An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.     

Histochemical studies on the rat submaxillary gland during post-natal development

Summary A post-natal development of the albino rat submaxillary gland was studied morphologically and histochemically. Throughout the developmental stages, conspicuous variations were observed morphologically; growth of acini, glandular tubules formation and advance of striated duct.For enzymatic histochemical observations, the localization and activity of hydrolytic and oxidative enzymes were apparently similar to those of the matured gland from about 5 weeks after birth in the intralobular striated ductal components. Granular tubules were clearly demonstrated from 7 weeks after birth showing almost the same activity as the adult gland.     

Enzyme-and immuno-histochemical localization of kallikrein

Summary Localization of kallikrein in the human parotid gland was investigated simultaneously by two markers: kallikrein-like (enzyme) activity and kallikrein antigenicity. Kallikrein-like activity was histochemically demonstrated by using a synthetic substrate, pro-phe-arg-naphthylester. Kallikrein antigenicity was demonstrated by an unlabelled antibody peroxidase-antiperoxidase method, where monospecific antiserum against highly purified urinary kallikrein was used as the primary antiserum. The results showed that kallikrein-like activity and kallikrein antigenicity were identical in their locations in the ductal cells, being localized in the luminal part of the striated ducts and to a lesser degree in the excretory ducts. This indicates the presence of active kallikrein in these regions. No enzyme activity nor antigenicity was observed either in acini or in intercalated ducts. Moreover, the peroxidase-antiperoxidase method reveated kallikrein antigenicity for the first time extracellularly in the basement-membrane region of acini and of ducts as well as in the interstitium surrounding ducts and major vessels.     

Enzyme- and immuno-histochemical localization of kallikrein. I. The human parotid gland

Localization of kallikrein in the human parotid gland was investigated simultaneously by two markers: kallikrein-like (enzyme) activity and kallikrein antigenicity. Kallikrein-like activity was histochemically demonstrated by using a synthetic substrate, pro-phe-arg- naphthylester . Kallikrein antigenicity was demonstrated by an unlabelled antibody peroxidase-antiperoxidase method, where monospecific antiserum against highly purified urinary kallikrein was used as the primary antiserum. The results showed that kallikrein-like activity and kallikrein antigenicity were identical in their locations in the ductal cells, being localized in the luminal part of the striated ducts and to a lesser degree in the excretory ducts. This indicates the presence of active kallikrein in these regions. No enzyme activity nor antigenicity was observed either in acini or in intercalated ducts. Moreover, the peroxidase-antiperoxidase method revealed kallikrein antigenicity for the first time extracellularly in the basement-membrane region of acini and of ducts as well as in the interstitium surrounding ducts and major vessels.     

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.     

Expression of transforming growth factor beta 1 in the submandibular gland of the rat

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.     

Morphological and histochemical studies on the parotid gland of Praomys (Mastomys) natalensis

The parotid gland of Praomys (Mastomys) natalensis follows the same anatomical pattern of Rodentia since no peculiar morphological and histochemical features were found in the present study that differentiate it from homologous glands in other rodents. It consists of serous secreting components and intralobular ducts that demonstrate no sexual dimorphism. Histochemical studies reveal that the glandular cells of the Praomys' parotid gland contain no acid mucosubstance, but that material of a neutral glycoprotein nature can be found in the secreting cells of acini, epithelium of the ducts, and the ductal lumina.     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Histology and histochemistry of the parotid and the principal and accessory submandibular glands of the little brown bat

The parotid and the principal and accessory submandibular glands of the little brown bat. Myotis lucifugus (Vespertilionidae), were examined using light microscopy and staining methods for mucosubstances. The parotid gland is a compound tubuloacinar seromucous gland. Parotid gland secretory cells contain both neutral and nonsulfated acidic mucosubstances. The principal and accessory submandibular glands are compound tubuloacinar mucus-secreting glands. They contain somewhat atypical mucus-secreting demilunar cells that often appear to be interspersed between mucous tubule cells. The mucous tubule cells in both the principal and accessory submandibular glands contain sulfonmucins. Demilunar cells of the principal submandibular gland contain moderate amounts of nonsulfated acidic mucosubstances, but the corresponding cells of the accessory submandibular gland contain considerable neutral mucosubstance with very little acid mucosubstance. Intercalated ducts composed of cuboidal or low columnar epithelial cells are present in all three glands. Striated ducts in all glands are composed of columnar cells whose apices bulge into the ductal lumina. Excretory ducts are composed of simple columnar epithelium, with occasional basal cells that suggest a possible pseudostratified nature. The cells of the excretory ducts also have bulging apices. All duct types contain apical cytoplasmic secretory material that is a periodic acid-Schiff positive, neutral mucosubstance. Ductal apical secretory material is more evident in intercalated and striated ducts than in excretory ducts.     

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     

Microscopic and ultrastructural study of the lymphatic system in the human parotid gland

The localization and fine structure of the lymphatic system vessels are examined in the human parotid gland. A network of lymphatic capillaries extends in the intralobular connective septa around the striated ducts. These lymphatics converge in collectors frequently bordering the excretory ducts. On the contrary, no lymphatics are present next to the intercalated ducts and adhenomers. Ultrastructurally, the lymphatic capillaries are characterized by a very thin endothelial wall and by slightly complicated intercellular adhesions. Open junctions are also present. The presence of numerous lymphatic capillaries bordering the striated ducts and their blood microvasculature is discussed in relation to the functional activities of the striated ducts in the modification of the saliva.     

Immunolocalization of PTHrP in the submandibular glands of three rodent species.

