Acid phosphatase and peroxidase in ‘resting’ acinar cells of the major salivary glands of cats and their possible movement into secretory granules

Synopsis After fixation by perarterial perfusion using an aldehyde mixture, salivary tissues were prepared for ultrastructural cytochemistry of acid phosphatase or peroxidase. Great variations in the distributions of the reaction products occurred, often within the same cell. Acid phosphatase staining occurred not only in lysosomes and sometimes in a GERL system, but a diffuse cytoplasmic component was also found in submandibular central acinar cells and to a lesser extent in parotid acini and variable staining occurred in the secretory granules of these cells. Peroxidase was variably associated with rough endoplasmic reticulum in submandibular demilunar cells, parotid acini, and more strongly in some sublingual cells. The secretory granules of the latter were darkly stained, but in parotid granules there was varibale staining and least staining occurred in the granules of submandibular demilunes.These results are thought to indicate that not all enzymes present in secretory granules have reached there by an elective secretory process. Sometimes they appear to have entered the granules haphazardly, possibly having been enzymes associated with intracellular cisternal channels for transport or metabolism of other secretory substances and ultimately to have passed into the cisternal channels by chance or as part of a natural removal of redundant material.     

Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands

Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although <10% of parotid secretory protein (PSP) aggregated below pH 6.0. To test aggregation directly in isolated granules, rat parotid secretory granules were permeabilized with 0.1% saponin in the presence or absence of calcium and in the pH range of 5.0 to 8.4. In contrast to the low-pH-dependent retention of amylase in exocrine pancreatic granules, amylase was quantitatively released and most PSP was released from parotid granules under all conditions. Both proteins were completely released upon granule membrane solubilization. Thus neither amylase nor PSP show low-pH- or calcium-induced aggregation under physiological conditions in the exocrine parotid secretory granules.     

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.     

Localization of cAMP-dependent protein kinase subunits along the secretory pathway in pancreatic and parotid acinar cells and accumulation of the catalytic subunit in parotid secretory granules following beta-adrenergic stimulation

Catalytic (C) and regulatory (RI and RII) subunits of cAMP-dependent protein kinases were localized by immunoelectron microscopy in cisternae of the rough endoplasmic reticulum (rER) and in the Golgi complex of rat pancreas or parotid cells. Zymogen granules of the exocrine pancreas showed C- and RI-immunoreactivity, secretory granules of parotid acinar cells only RII-immunoreactivity. Injection of rats with isoproterenol (IPR) increased in the parotid gland the number of acinar cells with RII-labeled granules. In addition, it led to the appearance of C-immunoreactivity in the condensing vacuoles and secretory granules with a maximum at 24 h after stimulation. This was confirmed by enzyme-linked immunosorbent assay (ELISA) determinations of C- and RII-subunits in secretory granules isolated from stimulated and control parotid glands. The amount of immunoreactive C-subunits in the secretory granules increased further following repeated injections of the beta-agonist. These findings suggest the existence of secretory forms of cAMP-dependent protein kinase R- and C-subunits and their separate regulation.     

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.     

Giant secretory granules in the ducts of the parotid and submandibular glands of the slow loris

The parotid and submandibular glands of a slow loris, a rare Southeast Asian primate, were obtained after the head had been perfused by fixative for a study of the brain. These tissues were processed by conventional means for electron microscopy. Glands also were obtained at autopsy from 2 other lorises, fixed by immersion in formalin, and subjected to a battery of tests for glycoconjugates. In the parotid gland, a short segment of the proximal striated duct lacks both basal striations and any sign of secretory activity. The major portion of the striated duct consists of tall cells that contain a spectrum of secretory granules, some larger than the nuclei (many granules are > 9 mum in diameter). These granules, which are delimited by a single membrane, are capable of chain exocytosis. Many of the giant granules have bundles of cytofilaments (4.5-6.5 nm) in apparent association with their surface. Occasional cells contain numerous small granules. Duct cells with or without granules lack basal striations. The granules contain neutral glycoconjugates but no acidic glycoconjugates. Some, but not all, interlobular excretory ducts also have secretory granules that run the gamut from tiny to giant. Exactly the same situation occurs in the submandibular gland. Unlike other primates, which may have duct cells that contain only a few tiny granules in their apices, the cells in both the striated and excretory ducts in the slow loris appear to be specialized for secretion rather than for transport. The biofunction of the giant granules is unknown.     

