Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, oamylase and Brimmunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.     

Liquid-diet-induced alterations of rat parotid acinar cells studied by electron microscopy and enzyme cytochemistry

After 1 day on liquid diet, the acinar cells were filled with secretory granules, and gland amylase content was approx. 50 per cent greater than chow-fed controls. After 3 days, the number of secretory granules was reduced, and amylase levels had fallen to 50 per cent of controls. Altered or degenerating secretory granules and autophagic vacuoles containing rough endoplasmic reticulum, secretory granules and mitochondria were observed with increasing frequency during the 1st week. These structures were cytochemically reactive for trimetaphosphatase, a lysosomal hydrolase. Concomitantly, macrophages invaded the acinar parenchyma and phagocytosed the degenerating acinar cell components. Lipid droplets and large aggregates of glycogen particles were present in the acinar cells after 7–10 days. After 3 weeks, the acinar cells were considerably reduced in size and contained few secretory granules, but their structure appeared otherwise normal. These results indicate that the acinar-cell lysosomal system and invading macrophages, as in other experimental conditions, play an important role in the parotid-gland atrophy induced by liquid diet.     

Cytoplasmic crystalloids in irradiated rat parotid glands

Cytoplasmic crystalloids were found in parotid acinar cells of rats given a large (6400 R) single exposure of X-rays to the head and neck. The crystalloids were first observed 1 day after irradiation and became numerous at 3-4 days. They were associated with autophagic vacuoles, which were seen in acinar cells as early as 3-6 h. Crystalloids sometimes appeared to be forming within autophagic vacuoles, which also contained membranous residues and apparently degenerating secretory material. They were bounded by a single smooth membrane and had a substructure consisting of dense, parallel longitudinal striations. They crystalloids were also seen in macrophages associated with the basal surface of acinar cells. At 3-4 days macrophages were numerous and many contained crystalloids, degenerated secretory droplets, and other cellular debris, which they apparently had phagocytosed. By 6-8 days crystalloids and macrophages were seen infrequently. Regarding mode of formation, removal by macrophages, and ultrastructure, the crystalloids resembled those described by others after ethionine intoxication. Ethionine-induced crystalloids have cytochemical characteristics consistent with a lysosomal identity. The crystalloids in irradiated parotid glands probably reflect a variant type of lysosome, which is a nonspecific manifestation of severe cellular injury and can be elicited by a variety of injurious agents.     

Endocytosis of proteins by salivary gland duct cells

The ability of the intralobular duct cells of the rat parotid gland to take up protein from the lumen was examined by retrograde infusion of exogenous proteins and by immunogold localization of endogenous secretory proteins. Small amounts of native horseradish peroxidase (HRP) were taken up by intercalated and striated duct cells, and were present in small vesicles, multivesicular bodies, and lysosomes. In contrast, HRP modified by periodate oxidation was avidly internalized by the duct cells and was present in large apical vacuoles that acquired lysosomal hydrolase activity. Native and cationized ferritin were taken up in a similar manner when infused at a high concentration (up to 10 mg/mL). At lower concentrations (0.3-1.0 mg/mL), endocytosis of cationized ferritin occurred mainly in small apical tubules and vesicles in striated duct cells. Little native ferritin was taken up at these concentrations. After stimulation of acinar cell secretion by isoproterenol, similar vacuoles were occasionally observed in both intercalated and striated duct cells. Labeling of thin sections with antibodies to amylase and to a 26,000-dalton secretory protein (protein B1), followed by protein A-gold, revealed the presence of these proteins in the vacuoles, indicating endocytosis of acinar secretory proteins by the duct cells. Although uptake of acinar proteins by duct cells occurs at a low rate in normal animals, previous work suggests that extensive endocytosis may occur in certain pathological conditions. This may be a mechanism for removing abnormal or modified proteins from saliva before it reaches the oral cavity.     

Parotid secretory granules: crossroads of secretory pathways and protein storage

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins-including amylase, proline-rich proteins, and parotid secretory protein (PSP)-to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.     

