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.     

Serial cultivation of epithelial cells from human and macaque salivary glands

Summary To study the regulation of human salivary-type gene expression we developed cell culture systems to support the growth and serial cultivation of salivary gland epithelial and fibroblastic cell types. We have established 22 independent salivary gland epithelial cell strains from parotid or submandibular glands of human or macaque origin. Nineteen strains were derived from normal tissues and three from human parotid gland tumors. Both the normal and the tumor-derived salivary gland epithelial cells could be serially cultivated with the aid of a 3T3 fibroblast feeder layer in a mixture of Ham’s F12 and Dulbecco’s modified Eagle’s media supplemented with fetal bovine serum, calcium, cholera toxin, hydrocortisone, insulin, and epidermal growth factor. Salivary gland epithelial cells cultured under these conditions continued to express the genes for at least two acinar-cell-specific markers at early passages. Amylase enzyme activity was detected in conditioned media from cultured rhesus parotid epithelial cells as late as Passage 5. Proline-rich-protein-specific RNAs were detected in primary cultures of both rhesus and human parotid epithelial cells. Neither amylase enzyme activity nor PRP-specific RNAs were detected in fibroblasts isolated from the same tissues. In addition, salivary gland epithelial cells cultured under our conditions retain the capacity to undergo dramatic morphologic changes in response to different substrata. The cultured salivary gland epithelial cells we have established will be important tools for the study of salivary gland differentiation and the tissue-specific regulation of salivary-type gene expression.     

Subcellular localization of epidermal growth factor in human submandibular gland

Epidermal growth factor in human submandibular gland was localized at the subcellular level by means of an immunogold staining method. Labelling was observed in serous acini and ducts. In the acini, gold particles were found within secretory granules, indicating that the growth factor is released into the saliva through granule exocytosis. In the ductal system, the most intense reactivity was revealed in the principal cells of striated ducts. In these cells, an abundant population of small cytoplasmic vesicles was specifically stained. Immunoreactive vesicles were found both apically and basally, suggesting that ductal cells can release their products not only into the saliva but also into the interstitium.     

Exocytosis couples to endocytosis of ferritin in parotid acinar cells from isoprenalin stimulated rats

Distribution of acid phosphatase as a marker enzyme for lysosomes was investigated in the isoprenalin stimulated rat parotid gland. The enzyme was localized in lipofuscin-like bodies as well as in non-discharged granules. The appearance of these bodies was correlated in time to the appearance of smooth vesicles and reduction of the acinar lumen. Ferritin, used as a tracer and introduced into the stimulated gland via cannulated parotid ducts, was found in smooth vesicles, vacuoles and lipofuscin-like bodies throughout the cytoplasm of the acinar cells. Very often ferritin-containing vesicles were found in the vicinity of the Golgi complex. In most cases the vesicles containing ferritin also showed acid phosphatase reaction product. A possible correlation between the lysosomal system and the process of recycling and degradation of membranes in the stimulated gland is discussed.     

Histological and histoquantitative study of the rat parotid gland after Trypanosoma cruzi infection

The present study deals with the morphology of the rat parotid gland and its changes after Trypanosoma cruzi infection. The glands of control and infected animals were analyzed by histologic and histoquantitative methods. After 18 days of infection with T. cruzi, a significant reduction of the density of the volume of the acini and duct system, as well as a significant increase in the amount of connective tissue was noted. In addition, these animals displayed an increase in the number of cells undergoing mitosis. In the 45 day infected rats, there was return to the normal pattern. It is suggested that in the infected animals the decrease in body weight could be responsible for retarded sexual maturity, leading to the lower level of testosterone. It can be assumed that decreased levels of epidermal growth factor (EGF) and neural growth factor (NGF) caused by the lack of testosterone in infected animals also contribute to the atrophy of the parotid gland and to the proliferation of the connective tissue.     

