Endocytosis of parotid salivary proteins by striated duct cells in streptozotocin-diabetic rats

Salivary gland striated duct cells play an important role in the modification of primary saliva by secretion and reabsorption of electrolytes, and secretion of glycoproteins. Recent observations have shown that in the rat parotid gland these cells are able to internalize exogenous proteins, e.g., horseradish peroxidase and ferritin, from the ductal lumen. In rats made diabetic by injection of streptozotocin, dense vacuoles and crystalloids are present in the apical cytoplasm of parotid striated duct cells. In this study we utilized electron microscopic immunocytochemistry to determine if these vacuoles and crystalloids contain acinar secretory proteins. At various times after induction of diabetes by streptozotocin (65 mg/kg), the parotid glands were fixed in a glutaraldehyde-formaldehyde mixture, postfixed in OsO4, and embedded in epoxy resin. Thin sections were immunolabeled with antibodies to protein B1 (Ball et al., 1988) and alpha-amylase (Baum et al., 1982) using a modification of the Protein A-gold technique (Bendayan and Duhr, 1986). With antibody to B1, label was localized in the secretory granules of acinar and intercalated duct cells of both normal and diabetic rats. In striated duct cells of diabetic rats, label was present over the electron-dense vacuoles but not over the crystalloids. Since crystalloids appear to form within the vacuoles, their lack of reactivity may indicate degradation of the internalized protein. The same distribution of label was found with antibody to amylase except for the intercalated duct granules, which were unlabeled in both control and diabetic animals. These results demonstrate that striated duct cells take up salivary proteins from the lumen and that the endocytosis of some secretory proteins from the saliva may be a significant function of these cells in certain pathological conditions.     

Morphological and cytochemical alterations of the Golgi apparatus and GERL in rat parotid acinar cells during ethionine intoxication and recovery

The present electron microscopic cytochemical investigation was undertaken to characterize the alterations in the Golgi apparatus and GERL of rat parotid acinar cells during ethionine intoxication and recovery. Although the Golgi apparatus and GERL were reduced in size, and some broadening of the Golgi saccules occurred as the result of ethionine treatment, the relative localization of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules, and acid phosphatase activity (AcPase) in GERL, remained unchanged. Shortly after ethionine treatment was stopped, a dramatic redistribution of enzyme activities was noted. Within the first 24 hours of recovery, the Golgi apparatus began to enlarge, and the content of secretory granules increased. By day 3 of recovery, cisternae morphologically identifiable as GERL and forming secretory granules possessed TPPase activity, while AcPase activity was virtually undetectable. After seven days of recovery, the Golgi apparatus and GERL appeared both morphologically and cytochemically normal. The enzyme modulation observed during recovery may be correlated with increased secretory granule production. Furthermore, the presence of TPPase activity in GERL and forming secretory granules lends support to the suggestion that GERL may be derived from the trans Golgi saccule.     

Effects of streptozotocin-induced diabetes on the rat parotid gland

The structure of the rat parotid gland was studied in male rats from 4 hours up to 1 year after induction of diabetes by the administration of streptozotocin (STZ, 65 mg/kg). Blood glucose and triglyceride levels were markedly increased, and plasma insulin levels were significantly decreased within 24 hours after STZ. The earliest detectable alteration in acinar cell structure was the presence of small lipid droplets in the basal cytoplasm at 24 hours. Lipid accumulation continued, reaching a peak at 4.5 months after STZ, when the acinar cells contained large lipid vacuoles. By 1 week the Golgi apparatus was enlarged and GERL was prominent at the trans Golgi face. Membrane-bound cytoplasmic crystalloids occurred in acinar cells 1 month after STZ administration and were numerous in older animals (10 to 12 months). These crystalloids occasionally were reactive for trimetaphosphatase, a cytochemical marker for lysosomes. Similar crystalloids as well as large, dense, phagosome-like bodies were also present in the striated duct cells. Macrophages containing acinar cell debris and crystalloids were frequently observed, often in association with the intercalated ducts. Focal alterations in the basement membranes, consisting of multiple layers, loops, and amorphous patches, and "redundant" folds, occurred as early as 2 months and were relatively frequent 10 to 12 months after STZ administration. These results suggest that maintenance of the normal structure and function of the rat parotid gland may, in part, be insulin dependent.     