Immunohistochemical localization of carbonic anhydrase isoenzymes VI, II, and I in human parotid and submandibular glands

Human salivary carbonic anhydrase (HCA VI) was purified by inhibitor affinity chromatography and its location in the human parotid and submandibular glands identified, using a polyclonal antiserum raised against the purified enzyme in rabbits in conjunction with the peroxidase-antiperoxidase complex method. The antibodies raised against the purified enzyme in rabbits did not crossreact with the HCA II or I. However, they slightly recognized human IgA; the antiserum was therefore absorbed with human IgA before immunohistochemical use. HCA VI-specific staining was detected in the cytoplasm and particularly in the secretory granules of the serous acinar cells of both parotid and submandibular glands, the staining of the secretory granules being most distinct in paraformaldehyde-fixed tissues. Some epithelial cells and the luminal content of the striated ducts also gave a specific HCA VI staining. Staining specific for HCA II was also found in the granules of the serous acinar cells, particularly in the submandibular gland when Carnoy fluid fixation was used. Slight HCA II-specific staining was also detected in the striated ductal cells in the Carnoy fluid-fixed specimens. No staining specific for HCA I was detected. The results indicate that the serous acinar cells in human parotid and submandibular glands contain abundant HCA II and HCA VI. Interestingly, only HCA VI is secreted into the saliva, although both enzymes appear to be located in structures resembling the secretory granules in the acinar cells. The enzymes probably form a mutually complementary system regulating the salivary buffer capacity.     

Changes in parotid acinar cells accompanying salivary secretion in rats on sympathetic or parasympathetic nerve stimulation

Morphological and secretory effects of stimulating autonomic nerves have been studied in parotid glands of rats. Sympathetic stimulation evoked a slow flow of saliva which had a high concentration of amylase. After long term sympathetic stimulation secretory granules were heavily depleted from the parotid acinar cells. Parasympathetic stimulation evoked a copious flow of saliva with a low concentration of amylase. However, at high frequency stimulation the total amount of amylase secreted on parasympathetic stimulation was as great or even greater than on symphatetic stimulation, nevertheless, any loss of secretory granules from the acinar cells was very small. It is concluded that secretion of parotid acinar granules in the rat is prinicipally a sympathetic function. Secretion of fluid is more effectively produced by parasympathetic stimulation and much of the amylase in such saliva appears to have arisen from sources other than the secretory granules.     

Cytochemical studies of GERL and its role in secretory granule formation in exocrine cells

Synopsis the structure and cytochemistry of GERL was studied in several different exocrine secretory cells, including the exorbital lacrimal gland, parotid, lingual serous (von Ebner's), submandibular, and sublingual salivary glands, and exocrine pancreas of the rat; the lacrimal, parotid and pancreas of the guinea-pig; and the lacrimal gland of the monkey. GERL was morphologically and cytochemically similar in all cell types studied. It was located in the inner Golgi region and consisted of cisternal and tubular portions. Immature secretory granules were in continuity with GERL through multiple tubular connections. Modified cisternae of endoplasmic reticulum, with ribosomes only on one surface, closely paralleled parts of GERL. GERL and immature granules were intensely reactive for acid phosphatase activity, while the inner Golgi saccules were reactive for thiamine pyrophosphatase and nucleoside diphosphatase activities. In the rat exorbital lacrimal and parotid glands, reaction product for endogenous peroxidase, a secretory enzyme, was present in the endoplasmic reticulum, Golgi saccules, immature and mature secretory granules. GERL was usually free of reaction product or contained only a small amount. The widespread occurrence of GERL in secretory cells, and its intimate involvement with the formation of granules, suggest that it is an integral component of the secretory process.     

Immunocytochemical localization of amylase in the parotid gland of developing and adult rats

An antiserum against purified rat parotid amylase was used to localize the protein in parotid glands of developing and adult rats. The unlabeled antibody peroxidase-antiperoxidase method and the protein A-gold colloid technique were used at the light and electron microscope levels, respectively. Immunoreactive amylase was detected in a few scattered cells in the glands of 2-day-old rats. During the following days the number of cells stained immunocytochemically for amylase increased rapidly; at 15 days of age all acinar cells revealed amylase, but the intensity of immunostaining varied from cell to cell. Electron microscopically, amylase was localized in the secretory granules, and by using a more concentrated antiserum, in the rough endoplasmic reticulum and Golgi complex. At early stages of development the acinar cells contained fewer and smaller secretory granules than in adult animals; the gold particles indicative of amylase were randomly distributed over the secretory granules. In the glands of adult rats, amylase was distributed inhomogeneously within the secretory granules. In the majority of secretory granules gold colloid particles were located over the electron-dense portions of the granules. However, secretory granules in which an amylase-rich shell surrounded an amylase-poor or amylase-negative "core" were not infrequent.     