Staurosporine-inhibitable protein kinase activity associated with secretory granule membranes isolated from rat submandibular gland cells

Protein kinases, such as protein kinase C, have been shown to be associated with secretory granules and to regulate the event of exocytosis in various tissues including parotid salivary acinar cells. However, in submandibular acinar cells that play an important role in the secretion of proteins into the oral cavity, kinase activity on the granule membrane has not been explored. Therefore, in the present study, we isolated the secretory granules from rat submandibular acinar cells and investigated the localisation of protein kinases on the granule membrane. Initially, we isolated and purified secretory granules from rat submandibular acinar cells. Addition of [gamma-32P] ATP to granule-membrane lysate phosphorylated the granule-membrane-associated 26, 32, 55 and 58kDa proteins, suggesting the presence of endogenous kinase activity on the membrane. Moreover, the phosphorylation of 26 and 32kDa proteins was inhibited by staurosporine and K252a, both non-specific protein kinase C inhibitors. However, the phosphorylation of 26 and 32kDa proteins was not inhibited by other protein kinase C inhibitors, such as calphostin C, GF109203X and chelerythrine, indicating that protein kinase C was not responsible for the phosphorylation. In addition, H-89, ML-9, KN-62 and genistein did not appear to inhibit this phosphorylation, indicating that protein kinase A, myosin light chain kinase (MLCK), Ca2+/calmodulin-dependent protein kinase II (CAMKII) and tyrosine kinase were not involved in the phosphorylation of 26 and 32kDa proteins. Moreover, Ca2+ had no effect on the kinase activity. Therefore, our results suggest that an unidentified, staurosporine-inhibitable protein kinase activity is associated with the secretory granule membrane of rat submandibular acinar cells.     

The effects of ductal obstruction on the acinar cells of the parotid of cat

Twenty-nine parotids ligated for between 1 and 365 days were examined by light and electron microscopy. Major changes in the acini were seen at 4 days and included vacuolation, disintegration, extravasation, apoptosis, phagy and a reduction in number and size of secretory granules. There was a further reduction in secretory granules from 7 to 12 days, but acinar cells persisted even up to 365 days, some contained a luminal concentration of small secretory granules and occasionally acinar cells of a similar appearance to normal were found. These findings contrast with a reported absence of acinar cells from the obstructed parotid of rat and show that parotid acinar cells are able to persist and retain an appearance indicative of secretory activity.     

Effect of cytochalasin D on acinar cell structure and secretion in rat parotid salivary glands in vitro

Cytochalasin D, a microfilament disrupting agent, considerably inhibited isoproterenol-stimulated amylase release from enzymatically dispersed parotid acini. Histologically cytochalasin D caused a loss of microvilli lining acinar lumina and luminal enlargement. Nearly empty vacuoles appeared near the luminal and lateral surface, and the membrane bordering on the vacuoles was often continuous with the plasma membrane. Therefore, the vacuolization probably resulted from an elongation of the membrane lining the lumen. Fluorescence staining with rhodamine-phalloidin showed that cytochalasin D caused disruption of microfilaments. When stimulating the cytochalasin D-treated cells with isoproterenol, the number of secretory granules in the cytoplasm diminished markedly and secretory material was observed in the vacuoles, indicating that inhibition of amylase release by cytochalasin D is not due to blocking of exocytosis but to the retention of amylase discharged into vacuoles. These findings suggest that microfilaments are essential in maintaining the parotid acinar structure but do not play a direct part in the movement of secretory granules and their fusion with the luminal membrane.     

Exocyst subunits are involved in isoproterenol-induced amylase release from rat parotid acinar cells

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.     