Ethionine induced degeneration and regeneration in the rat parotid gland: An electron microscope study

The object of the present study was to establish a model for the study of parotid gland regeneration. Adult female Sprague-Dawley rats were placed for 11 days on a protein-free diet with daily intraperitoneal doses of aqueous DL-ethionine equivalent to 0.5 mg/gm body weight, and returned to a normal diet on day 12. At varying intervals, both during and after intoxication, animals were sacrificed and the parotid glands prepared for study with the transmission electron microscope. Ultrastructural observations indicated that damage was essentially limited to the acinar cells, in which the protein synthetic apparatus was the focus of injury. The rough endoplasmic reticulum displayed atypical configurations, loss of attached ribosomes and membrane fragmentation. In the Golgi region, an atypical structure, a “crystalloid” arose during intoxication. Because of the morphology and apparent formation of the “crystalloid” it was assumed to be an abnormal secretion product. Resumption of a normal diet resulted in the rapid restitution of the normal cytoarchitecture. During the first week of recovery, there was prominent mitotic activity in mature acinar cells. It was concluded that the primary effect of ethionine upon the parotid gland is interference with the function of the protein-synthetic apparatus, leading to morphologic alteration of the acinar cells. The mitotic activity observed during recovery indicated that in the adult rat, acinar cells retain the potential for proliferation.     

The effects of reserpine on the ultrastructure and secretory responses of rat exocrine pancreas.

The effects of chronic reserpine administration on rats mimic in several respects the exocrine dysfunction observed in cystic fibrosis. This drug treatment has been proposed as a model for the human disease. In cystic fibrosis, the pancreas is usually a major organ involved pathologically. The present study was designed to investigate the fine-structural morphology and secretory responses of the pancreas of rats treated chronically with reserpine. Reserpine treatment resulted in increased storage of zymogen granules in pancreatic acinar cells and an apparently reduced ability to discharge these granules following stimulation with cholecystokinin. In addition, granule storage may have produced feedback inhibition on the protein synthesizing machinery as manifested by a slight reduction of rough endoplasmic reticulum. Many acinar cells also had autophagic bodies, suggesting the degradation of excess secretory material. Cholecystokinin stimulation of both control and reserpine-treated rats resulted in the appearance of large vacuoles containing myelin figures and granular material, numerous autophagic bodies (cytosegresomes), and a slight decrease of rough endoplasmic reticulum in pancreatic acinar cells. Secretin stimulation did not cause large vacuole formation, but did cause cytosegresome formation in acinar cells. Large vacuoles in intralobular ducts were noted after reserpine treatment, but were also present after stimulation of untreated pancreas with cholecystokinin.     

Secretory protein expression patterns during rat parotid gland development

The rat parotid gland produces a number of well-characterized secretory proteins. Relatively little is known, however, about the onset of their synthesis and cellular localization during gland development. Secretory protein expression was studied in parotid glands of fetal and postnatal rats using light and electron microscopic immunocytochemistry and Northern blotting. Amylase, parotid secretory protein (PSP), common salivary protein-1 (CSP-1), and SMGB were first detected by immunofluorescence in parotid glands of 18 day fetuses. By 5 days after birth, light and electron microscopic immunolabeling localized all of these proteins to the secretory granules of developing acinar cells. Labeling of acinar cells for DNAse I, however, was not observed until 18 days after birth. Between 9 and 25 days, CSP-1 and SMGB reactivity of acinar cells declined, but increased in intercalated duct cells. After 25 days, CSP-1 and SMGB were found only in intercalated ducts, and amylase, PSP, and DNAse I were restricted to acinar cells. Levels of CSP-1 and SMGB mRNA were relatively constant through 21 postnatal days, but declined significantly after that. Amylase and PSP mRNA increased rapidly and continuously from five days after birth to the adult stage. In contrast, DNAse I mRNA was not detectable until 18 days after birth. The immunocytochemical and molecular analyses define three basic patterns of protein expression in the rat parotid gland: proteins whose synthesis is initiated early in development and is maintained in the acinar cells, such as amylase and PSP; proteins that are initially synthesized by immature acinar cells but are restricted to intercalated ducts in the adult gland, such as CSP-1 and SMGB; and proteins that are synthesized only by mature acinar cells and first appear during the third postnatal week, such as DNAse I. The parotid gland exhibits four distinct developmental stages: prenatal, from initiation of the gland rudiment until birth; neonatal, from 1 day up to about 9 days postnatal; transitional, from 9 days to 25 days of age; and adult, from 25 days on. Although differences exist in timing and in the specific proteins expressed, these developmental stages are similar to those seen in the rat submandibular gland. Additionally, the results support the suggestion that intercalated ducts may differentiate from the neonatal acini. Anat. Rec. 252:485–497, 1998. © 1998 Wiley-Liss, Inc.     