The present study deals with immunohistochemical localization of PTHrP in bank vole, pine vole and white mouse submandibular glands. PTHrP immunoreactivity was observed in epithelial cells of all ductal segments (intercalated, striated, interlobular and main excretory ducts) of the salivary glands in all the three animal species tested. We also found PTHrP expression in myoepithelial cells surrounding the mucous alveoli of submandibular glands in those animals. The reaction was less intense than that found in the epithelial cells of excretory ducts. We occasionally observed a very slight positive reaction for PTHrP in smooth muscle cells of small blood vessels. We also found PTHrP expression in the neurons of ganglion in the submandibular gland.     

Microstereological and histochemical studies of the salivary glands of the giant rat (Cricetomys gambianus, waterhouse)

The histology and histochemistry of the parotid, submandibular and sublingual glands were studied. The submandibular gland contained only serous acini as in the guinea pig, but unlike in many other mammals. The parotid gland contained only serous acini while the sublingual gland was mixed, mucous acini being the predominant secretory tissue interspersed by a few serous acini. Serous demilunes also commonly formed caps on the mucous acini. The ducts of the gland contributed over 30% of the volume of the submandibular gland, while those of the parotid and sublingual glands formed about 12 and 10% of the gland, respectively. The secretions of the parotid gland, as judged by histochemical methods, contained neutral mucins and some sialomucins. Neutral mucins, sulphomucins and sialomucins were detected in both the submandibular gland and sublingual gland.     

On the fine structure of the parotid gland of tufted capuchin monkey, Cebus apella

The parotid gland of the tufted capuchin monkey (Cebus apella) was examined by scanning and transmission electron microscopy. In general the ultrastructural morphology of this gland appears similar to that described in spider and squirrel monkeys. This study has established that the gland is serous. Acinar cells, which contain a well developed granular endoplasmic reticulum and a prominent GOLGI complex, produce a single secretory material. Myoepithelial cells are present around acini and around intercalated ducts. The intercalated ducts, which are composed of cells that show no evidence of secretory activity, lead into striated ducts.     

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.     

Immunohistochemical localization of kallikrein in human pancreas and salivary glands

The localization of kallikrein in human exocrine organs was studied with a direct immunofluorescence method. In the submandibular and parotid salivary glands, kallikrein was found apically in the striated duct cells whereas it was absent from the main excretory ducts or present only as a weak luminal rim. Kallikrein was not found in the acinar cells or in cells of the intercalated ducts. In the pancreas, kallikrein-specific fluorescence was seen in the granular portion of the acinar cells, whereas the islets of Langerhans and ductal cells were unstained.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Ultrastructure of bovine parotid glands

Bovine parotid glands exhibit outstanding structural differences when compared with those of non-ruminant mammals. The acini are tortuous, branched and lined with cells of different heights, imparting a scalloped appearance to acinar lumina. Numerous microvilli, ca. 1.5 μ in length, extend into the lumina and intercellular canaliculi. Intercellular canaliculi measure ca. 3 μ in diameter and interweave in close association with intercellular tissue spaces. Intercellular tissue spaces are separated from the extraacinar spaces across a basal lamina only, whereas junctional complexes guard canaliculi from direct continuity with tissue spaces and/or extraacinar spaces. Flattened cytoplasmic lamellae extend from adjacent acinar cells and loosely interdigitate with one another across the tissue spaces. Acinar cells contain more mitochondria and less granular endoplasmic reticulum than parotid glands of non-ruminant mammals. Two types of secretory material, in the form of inclusions which vary in size and electron density, are present in the acinar cells. Intercalated ducts connect acini with striated ducts which in turn, empty into collecting ducts located between gland lobules. In terms of frequency of “basal infoldings” and numbers of mitochondria, striated ducts of calf parotid glands are not as well developed as those of certain other salivary glands. Myoepithelial cells are most often present at junctions of acini and intercalated ducts where they may attach to both acinar and ductal epithelium. Nerve “terminals” were not observed on the epithelial side of basement membranes in relation to the secretory cells.     

Occurrence and Nuclear Localization of cAMP Response Element- Binding Protein in the Post-natal Development of the Rat Submandibular Gland

Cyclic AMP response element-binding protein (CREB) is a 43-kDa polypeptide that binds a cAMP response element located at the 5 promoter region of cAMP regulatory genes. The spatial and temporal distribution of CREB in the post-natal development of the rat submandibular gland was investigated using immunohistochemistry with a specific antibody. At birth, cells of the terminal tubules and ducts in the submandibular gland showed a nuclear CREB immunoreactivity of moderate intensity. At 1–2 weeks after birth, an intense CREB immunoreactivity was localized primarily to acinar cells. When the r352;-adrenergic agonist isoproterenol was administered to 2-week-old rats, a twofold transient increase in the number of immunoreactive acinar cells was induced. Beginning 3 weeks after birth, CREB immunoreactivity shifted from acini to the duct system and showed a clear localization in the cells of the intercalated ducts and distal portions of striated ducts, where the granular convoluted tubule develops after 4 weeks. Immunopositive materials were localized exclusively in the nuclei of both acinar and ductal immunoreactive cells. After the development of the granular convoluted tubules, CREB immunoreactivity was absent in the tubule cells and was gradually reduced in intensity over the entire gland. In order to examine a hypothesis that CREB is involved in the initial differentiation of the granular convoluted tubular cells, testosterone was administered to hypophysectomized adult rats. Whereas the tubular cells of hypophysectomized rats showed a complete regression, and no CREB immunoreactivity was found in any acinar or duct cells, administration of testosterone for a few days induced an intense CREB immunoreactivity in the nuclei of duct cells, followed by their differentiation into the granular convoluted tubular cells. These results suggested that CREB is involved not only in the growth and differentiation of acinar ce lls that are regulated by r352;-adrenergic nerves but also in those of the duct system, and especially in the androgen-regulated differentiation of the granular convoluted tubular cells, during the post-natal development of the rat submandibular gland.