Redistribution of small GTP-binding protein, Rab27B, in rat parotid acinar cells after stimulation with isoproterenol

Small GTP-binding protein, Rab27, has been implicated in the regulation of different types of membrane trafficking, including melanosome transport in melanocytes and regulated secretion events in a wide variety of secretory cells. We have previously shown that Rab27 is involved in the control of isoproterenol (IPR)-induced amylase release from rat parotid acinar cells. Although Rab27 is predominantly localized on secretory granules under resting conditions, changes to its intracellular localization after β-stimulation have never been elucidated. The present study investigated IPR-induced redistribution of Rab27B in the parotid acinar cells, revealing translocation from secretory granules to the subapical region after 5 min of IPR treatment and then diffusion into the cytosol after 30 min of IPR treatment. Dissociation of Rab27B from the apical plasma membrane is probably mediated through the Rab GDP dissociation inhibitor (GDI) in the cytosol extracting GDP-bound Rab protein from membranes, as a dramatic increase in the amount of the Rab27B–GDI complex in the cytosol was observed 30 min after stimulation with IPR. These results indicate that, in parotid acinar cells, Rab27B is translocated, in a time-dependent manner, from secretory granules into the apical plasma membrane as a result of exposure to IPR, and then into the cytosol through binding with the GDI.     

Immunocytochemical analysis of cyclic AMP receptor proteins in the developing rat parotid gland

Previous studies showed that regulatory subunits of type II cyclic AMP-dependent protein kinase (RII) are present in adult rat parotid acinar cells, and are secreted into saliva. If the synthesis and intracellular distribution of RII exhibit developmental specificity, then RII can be an indicator of secretory and regulatory activity of salivary glands. OBJECTIVE: To determine the expression and distribution of RII in the rat parotid at specific ages representing defined developmental stages. METHODS: Parotid glands of fetal, neonatal and adult rats were prepared for morphologic and immunocytochemical study. The cellular distribution of RII was studied using light microscopic immunogold silver staining with anti-RII, and its intracellular distribution using electron microscopic immunogold labeling. RESULTS: In utero, parotid RII levels were low; 5-18 days after birth, labeling of secretory granules and cytoplasm rose to a peak, followed by a rapid decrease in both compartments at 25 days. At 60 days, granule labeling increased to levels near those at 18 days, whereas cytoplasmic labeling remained low. Nuclear labeling was highest during the first 3 weeks after birth, and then declined. CONCLUSIONS: The higher nuclear and cytoplasmic labeling during the neonatal period may reflect RII involvement in acinar cell differentiation. The accumulation of RII in secretory granules is similar to the pattern of the major salivary proteins, amylase and PSP. The redistribution of RII in these compartments during development may reflect changing gene expression patterns, and may be useful for identification of genetic or metabolic abnormalities.     

Radioprotection of the mice parotid gland by isoproterenol: study on morphometry of secretory granules and on autoradiography

Objectives The aim of the present study was to investigate the effect of radiosensitivity on acinar cells in the parotid gland when the secretory granules were released. Methods The parotid glands of mice were exposed to 10 Gy of X-radiation when the acinar cells were degranulated with isoproterenol (IPR). Three days later, morphological images and number and area of secretory granules within the acinar cells in the parotid glands were obtained and light microscope autoradiography (LMARG) was performed using ³H-leucine. Results The light microscopy images showed a disorderly arrangement and pycnosis of acinar cells and cellular atrophy in irradiated groups. The changes were milder in IPR-administered groups than in non-IPR-administered groups. The number of secretory granules in irradiated groups, which included both IPR-administered and non-IPR-administered sets, was significantly less than that in nonirradiated groups. The number of silver grains within acinar cells obtained by LMARG in the non-IPR-administered set of irradiated groups was significantly lower than that in the nonirradiated group or the IPR-administered set after 30 min of radioisotope administration, and it was significantly higher than that of the nonirradiated group after 240 min. Conclusions When the secretory granules of acinar cells in mouse parotid gland were degranulated by isoproterenol, alleviation of the effects of radiation exposure on morphological change as well as the ingestion and egestion of secretory substances were indicated.     