Mitogen-activated protein kinase up-regulation and activation during rat parotid gland atrophy and regeneration: role of epidermal growth factor and beta2-adrenergic receptors

Abstract The rat secretory ductal obstruction model has been widely used to assess salivary gland injury, growth, and differentiation. In this study, a novel ductal obstruction and release procedure was used to explore the signaling pathways leading to salivary gland regeneration. Rats underwent bilateral parotid ductal obstruction in which the duct was occluded against a plastic disk subcutaneously and released by external ligature removal. This ductal obstruction/release procedure was validated to produce glandular atrophy and regeneration with histological analysis and periodic acid-Schiff staining. Immunoblot analysis indicated that during ductal obstruction and the early post-release period (day 7), expression of immunoreactive proliferating cell nuclear antigen and vimentin was increased in the parotid glands compared with sham-operated animals. Immunohistochemical staining and immunoblots revealed up-regulation of the mitogen-activated protein kinases (MAPKs), extracellular signal-regulated receptor kinase (ERK)1/2, and p38 during the atrophic and regeneration phases of ductal obstruction/release. Similarly, increases in activated, i.e., phosphorylated, ERK1/2 (pERK1/2) and p38 (phospho-p38) were demonstrable in both ductal and recovering acinar cells, with pERKs expressed predominantly in the nuclei and phospho-p38 distributed throughout the cells. Furthermore, levels of epidermal growth factor (EGF) receptor and β2-adrenergic receptor (β2-AR) were elevated in the ligated glands and at day 7 post-release; β1-AR levels did not change over the same time period. These results support the view that cell proliferation is involved in duct ligation-induced atrophy of the rat parotid gland and gland recovery upon ligature removal. Up-regulation of ERKs and p38, and the activation of these MAPKs by up-regulated EGF and β2-ARs, may be important signaling components underlying glandular atrophy and subsequent regeneration.     

Epidermal growth factor (EGF) expression in human salivary glands. An immunohistochemical study.

Epidermal growth factor (EGF) is a biologically active peptide involved in differentiation, growth, regeneration and repair of human and animal tissues. Quantitative biochemical studies showed in man the highest concentration of EGF in the parotid gland. The aim of the present study was to define EGF immunolocalization in the individual segments of the human major salivary glands (salivon). The material consisted of sections obtained from the surgically removed salivary glands: parotid, submaxillary and sublingual. Immunohistochemical studies were performed by PAP method using monoclonal antibody against human epidermal growth factor. EGF expression was found almost exclusively in the efferent pathways of the salivary glands, mostly in the intercalated ducts and Pflüger salivary tubules. These segments of the salivon are most developed in the parotid gland in which the staining was stronger than in other salivary glands.     

Mouse submandibular salivary epithelial cell growth and differentiation in long-term culture: Influence of the extracellular matrix

Summary The adult mouse submandibular salivary gland provides a good model system to study gene regulation during normal and abnormal cell behavior because it synthesizes functionally distinct products ranging from growth factors and digestive enzymes to factors of relevance to homeostatic mechanisms. The present study describes the long-term growth and differentiation of submandibular salivary epithelial cells from adult male mice as a function of the culture substratum. Using a two-step partial dissociation procedure, it was possible to enrich for ductal cells of the granular convoluted tubules, the site of epidermal growth factor synthesis. Long-term cell growth over a period of 2 to 3 mo. with at least 3 serial passages was obtained only within three-dimensional collagen gels. Cells grew as ductal-type structures, many of which generated lumens with time in culture. Electron microscopic analysis in reference to the submandibular gland in vivo revealed enrichment for and maintenance of morphologic features of granular convoluted tubule cells. Reactivity with a keratin-specific monoclonal antibody established the epithelial nature of the cells that grew within collagen. Maintenance of cell differentiation, using immunoreactivity for epidermal growth factor as criterion, was determined by both cytochemical and biochemical approaches and was found to be dependent on the collagen matrix and hormones. Greater than 50% of the cells in primary collagen cultures contained epidermal growth factor only in the presence of testosterone and triiodothyronine. In contrast, cells initially seeded on plastic or cycled to plastic from collagen gels were virtually negative for epidermal growth factor. Biochemical analysis confirmed the presence of a protein with an apparent molecular weight of 6000 which comigrated with purified mouse epidermal growth factor. Epidermal growth factor was also present in detectable levels in Passage 1 cells. This culture system should permit assessment of whether modulation of submandibular gland ductal cell growth can be exerted via a mechanism that in itself includes epidermal growth factor and its receptor and signal transduction pathway. This work was supported by Public Health Service grant DE07766 from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Prelysosomal divergence of transferrin and epidermal growth factor during receptor-mediated endocytosis

