demonstrated by the tannic acid-uranyl acetate technique. Hexose residues of glycoconjugates were identified using ferritin conjugated withCanavalia ensiformis agglutinin (Con A),Triticum vulgare or wheat germ agglutinin (WGA),Ricinus communis I agglutinin (RCA-I),Phaseolus vulgaris agglutinin (PHA-E) andArachis hypogaea agglutinin (PNA). Whereas qualitative and quantitative differences were observed in sugar residues of secretory granules in intercalated duct and acinar cells, apical plasmalemmae were labelled sparsely and similarly. This indicates that the glycocalyx composition of apical plasma minae in the parotid acinar and intercalated duct cells is little influenced by secretory granule composition.     

Detection of Cell Surface Antigens in Tissue Sections by Means of Pre-Embedding Immunogold Staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualise cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that it is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Detection of cell surface antigens in tissue sections by means of pre-embedding immunogold staining

The immunogold technique has been used in electron microscopy to detect cytoplasmic and extracellular antigens by postembedding techniques. It has also been used to detect plasma-membrane-associated molecules on suspended cells and, recently, to visualize cell surface antigens in ultrathin sections of Lowicryl embedded specimens. In the present study, cell surface antigens of rat kidney and human skin were identified in tissue sections by using pre-embedding immunogold labeling. Brush border microvillar antigens and dermal lymphocyte antigens both bound numerous gold particles. The immunogold staining described here has the advantage over immunoperoxidase procedures that is not subject to diffusion or reabsorption artifacts, and allows estimation of the antigen density on labeled cells. Furthermore, this pre-embedding immunogold technique is ideally suited to detecting cell surface-associated antigens since it preserves antigenicity, allows gold particle penetration and enhances cell membrane profiles.     

Endocytosis of native and glycosylated bovine serum albumin by duct cells of the rat parotid gland

Summary The ability of duct cells of the rat parotid gland to internalize bovine serum albumin (BSA) and several glycosylated albumins (glucosamide, galactosamide, fucosamide, lactosyl, p-aminophenyl-N-acetyl-D-glucosamide, p-aminophenyl-N-acetyl-D-mannopyranoside, p-aminophenyl-N-acetyl-D-galactosamide) was investigated. The various BSA preparations were infused into the gland via the main excretory duct, after which the tissues were fixed and prepared for light and electron microscopy. Immunolocalization of native BSA, as well as the glycosylated BSAs, was performed on thin sections, using an unlabeled antibody to BSA followed by protein A-colloidal gold. Gold particles were present over the lumina of both intercalated ducts and striated ducts, and over small endocytic structures and large vacuoles in the apical cytoplasm of both duct cell types. Endocytosis of the glycosylated BSAs by duct cells was greater than native BSA. Fucosylamide-BSA and mannopyranoside-BSA were taken up to a greater extent than the other glycosylated BSAs. Uptake by intercalated duct cells was greater than by striated duct cells, was independent of the concentration of the glycosylated BSA, and was reduced by an excess of the corresponding sugar. Striated duct cells showed some damage by the glycosylated BSAs that was concentration-dependent, and which was reduced in the presence of an excess of the corresponding sugar. These results suggest that endocytosis by salivary gland duct cells may involve specific recognition of carbohydrate residues and that the endocytosis of acinar secretory proteins observed in certain conditions may be due to increased and/or altered protein glycosylation.     

Plasma membrane of the rat parotid gland: preparation and partial characterization of a fraction containing the secretory surface

A plasma membrane fraction from the rat parotid gland has been prepared by a procedure which selectively enriches for large membrane sheets. This fraction appears to have preserved several ultrastructural features of the acinar cell surface observed in situ. Regions of membrane resembling the acinar luminal border appear as compartments containing microvillar invaginations, bounded by elements of the junctional complex, and from which basolateral membranes extend beyond the junctional complex either to contact other apical compartments or to terminate as free ends. Several additional morphological features of the apical compartments suggest that they are primarily derived from the surface of acinar cells, rather than from the minority of other salivary gland cell types. Enzymatic activities characteristically associated with other cellular organelles are found at only low levels in the plasma membrane fraction. The fraction is highly enriched in two enzyme activities--K+ -dependent p-nitrophenyl phosphatase (K+ -NPPase, shown to be Na+/K+ adenosine triphosphatase; 20-fold) and gamma-glutamyl transpeptidase (GGTPase; 26-fold)--both known to mark plasma membranes in other tissues. These activities exhibit different patterns of recovery during fractionation, suggesting their distinct distributions among parotid cellular membranes. Secretion granule membranes also exhibit GGTPase, but no detectable K+ -NPPase. Since Na+/K+ adenosine triphosphatase and GGTPase, respectively, mark the basolateral and apical cellular surfaces in other epithelia, we hypothesize that these two enzymes mark distinct domains in the parotid plasmalemma, and that GGTPase, as the putative apical marker, may signify a compositional overlap between the two types of membranes which fuse during exocytosis.     