Expression of Ca2+-dependent synaptotagmin isoforms in mouse and rat parotid acinar cells

Synaptotagmin is a Ca2+ sensing protein, which triggers a fusion of synaptic vesicles in neuronal transmission. Little is known regarding the expression of Ca2+-dependent synaptotagmin isoforms and their contribution to the release of secretory vesicles in mouse and rat parotid acinar cells. We investigated a type of Ca2+-dependent synaptotagmin and Ca2+ signaling in both rat and mouse parotid acinar cells using RT-PCR, microfluorometry, and amylase assay. Mouse parotid acinar cells exhibited much more sensitive amylase release in response to muscarinic stimulation than did rat parotid acinar cells. However, transient [Ca2+]i increases and Ca2+ influx in response to muscarinic stimulation in both cells were identical, suggesting that the expression or activity of the Ca2+ sensing proteins is different. Seven Ca2+-dependent synaptotagmins, from 1 to 7, were expressed in the mouse parotid acinar cells. However, in the rat parotid acinar cells, only synaptotagmins 1, 3, 4 and 7 were expressed. These results indicate that the expression of Ca2+-dependent synaptotagmins may contribute to the release of secretory vesicles in parotid acinar cells.     

Origin of acinar cell regeneration after atrophy of the rat parotid induced by duct obstruction

Acinar cell regeneration in the rat parotid gland after atrophy induced by a one week period of duct obstruction was examined using histology, immunohistochemistry and transmission electron microscopy (TEM). For immunohistochemistry, antibodies to 5-bromo-2'-deoxyuridine (BrdU), injected one hour before tissue collection, and cytokeratin were employed. When clips were removed from the duct, only ductal epithelial cells remained; all acinar cells had been deleted. Some duct cells were BrdU positive. After three days, newly-formed acini comprising immature acinar cells had appeared; many of the cells were BrdU positive and mitotic figures were readily identified. Thereafter progressive acinar cell maturation and proliferation occurred, parotid gland weight returning to control levels by 7 days. Peak BrdU labelling indices for duct and acinar cells were on days 0 and 4, respectively. By TEM, cytoplasmic organelles in epithelial cells of transitional duct-acinar structures seen at 2 days were poorly developed. Immature acinar cells seen on day 3 contained zymogen granules and had increased endoplasmic reticulum and mitochondria. By day 5, maturing acinar cells had abundant endoplasmic reticulum and zymogen granules, resembling acinar cells in control glands. These observations indicated origin of acinar cell precursors from duct cells during regeneration of the acinar cell-free atrophic gland. Subsequent expansion of the acinar cell population was dependent on maturation and proliferation of these newly-formed cells.     

Changes in the number and distribution of myoepithelial cells in the rat parotid gland during postnatal development

The mature rat parotid gland shows hardly any cell bodies of myoepithelial cells around the acini, only a few cell processes being visible. However, in the early postnatal period, the rat parotid gland shows many myoepithelial cell bodies around the acini, including the intercalated ducts. In order to clarify the reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development, changes in the number and distribution of myoepithelial cells in the rat parotid gland were examined histochemically and chronologically, with particular reference to cell proliferation and cell death. From day 7 to day 14, many myoepithelial cells showing a positive reaction with anti-actin antiserum were found around the acini and intercalated ducts, but thereafter the number of such cells decreased gradually, particularly around the acini, and had almost disappeared after day 35. BrdU/PCNA-positive myoepithelial cells surrounding the acini were easily detected on day 14, but disappeared by day 21, whereas BrdU/PCNA-positive acinar cells remained numerous even after day 21. TUNEL/ISEL staining showed no positive myoepithelial cells throughout the observation period. Transmission electron microscopy also demonstrated no myoepithelial cells with chromatin condensation characteristic of apoptosis through the observation period. These findings suggest that the main reason for the disappearance of myoepithelial cells from the area around the acinus during postnatal development is the large difference between the number of myoepithelial cells and that of acinar cells, because the acinar cells retain their proliferative activity even after myoepithelial cells have become quiescent.     