The routes followed by epidermal growth factor and transferrin during their endocytosis by human epithelial cells were compared in double-label studies by using density gradient centrifugation of cell homogenates and fluorescence microscopy with intact cells. Gradient centrifugation studies of cells incubated with radioactively labeled epidermal growth factor and transferrin indicated that both ligands initially were associated with a class of vesicles having a density of 1.037 g/mL and then were rapidly transferred to a membrane compartment having a slightly higher density (1.039 g/mL). Subsequently, the two ligands diverged. Epidermal growth factor ultimately was transferred to a membranous compartment containing lysosomal enzymes (density (1.08 g/mL) where it was degraded. Transferrin was released intact from the cells; very little was transferred to lysosomes. Using fluorescently labeled ligands, it was observed that after cells were warmed to 37 degrees C for 5 min, transferrin and epidermal growth factor gave coincident, punctate fluorescent patterns, strongly suggesting they were localized within the same endocytic vesicles. Subsequently, the epidermal growth factor signal was observed in lysosomes whereas the transferrin signal became weaker and diffuse and did not coincide with the punctate epidermal growth factor fluorescence. The time course of the divergence of the radioactive and fluorescent ligands coupled with the previous morphologic studies on the pathway of epidermal growth factor internalization [Willingham, M. C., & Pastan, I. (1982) J. Cell Biol. 94, 207-212] suggests that the sorting process is prelysosomal and possibly Golgi associated.     

Purification and tissue distribution of rat epidermal growth factor.

1. Two forms of rat epidermal growth factor, EGF-I and EGF-II, were purified to homogeneity from male rat submandibular glands. 2. The mol. wts of EGF-I and -II were estimated to be 5200 and 5400, respectively, both of them having an apparent biological activity. 3. The antiserum against EGF-II strongly cross-reacted with EGF-I; however, it did so only slightly with mouse or human EGF. 4. EGF was detected by radioimmunoassay in various tissues of male and female rats, and the concentrations of rat EGF in the submandibular gland, parotid gland, sublingual gland, and liver were significantly higher in the male than in the female.     

Possible physiological role of epidermal growth factor in the development of the mouse mammary gland during pregnancy

Epidermal growth factor stimulated cell proliferation in a primary mammary epithelial cell culture derived from mice at different stages of pregnancy. Moreover, the peptide hormone inhibited casein production induced by the synergistic actions of insulin, cortisol and prolactin. The inhibitory effect of epidermal growth factor was influenced by the gestational stages of the mammary gland. These effects of epidermal growth factor were exerted at physiological concentrations. The dual actions of epidermal growth factor on mammary cells implicate its participation in regulation of the growth and differentiation of the mammary gland during pregnancy.     

The relationship between synthesis of secretory products and reducing capacity in pancreas and parotid acinar cells

The secretory products in exocrine pancreas acinar cells in utero were found to reduce osmium tetroxide. This reducing capacity was also exhibited by adult pancreas and parotid glands in different phases of synchronized secretion, and after single or chronic administration of a secretagogue, pilocarpine or isoprenaline. In utero, the reducing capacity appeared in the pancreas concomitantly with the synthesis of secretory products, and was limited to the transitional vesicles on the cis Golgi side. After birth, osmium staining occurred in the cis Golgi vesicles and cisternae of both glands. In the chronically-treated parotid gland, where the occupational programme for secretory proteins had been altered, the reducing capacity was diminished, resembling that in embryonic exocrine pancreas.     