The influence of fixation on the carbohydrate cytochemistry of rat salivary gland secretory granules

Synopsis A series of studies was performed to assess the optimum fixation conditions for staining of carbohydrate-containing constituents of rat salivary gland secretory granules. In the parotid and submandibular salivary glands of the rat, the reactivity of secretory granules, at both the light and electron microscopic level, with routine stains and with cytochemical reagents was highly dependent upon the nature of the fixative employed. At the light microscopic level, secretory granules in rat parotid gland were periodic acid-Schiff (PAS) positive if fixed with buffered formalin fixatives. However, if the gland was fixed with lipid-solvent-containing fixatives, or with formalin at a very acid pH (as in Bouin's fixative), the PAS reactivity of the granules was lost. In the submandibular gland of rats, the acinar cells and granular tubules behaved similarly after such fixation in terms of their PAS reactivity, particularly in males; acinar cells of the female submandibular gland stained only lightly with PAS. At the fine structural level, the morphology of secretory granule constituents depended on the buffer used (cacodylate, phosphate or collidine) and on whether or not tissue was post-osmicated. Post-osmication considerably reduced the reaction of secretory granule components with stains for carbohydrates.The experimental evidence indicated that the carbohydrate-containing components of both parotid and submandibular gland secretory granules were not typical long-chain neutral or acidic mucins, but were rather glycolipids or lipophilic glycoproteins that were solubilized by lipid solvents or at acidic pH and were lost or destroyed in the presence of strong oxidants.     

Transferrin secretory pathways in rat parotid acinar cells

Transferrin is the major iron transporter in blood plasma, and is also found, at lower concentrations, in saliva. We studied the synthesis and secretion of transferrin in rat parotid acinar cells in order to elucidate its secretory pathways. Two sources were identified for transferrin in parotid acinar cells: synthesis by the cells (endogenous), and absorption from blood plasma (exogenous). Transferrin from both sources is secreted from the apical side of parotid acinar cells. Endogenous transferrin is transported to secretory granules. It is secreted from mature secretory granules upon stimulation with a β-adrenergic reagent and from smaller vesicles in the absence of stimulation. Exogenous transferrin is internalized from the basolateral side of parotid acinar cells, transported to the apical side by transcytosis, and secreted from the apical side. Secretory processes for exogenous transferrin include transport systems involving microfilaments and microtubules.     

Immunogold localization of the type II regulatory subunit of cyclic AMP-dependent protein kinase. Monoclonal antibody characterization and RII distribution in rat parotid cells

A mouse monoclonal antibody of the IgM class, MAb BB1, specific for the type II regulatory subunit (RII) of cyclic AMP-dependent protein kinase (cAPK), was produced using a purified subcellular protein fraction from rat parotid gland as the original antigen. The antibody immunoprecipitated radioactivity labeled RII from bovine heart cAPK, and from rat and human parotid saliva. Western blot analysis revealed specific binding of the antibody to proteins of 52 and 54 KD in extracts of rat parotid tissue, parotid saliva, and bovine heart cAPK. Immunogold labeling of thin sections of rat parotid gland revealed specific labeling of acinar cell nuclei (especially the heterochromatin), cytoplasm (particularly in areas containing granular endoplasmic reticulum), and the content of secretory granules. Labeling was greatly reduced (approximately 84%) when the antibody was pre-absorbed with an excess of bovine heart cAPK. In duct cells the cytoplasm and nuclei were also labeled, but few gold particles were present over secretory granules. These results provide additional evidence for the presence of nuclear cAPK in rat parotid cells, and confirm previous observations on the presence of cAPK regulatory subunits in acinar secretory granules and saliva. The hybridoma reagent will be used for studies of stimulus responses in the parotid and for immunocytochemical analyses of RII distribution in other secretory tissues.     

Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway

Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins. amylase; parotid secretory protein; polarized secretion     

Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells

The structure and cytochemistry of the Golgi apparatus and GERL of rat parotid acinar cells was studied after in vivo secretory stimulation with isoproterenol. Discharge of mature secretory granules was complete within 1 hr after isoproterenol injection, but immature granules in the Golgi region or near the lumen were not released. At early times (1-5 hr) after isoproterenol, acid phosphatase (AcPase) activity was markedly increased in GERL and immature secretory granules compared to uninjected controls. GERL appeared increased in extent and numerous continuities with immature granules were observed. Reaccumulation of mature secretory granules was first evident at 5 hr, and was almost complete by 16 hr after isoproterenol. Thiamine pyrophosphatase (TPPase) activity, normally restricted to the trans Golgi saccules, was frequently present in immature granules during this time. Narrow cisternae resembling GERL, occasionally in continuity with immature granules, also contained TPPase reaction product. By 16-24 hr after stimulation, the activity and distribution of AcPase and TPPase were similar to control cells. These results demonstrate the dynamic nature of the Golgi apparatus and GERL in parotid acinar cells, and emphasize the close structural and functional relationship between these two structures.     