Changes in the properties of secretory granules in the palatine gland acinar cells of the postnatally developing rat

This study was designed to examine whether or not phospholipid is contained in the secretory granules of the rat palatine gland acinar cells, and if present, to examine the movements of phospholipid in the secretory granules during postnatal development. The palatine glands of male Wistar rats aged 0 to 56 days were used. Acid-hematin staining showed a few positive acinar cells with a faint reaction in the acini on day 0, numerous positive cells with an intense reaction on day 7, a weakening reaction in the cells on day 14, and almost no reactivity on day 35 and after. In contrast, alcian blue staining showed acinar cells with a weak reaction on day 7, a gradual increase in the reaction from day 14, and the presence of many cells with an intense reaction on day 28 and after. Electron probe microanalysis (EPMA) revealed a higher density of phosphorus in samples on day 7 than on day 56. These findings suggest that developing rat palatine gland acinar cells contain phospholipid in the secretory granules, being particularly more conspicuous around postnatal day 7, but that the amount of phospholipid decreases as the cells change to mature mucous cells.     

The ultrastructure of rapid-frozen,substitution fixed parotid gland acinar cells of the mongolian gerbil (Meriones meridianus)

The ultrastructure of gerbil parotid gland acinar cells studied by rapid freezing in liquid nitrogen and freeze-substituting with OsO₄ was similar in general to conventionally prepared tissue, but different in several aspects. Acceptable preservation was limited to about 10 μm of the surface; the secretory granule membrane was a single dense line, while all others were trilaminar; the dense-cored secretory granule had a rim of low density which was homogeneous; there were many continuous profiles between the Golgi lamellae, vesicles, and vacuoles; and mitochondria were long and arborized.     

Changing myoepithelial cell distribution during regeneration of rat parotid glands

The distribution of the myoepithelial cells during regeneration of the rat parotid gland after atrophy induced by one week of parotid duct ligation was investigated by immunohistochemistry for actin and transmission electron microscopy (TEM). Immunohistochemically, residual ducts were surrounded by actin-positive cells when clips were removed from the duct. Three days later, most of the newly formed acini originating from the residual ducts were also embraced by actin-positive cells. After 10 days, actin-positivity tended to be seen as dots around acini that decreased in number day by day. On day 21 actin-positive cells mainly surrounded intercalated ducts with only a few positive reactions identified at the acinar periphery. Electron microscopically, residual ducts and newly formed acini were peripherally embraced by myoepithelial cells before day 5. After day 7, shift of myoepithelial cells from the periphery of acini to the duct-acinar junctional region was identified. Then few myoepithelial cells were identified at the periphery of acini. These observations indicate that myoepithelial cells migrate from the acinar periphery to the duct-acinar junctional region during rat parotid regeneration, and that such behaviour is closely related to that seen during rat parotid development.     

Developmental changes of sugar residues and secretory protein in mucous cells of the early postnatal rat parotid gland.

Mucous cells have been identified in the terminal portions of the early postnatal parotid gland in human and rat, although mature parotid gland acini are composed of serous cells or seromucous cells. Previously, Ikeda et al. demonstrated that mucous cells are present in the rat parotid gland on days 1 to 8 after birth and that the secretory granules within these mucous cells share some histochemical characteristics with mature serous cells. However, it is still not clear whether the mucous cells change into serous cells as the gland develops. The purpose of this study was to determine whether the mucous cells that appear in the early postnatal rat parotid gland change into serous cells. Parotid glands were obtained from male or female Wistar rats (aged 0-14 days and adults). Fixed tissue sections were reacted with soybean agglutinin (SBA) and wheat germ agglutinin (WGA) to detect glycoconjugates, or were stained using an anti-neonatal submandibular gland protein B1 (SMG-B1) antibody to identify serous acinar cells. The sections were observed by transmission electron microscopy. Electron microscopy revealed that cells with characteristics intermediate between those of mucous and serous cells (transitional cells) appeared around day 8 and that the nuclei of these cells did not show chromatin condensation, a characteristic of apoptotic cells. Lectin histochemistry showed that the mucous cells had the same sugar residues as the serous cells, which appeared after day 10. Immunohistochemistry with an anti-SMG-B1 antibody gave a positive reaction not only in the cells with highly electron-dense granules but also in the electron-dense cores of bipartite or tripartite granules in the transitional cells. Cells with morphological characteristics intermediate between those of mucous and serous cells (transitional cells) appearing in the early postnatal rat parotid gland begin to produce B1-immunoreactive protein common to serous acinar cells during development of the gland.     