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.     

Effects of selective denervation and nerve growth factor on activity-mediated growth of rat parotid gland.

A dietary change from all liquid to solid food is followed by an average increase of 200% in [3H]thymidine uptake into the parotid gland of rat. However, removal of either the parasympathetic (Px) or the sympathetic (Sx) innervation to the parotid gland prior to the dietary change resulted in a partial inhibition of the increase; values for the parasympathectomized gland were 51% of those of the innervated gland, and values of the sympathectomized parotid gland were 42% of those of the innervated gland. Removal of both autonomic nerves caused a complete inhibition. Initiation of nerve growth factor (NGF) injection (1 microgram/kg body wt, two times daily for the 2 days of chow refeeding) at the time of chow refeeding had no effect on completely or partially denervated glands, and thymidine values for the denervated parotid gland of rats given NGF did not differ statistically from those of rats not given NGF. With parasympathectomy, sympathectomy, and complete denervation, weight of parotid gland was decreased from that of innervated glands, and administration of NGF had no effect on the denervation-induced decreases. The data show that both branches of the innervation to parotid gland must be intact to ensure a maximal increase in thymidine uptake with the dietary change from liquid to solid food. The level of the enzyme, beta 1-4 galactosyltransferase, involved in proliferation, also depended on the presence of intact nerves. Enzyme activity of innervated parotid gland showed an average increase of 200% with chow refeeding of rats previously on liquid diet, but with Px, the average increase was 51% of that of the innervated parotid, and with Sx, it was 41%. NGF administration did not cause any change in levels of this enzyme in any Px or PxSx parotid gland and only a small change in Sx parotid; it did increase levels of this enzyme in parotid of rats without submandibular-sublingual glands.     

Secretion of epidermal growth factor. The role of calcium in stimulcule during secretion.

Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca2+ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of alpha-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth facto; occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.     

Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells

In parotid acinar cells, horseradish peroxidase (HRP) administered via the main excretory duct is endocytosed from the apical cell surface in smooth C- or ring-shaped vesicles (Oliver, C. and A. R. Hand. 1979. J. Cell Biol. 76:207). These vesicles ultimately fuse with lysosomes adjacent to the Golgi apparatus. The present investigation extends these findings and examines the uptake and fate of intravenously injected HRP from the lateral and basal cell surfaces of resting and stimulated parotid and pancreatic acinar cells from rats and mice. Isoproterenol and pilocarpine were used to stimulate the parotid gland and the pancreas, respectively. HRP was internalized in smooth and coated vesicles primarily in areas of membrane infoldings. Both the number of coated vesicles and the amount of tracer internalized increased markedly following secretagogue administration. In both resting and stimulated cells, the HRP was rapidly sequestered in a unique system of basally located lysosomes that possess trimetaphosphatase activity, but not acid phosphatase activity. At 1-3 h after HRP administration, reaction product was also found in multivesicular bodies, vesicles, and lysosomes adjacent to the Golgi apparatus. With time, more HRP was localized in Golgi-associated lysosomes. By 6-7 h, tubules in the apical cytoplasm of stimulated cells contained HRP reaction product. When native ferritin was administered retrogradely and HRP injected intravenously, both tracers could be localized in the same lysosome after 4-5 h, indicating that material taken in from all cell surfaces mixes in Golgi-associated lysosomes. The results of this study suggest that two separate and distinct endocytic pathways exist in exocrine acinar cells: one involves membrane retrieval from the apical cell surface; and the other is a stimulation-dependent process at the lateral and basal cell surfaces.     

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.     

Secretion of epidermal growth factor The role of calcium in stimulus-secretion coupling and structural modification of the growth factor molecule during secretion

Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca²⁺ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of α-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth factor occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecular weight molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.