Electron microscopy of watery vacuole formation in rat parotid acinar cells during reflex stimulation

Reflex stimulation from eating hard chow caused variable degrees of watery vacuolation, most conspicuously in animals that had eaten the most food; this was accentuated by eating in the cold. Some vacuolation occurred in the absence of sympathetic impulses but there was much more in glands receiving both parasympathetic and sympathetic impulses. Vacuole formation was often associated with, but not dependent upon, a degree of acinar degranulation. The vacuoles appeared to originate either from the inner side of the Golgi apparatus, near condensing vacuoles, or from ballooning within the rough endoplasmic reticulum; some vacuoles appeared to arise close to secretory granules. Certain cellular components, including secretory granules, entered the contents of vacuoles, which also often had connections with lumina. Many basally-located vacuoles were large and bounded only by a tenuous layer of cytoplasm which was contained by the basal lamina. Vacuole formation may be related to strong stimulation of susceptible cells, which occurs to a variable extent during natural reflex secretion, as well as during artificial stimulation. Thus, under normal conditions, certain macromolecular components might enter saliva via this route and not solely by exocytosis. Leakages from acinar vacuoles could also occur into the glandular interstices, accounting for some of the parotid amylase found in peripheral blood.     

Inhomogeneities in glycoprotein cytochemistry of secretory granules in rat-parotid acinar cells after selective β1-adrenoceptor stimulation

The distribution of periodate-positive glycoproteins was studied in parotid acinar cells of the rat after stimulation with the specific β₁-adrenoceptor agonist prenalterol. After two weeks of daily injections, the majority of secretory granules were larger, less electron dense, and often exhibited a bipartite or pleomorphic PA-CrA-silver staining pattern. A great variation in the staining pattern of the secretory granules was seen among different cells but, within any individual cell, most granules displayed a similar pattern. The staining reaction suggests a mucoid transformation of the granules, or a reorganization of the glycoproteins within the granules. The variation among cells in the staining pattern of granules may suggest that there are different populations of acinar cells or of secretory granules.     

Morphological and Histochemical Changes in the Parenchyma of the Rat Parotid and Sublingual Glands with Growth and Aging

The purpose of this review is to provide an overview of the differences in parenchymal structures that occur between the parotid and sublingual salivary glands of the rat from prior to birth to old age.Although the mature rat parotid gland is classified as serous, mucous cells have been identified in the acini during the early postnatal period. Mucous cells contained secretory granules including mucins and secretory proteins common to those of serous granules. Moreover, these cells morphologically showed transformation from mucous to serous type, and lack of apoptosis. When these findings are considered together, they suggest the possibility that mucous cells are converted into serous cells.Many myoepithelial cells around the acini were observed during the early postnatal period, whereas the mature rat parotid gland showed hardly any myoepithelial cells around the acini, but had such cells investing the intercalated ducts. The main reason for the disappearance of myoepithelial cells surrounding the acini during postnatal development is not apoptosis, but rather the large difference in cell number between myoepithelial cells and acinar cells, because the rate of proliferation of acinar cells declined more slowly than that of myoepithelial cells.Transient cells also occurred in the sublingual gland acini during the perinatal period, but these transformed into mucous cells, the opposite of transient cells in the developing parotid gland.     

Cytoplasmic crystalloids in irradiated rat parotid glands

Abstract Cytoplasmic crystalloids were found in paroid aeinar cells of rats given a large (6400 R) single exposure of X-rays to the head and neck. The crystalloids were first observed 1 day after irradiation and became numerous at 3–4 days. They were associated with autophagie vucuoles, which were seen in acinar cells as early as 3–6 h. Crystalloids sometimes appeared to be forming within autophagic vacuoles, which also contained membranous residues and apparently degenerating secretory material. They were bounded by a single, smooth membrane and had a substructure consisting of dense, parallel longitudinal striations. The crystalloids were also seen in macrophages associated with the basal surfaces of acinar cells. At 3–4 days macrophages were numerous and many contained crystalloids, degenerated secretory droplets, and other cellular debris, which they apparently had phagocytosed. By 6–8 days crystalloids and macrophages were seen infrequently. Regarding mode of formation, removal by macrophages, and ultrastructure, the crystalloids resembled those described by others after ethionine intoxication. Ethionine-induced crystalloids have cytochemical characteristics consistent with a lysosomal identity. The crystalloids in irradiated parotid glands probably reflect a variant type of lysosome, which is a nonspecific manifestation of severe cellular injury and can be elicited by a variety of injurious agents.     