Involvement of Aquaporin-5 Water Channel in Osmoregulation in Parotid Secretory Granules

Aquaporins (AQPs) are a family of channel proteins that allow water or very small solutes to pass, functioning in tissues where the rapid and regulated transport of fluid is necessary, such as the kidney, lung, and salivary glands. Aquaporin-5 (AQP5) has been demonstrated to localize on the luminal surface of the acinar cells of the salivary glands. In this paper, we investigated the expression and function of AQP5 in the secretory granules of the rat parotid gland. AQP5 was detected in the secretory granule membranes by immunoblot analysis. The immunoelectron microscopy experiments confirmed that AQP5 was to be found in the secretory granule membrane. Anti-AQP5 antibody evoked lysis of the secretory granules but anti-aquaporin-1 antibody did not and AQP1 was not detected in the secretory granule membranes by immunoblot analysis. When chloride ions were removed from the solution prepared for suspending secretory granules, the granule lysis induced by anti-AQP5 antibody was inhibited. Furthermore, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, an anion channel blocker, blocked the anti-AQP5 antibody-induced secretory granule lysis. These results suggest that AQP5 is, expressed in the parotid gland secretory granule membrane and is involved in osmoregulation in the secretory granules.     

Rab3D is not required for exocrine exocytosis but for maintenance of normally sized secretory granules

Rab3D, a member of the Rab3 subfamily of the Rab/ypt GTPases, is expressed on zymogen granules in the pancreas as well as on secretory vesicles in mast cells and in the parotid gland. To shed light on the function of Rab3D, we have generated Rab3D-deficient mice. These mice are viable and have no obvious phenotypic changes. Secretion of mast cells is normal as revealed by capacitance patch clamping. Furthermore, enzyme content and overall morphology are unchanged in pancreatic and parotid acinar cells of knockout mice. Both the exocrine pancreas and the parotid gland show normal release kinetics in response to secretagogue stimulation, suggesting that Rab3D is not involved in exocytosis. However, the size of secretory granules in both the exocrine pancreas and the parotid gland is significantly increased, with the volume being doubled. We conclude that Rab3D exerts its function during granule maturation, possibly by preventing homotypic fusion of secretory granules.     

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.     

Nerve-induced secretion of parotid acinar granules in cats

Summary Electron microscopy of cat parotid glands revealed great heterogeneity in the secretory granules of normal unstimulated acinar cells. Electrical stimulation of the parasympathetic nerve to the gland evoked a copious flow of parotid saliva which was accompanied by an extensive depletion of the secretory granules from the acinar cells. Exocytosis was captured as it was occurring by means of perfusion-fixation, and showed that the events occur in a conventional manner. Stimulation of the sympathetic nerve caused only a very small flow of saliva, and no acinar degranulation was detected. It can be concluded that the parasympathetic secretomotor axons provide the main drive for parotid acinar degranulation in the cat. This contrasts with the rat in which sympathetic impulses provide the main stimulus for parotid acinar degranulation. These dissimilarities serve to emphasise how extensively species differences may influence autonomic responses in salivary glands.     

Enzyme modulation of the Golgi apparatus and GERL: a cytochemical study of parotid acinar cells

The distribution of thiamine pyrophosphatase (TPPase) and acid phosphatase (AcPase) has been examined in resting parotid acinar cells as well as during decreased and increased secretory granule production. In resting acinar cells, TPPase activity was restricted to the trans Golgi saccules and AcPase activity was localized in GERL and immature secretory granules. Although secretory granule production is diminished during ethionine intoxication, no significant alteration in the distribution of either TPPase or AcPase was noted. However, marked changes in enzyme localization, especially of TPPase, occurred during accelerated secretory granule production. The alterations were essentially the same for all of the conditions studied (recovery from ethionine treatment, recovery from a protein depletion diet, secretory stimulation with isoproterenol, and postnatal maturation of the parotid gland). During maximal secretory granule production, TPPase activity was localized not only in the trans Golgi saccules, but also in GERL-like cisternae and immature secretory granules. The immature secretory granules were often in continuity with the GERL-like cisternae. At the same time that the TPPase activity was increased, the AcPase activity was frequently diminished. These modulations in enzyme activity provide evidence that GERL is derived from the trans Golgi saccule.