Cell death and cell proliferation during atrophy of the rat parotid gland induced by duct obstruction

Rat parotid gland atrophy after unilateral duct ligation was studied by light and electron microscopy. Death of secretory acinar cells, which took the form of apoptosis, resulted in their complete disappearance within 5 days. The remnants of the dying cells were mostly phagocytosed and degraded by macrophages within the glandular epithelium; a few were taken up by adjoining epithelial cells. The acinar cell deletion was accompanied by increased mitosis of striated and intercalated duct epithelial cells. However, over many weeks, there was enhanced apoptosis of duct cells, which eventually led to marked shortening of intercalated ducts. Apoptosis of capillary endothelial cells was observed and may account for the reduction in the capillary bed known to accompany gland atrophy. The end-stage lesion comprised small numbers of ducts in a condensed stroma. Compensatory hyperplasia, involving proliferation of duct and acinar cells, was demonstrated in the contralateral glands.     

Studies on Cellular Autophagocytosis: The Relationship of Autophagocytosis to Protein Synthesis and to Energy Metabolism in Rat Liver and Flounder Kidney Tubules in Vitro

The purpose of the present study was to elucidate the metabolic requirements of autophagocytosis. Two model systems were used for this purpose: a) glucagon-induced autophagocytosis in the rat liver, and b) the wave of autophagocytosis which occurs when isolated flounder kidney tubules are incubated in vitro. In the rat liver, protein synthesis was inhibited by the administration of cycloheximide (1.5 mg/kg) to rats 2 hours prior to glucagon injection. In flounder kidney tubules, protein synthesis was inhibited at least 90% by adding cycloheximide, actinomycin D, pactamycin and puromycin to the medium. In both systems the inhibition of protein synthesis failed to inhibit the formation of autophagic vacuoles or their subsequent transformation into autolysosomes, as depicted from electron microscopic histochemical preparations. In flounder kidney tubules no differences were found in the levels of p-nitrophenylphosphates, β-DL-glycerophosphatase, N-acetyl-β-D-glucosaminidase, arylsulphatase, β-D-galactosidase or acid proteinase when tubules were incubated up to 5 hours in the presence or absence of protein synthesis inhibitors. When ethionine was administered to rats 2 hours before glucagon injection, a decrease of approximately 75% in the ATP levels was observed. After ethionine administration, glucagon failed to induce the formation of autophagic vacuoles. The incubation of flounder kidney tubules in the presence of cyanide or in a nitrogen atmosphere decreased the ATP levels to less than 10% of controls and blocked autophagy. On the other hand, cyanide had little effect on acid hydrolase levels at 1 hour of incubation. A wide variety of other inhibitors were also shown to block autophagy. These results further support the hypothesis that, in the formation of antophagic vacuoles, preexisting enzyme and membrane pools are utilized. On the other hand, the esotropy-exotropy membrane conformational changes occurring in the formation of autophagic vacuoles seem to be energy dependent and can therefore be blocked by lowering intracellular ATP levels.     

Changes in the secretory acinar cells of the rat parotid gland during aging

The secretory acinar cells of parotid glands from rats of varying ages have been examined by electron microscopy to determine what age-related changes occur in these cells. The most prominent change noted in these cells is the progressive increase in the amount of lipofuscin granules with age. Lipofuscin granules are membrane-bound structures consisting of lipids, other subcomponents, and a matrix. In addition, these cells contain lipid droplets that are not associated with any other components and tend to accumulate at the base of the cells in older rats. Also, many acinar cells in the glands of old rats contain altered secretory granules which appear to be in the process of degeneration. The accumulation of lipid and degenerating secretory granules appears to be related to the reduced level of cellular secretory activity in the glands of older rats. It is possible that these two types of inclusions contribute to the formation of lipofuscin granules. Lipofuscin and degenerating secretory granules are associated with acid phosphatase, which is demonstrated cytochemically, indicating that these granules are lysosomal structures.     