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.     

Immunohistochemical demonstration of lysozyme and lactoferrin in salivary pleomorphic adenomas

Immunohistochemical identification of lysozyme and lactoferrin was made in salivary pleomorphic adenomas (147 cases) and the staining patterns were evaluated with respect to the histological features and histogenesis. In normal salivary glands, the intercalated duct cells gave positive staining for lysozyme in major glands, and serous acinar cells, demilune cells, and interlobular duct cells were positive in minor glands. Lactoferrin staining was irregularly positive in serous cells and ductal epithelium. In pleomorphic adenomas, the reaction for lysozyme was positive in 14% (21/147) of the cases, and was confined to luminal cells of tubulo-ductal structures. Lactoferrin in pleomorphic adenomas was distributed in luminal tumor cells (51%; 75/147), in outer tumor cells (3%; 4/147), and in both luminal and outer tumor cells (5%; 7/147) in tubulo-ductal structures; it was also detected in plasmacytoid myoepithelial cells (5%, 8/147). However, modified myoepithelial cells and other types of neoplastic myoepithelial participants were negative for lactoferrin staining. The occurrence of both lysozyme and lactoferrin in salivary pleomorphic adenomas suggests their participation in the local defense mechanism in the tumor.     

Parotid salivary gland ultrastructure in an omnivorous neotropical bat: evolutionary diversity at the cellular level

The parotid salivary gland in an omnivorous neotropical bat, Carollia perspicillata (L.), was studied by transmission electron microscopy and compared to the parotid gland in other species of bats and in mammals in general. The parotid acinar secretory granules were found to be unique among mammals: they consist of a finely punctate matrix containing a variety of electron dense inclusions ranging from punctate densities to complex cage-like geodesic structures. The parotid acinar product in Carollia perspicillata is intermediate in morphology between that formed by homologous secretory cells in insectivorous/carnivorous bats and that in frugivorous bats. Both the intercalated and striated ducts probably release additional secretory products into the saliva.     

Ultrastructure and complex carbohydrate ultracytochemistry of the cat parotid gland

The structure and glycoconjugate content of the cat parotid gland were analyzed at electron microscopic level by applying morphological techniques and three ultrastructural histochemical methods - HID-TCH-SP, LID-TCH-SP and PA-TCH-SP. This gland appeared as a typical salivary gland composed of acinar secretory cells, intercalated ducts, striated ducts and excretory ducts. The most common configuration of secretory granules consisted of a dense core surrounded by a variable electron-lucent halo. All ductal segments were characterized by the presence of different cell populations and small apical granules greatly different from those localized in the acinar cells. By using HID-TCH-SP we were able to demonstrate that in a few acinar cells there are sulphated sites, whereas PA-TCH-SP staining revealed the presence of vic-glycol radicals in all acinar cells preferentially located on the halo of secretory granules.     

Electron microscopic immunocytochemical localization of proline-rich proteins in normal mouse parotid salivary glands

Summary Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with -amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.     

Localization of nuclear subunits of cyclic AMP-dependent protein kinase by the immunocolloidal gold method

An immunocolloidal gold electron microscopy method is described allowing the ultrastructural localization and quantitation of the regulatory subunits RI and RII and the catalytic subunit C of cAMP-dependent protein kinase. Using a postembedding indirect immunogold labeling procedure that employs specific antisera, the catalytic and regulatory subunits were localized in electron-dense regions of the nucleus and in cytoplasmic areas with a minimum of nonspecific staining. Antigenic domains were localized in regions of the heterochromatin, nucleolus, interchromatin granules, and in the endoplasmic reticulum of different cell types, such as rat hepatocytes, ovarian granulosa cells, and spermatogonia, as well as cultured H4IIE hepatoma cells. Morphometric quantitation of the relative staining density of nuclear antigens indicated a marked modulation of the number of subunits per unit area under various physiologic conditions. For instance, following partial hepatectomy in rats, the staining density of the nuclear RI and C subunits was markedly increased 16 h after surgery. Glucagon treatment of rats increased the staining density of only the nuclear catalytic subunit. Dibutyryl cAMP treatment of H4IIE hepatoma cells led to a marked increase in the nuclear staining density of all three subunits of cAMP-dependent protein kinase. These studies demonstrate that specific antisera against cAMP-dependent protein kinase subunits may be used in combination with immunogold electron microscopy to identify the ultrastructural location of the subunits and to provide a semi-quantitative estimate of their relative cellular density.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.