Immunolocalization of E-cadherin and alphaE-catenin in rat parotid acinar cell under chronic stimulation of isoproterenol

E-cadherin and alphaE-catenin were localized in normal and chronically isoproterenol-treated acinar cells of rat parotid gland by means of immunogold labelling of Lowicryl embedded sections. Immunostaining of both experimental groups with polyclonal antibodies to E-cadherin and alphaE-catenin was mainly restricted to the areas of adherens junctions. Surprisingly, in isoproterenol-treated cell alphaE-catenin was also found on the secretory granules periphery and appeared to encircle a secretory vesicle. In isoproterenol-induced cell hyperproliferation, the maintened presence of adherens junctions components, such as E-cadherin and alphaE-catenin molecules, should be an essential prerequisite for tissue integrity. Our data suggest the presence of a correlation between the organization of actin and the localization of alphaE-catenin in the chronically isoproterenol-treated acinar cell of rat parotid gland.     

Relation of Rab26 to the amylase release from rat parotid acinar cells

Amylase secretion from rat parotid acinar cells is induced by the accumulation of cAMP in response to beta-adrenergic agonists as well as by the elevation of intracellular Ca2+ in response to muscarinic cholinergic stimulation. Several proteins including the low molecular weight GTP-binding protein Rab may participate in these exocytic processes. In the current studies, we investigated the role of Rab26 in the process of amylase secretion. Secretory granules were separated by centrifugation on a Percoll-sucrose density gradient into mature and immature granule fractions. Rab26 and two other type III Rab proteins, Rab3D and Rab27, were present in the mature granule membrane fraction. Also, Rab26 was absent in immature granule membrane fractions. Isoproterenol-induced amylase release from streptolysin-O-permeabilised acinar cells was inhibited by an anti-Rab26 antibody, but this antibody had no effect on the Ca2+-induced release of amylase. Finally, in the early stage of beta-adrenergic stimulation, Rab26 was condensed in the secretory granule membrane. These results indicate that Rab26 is involved in the recruitment of mature granules to the plasma membrane upon beta-adrenergic stimulation.     

Regulatory aspects of N-linked glycoproteins

Activation of beta-adrenoreceptors in rat parotid acinar cells leads to copious exocrine protein secretion. Additionally, beta-adrenergic stimulation dramatically increases specific secretory protein synthesis and enhances N-linked glycosylation of secretory glycoproteins. Recently, efforts have been directed toward understanding the mechanisms underlying these biosynthetic events. We have been particularly interested in the receptor-mediated regulation of glycosylation. In this report, we evaluate available mechanistic information from the rat parotid gland and present initial data examining the ability of various regulatory agents to modulate N-linked glycosylation in enzymatically-dispersed cell aggregates from surgical specimens of human parotid glands. We conclude that glycosylation of human parotid N-linked glycoproteins may be regulated by extracellular signaling similar to that operative in the rat parotid gland.     

Presence of cytoskeleton proteins in parotid glands and their roles during secretion

Amylase secretion is induced by the accumulation of cAMP in response to β-adrenergic stimulation and by the augmentation of intracellular Ca²⁺ in response to muscarinic-cholinergic stimulation in rat parotid glands. The roles of cytoskeleton and motor proteins in the secretory process are not yet known. We examined the effects of cytoskeleton-modulating reagents on the amylase release induced by isoproterenol (IPR) and carbamylcholine (Cch) in rat parotid acinar cells. The amylase release induced by Cch was decreased by the microtubule-disrupting reagent colchicine (Colch) and the myosin ATPase inhibitor 2,3-butanediene monoxime (BDM), but the release induced by IPR was not. The actin filament-stabilizing reagent jasplakinolide (Jasp) and actin filament-disrupting reagent cytochalasin D (CytoD) decreased the amylase release induced by both the β-adrenergic and the muscarinic-cholinergic stimulants. Pretreatment with CytoD affected the shape of the acinar cells, which showed an intermediate state between the fusion of the secretory granules with the apical membrane and the retrieval of the membranes only after stimulation with IPR. Myosin and Dynein/dynactin complex were detected in the secretory granule membrane fraction. We concluded from this study that the cytoskeleton played different roles in the β-adrenergic and the muscarinic-cholinergic secretory processes.