Effects of vinblastine, leucine, and histidine, and 3-methyladenine on autophagy in Ehrlich ascites cells

The microtubule inhibitor vinblastine causes accumulation of autophagic vacuoles in many cell types. In hepatocytes, many of the accumulated vacuoles are nascent, which has been interpreted to suggest that vinblastine acts by inhibiting the fusion of hydrolase-containing lysosomes with early autophagic vacuoles. However, our previous results suggested that, in Ehrlich ascites cells, vinblastine causes accumulation mainly of older autophagic vacuoles (AVs). This study was undertaken to further characterize the mode of action of vinblastine in these cells. The vinblastine-accumulated AVs were quantified by electron-microscopic morphometry. In addition, the effects of inhibitors of autophagic segregation (leucine, histidine, and 3-methyladenine) on the vinblastine-induced accumulation of autophagic vacuoles were studied. Protein degradation was measured using [14C]valine. Vinblastine caused accumulation of advanced autophagic vacuoles but did not increase the rate of protein degradation. The volume density of early vacuoles remained at the control level. The amino acids retarded but did not prevent the accumulation of autophagic vacuoles, whereas 3-methyladenine almost completely prevented the accumulation. The results suggest that in Ehrlich ascites cells vinblastine acts by inhibiting the maturation of advanced autophagic vacuoles into residual bodies and by stimulating the formation of new autophagic vacuoles. However, 3-methyladenine almost completely prevents the formation of new autophagic vacuoles in the presence of vinblastine. In conclusion, in Ehrlich ascites cells, vinblastine does not prevent the entry of hydrolases into autophagic vacuoles. This calls into question the importance of microtubules in the transport of lysosomal enzymes into autophagic vacuoles.     

Intercalated duct cells in the rat parotid gland may behave as tissue stem cells

This study was conducted to determine whether intercalated duct cells in the rat parotid gland have the properties of tissue stem cells. After induction of cellular proliferation by repeated administration of isoproterenol (IPR), a β-adrenergic agonist, proliferation activity in acinar, intralobular, and intercalated ductal cells was quantified using Ki-67 immunohistochemistry. The total number of each type of component cell in a gland was also estimated in the course of IPR treatment. IPR was found to induce proliferation of acinar and intercalated duct cells, but not intralobular duct cells. The total number of acinar cells in a gland on day 5 of IPR treatment was 1.6 times of that at day 0 (baseline). In contrast, the total numbers of intercalated and intralobular duct cells did not change from baseline, indicating a high possibility that the proliferated intercalated duct cells differentiated into acinar cells. On days 2 to 3 of IPR treatment, intercalated duct cells with amylase-positive secretory granules were recognized in a region very close to the acini, and were suspected of being transitional cells from intercalated duct to acinar cells. This quantitative study indicates that intercalated duct cells may have the properties of tissue stem cells upon IPR stimulation.     

Electron microscopic studies of viral pancreatitis in coxsackie B4 virus infected mice.

Using a high titer culture of Coxsackie B₄ virus, cytonecrosis consistent with picornaviral infection and cytopathic changes indicative of viral pancreatitis were produced in the pancreas of newborn mice. Virocytonecrosis was manifested within acinocytes by formation of characteristic membrane-vesicle complexes, dilatation of rough endoplasmic reticulum, margination of nuclear chromatin, pyknosis of nuclei, and acute inflammation. Viral pancreatitis was characterized by formation of cytosegresomes, fibroid bodies, and sloughed damaged acinar cytoplasm apparent in autophagic vacuoles of macrophages. Immature leucocytic cells were closely aligned to the wall of acinar ducts. Beta cell damage was also observed. Of significance was the observation of aggregates of Coxsackie B₄ virus particles in various pancreatic cells of 11 of the 23 mice studied. These aggregates appeared in the typical crystalline form with cubic and hexagonal lattice configurations and as viral particles circularly arranged into eight-sided and ten-sided “rosettes.” These findings strongly support the concept that viruses are etiologic agents in pancreatitis and diabetes mellitus.