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.     

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.     

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.     

An Unusual Sexually Dimorphic Mosaic Distribution of a Subset of Kallikreins in the Granular Convoluted Tubule of the Mouse Submandibular Gland Detected by an Antibody with Restricted Immunoreactivity

The granular convoluted tubule of the mouse submandibular gland contains a wide variety of biologically active proteins, including several kallikreins. The tubule is under multihormonal regulation, and is sexually dimorphic, being larger in males than in females. Correspondingly, levels of its various protein secretory products are more abundant in males than in females. However, isoelectric focussing studies show that the true tissue kallikrein, mK1, is more abundant in the female than in the male submandibular gland. In this study, an antiserum was prepared with restricted immunoreactivity for mouse mK1, and possibly other kallikrein family members of low abundance in the mouse submandibular gland, and used for the immunocytochemical staining of the granular convoluted tubule cells in the submandibular gland of adult male and female mice, by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. The distribution of immunoreactive tubule cells showed an unusual sexual dimorphism. In males only a few scattered slender tubule cells were strongly stained, while the more typical large tubule cells were only occasionally weakly positive, and many of them were not stained. By contrast, in females slender tubule cells were not seen, and about two thirds of the more typical tubule cells showed moderate to strong immunostaining. Immunoelectron microscopy revealed that immunostaining was confined to the secretion granules in granular convoluted tubule cells in both sexes. The slender tubule cells of males had many strongly stained small apical secretion granules and occasional basal infoldings; in the weakly positive larger more typical tubule cells not all secretion granules were positive, and there was intergranular variation in the intensity of staining of positive granules. In females, although more tubule cells were stained, intergranular variations in staining intensity were also noted. In both sexes, many tubule cells did not contain any secretion granules that showed immunogold labeling for kallikreins. These findings establish that, in contrast to the situation for the majority of granular convoluted tubules proteins, mK1 and possibly other minor kallikrein family members are more abundant in the granular convoluted tubules of female mice, and that there is considerable variation in the content of these kallikreins not only between different tubule cells, but also in individual secretion granules in any given tubule cell in either sex.     

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.     

Histochemical and biochemical determination of calcium in salivary glands of cat

Although feline salivary glands have been used in investigations on secretion and microlithiasis and both processes involve calcium, nothing is known about its distribution in these glands. Therefore we have demonstrated the presence of calcium by a histochemical technique using glyoxal bis(2-hydroxyanil) and a biochemical technique using dry ashing. The histochemical technique stained serous acinar cells weakly and rarely found mucous acinar cells strongly in the parotid gland, mucous acinar cells moderately to strongly and serous acinar cells weakly in the sublingual gland, and central and demilunar acinar cells moderately to strongly in the submandibular gland. The biochemical technique revealed less calcium in the parotid than in the submandibular and sublingual glands. Both techniques revealed a decrease of calcium in submandibular and sublingual glands following parasympathetic stimulation. The histochemical distribution of calcium, which corresponds to that of acinar secretory glycoprotein, and the loss of calcium following parasympathetic stimulation, which causes release of secretory granules, indicate the presence of calcium in secretory granules. The concentration of calcium in the different types of acinar cell corresponds to the acidity of the secretory glycoprotein and suggests that calcium is present as a cationic shield to allow the condensation of polyionic glycoprotein in secretory granules.     

Histochemistry of hydrolytic enzymes in resting submandibular glands of rabbits

Summary The distribution of several hydrolytic enzymes was investigated in rabbit submandibular glands at both the light and electron microscopical levels. Glands were fixed by either immersion or perfusion fixation with a variety of fixatives containing 1–2% glutaraldehyde and 2–4% formaldehyde in 0.1m cacodylate buffer at pH 7.2. Light microscopically, the acinar cells showed some staining for ATPase, acid phosphatase and nonspecific esterases but showed weak staining for thiamine pyrophosphatase. Acid phosphatase staining occurred most strongly in granular tubule cells. Staining for esteroproteases was confined to the periluminal rims of intercalary and striated ducts. Alkaline phosphatase was very sensitive to glutaraldehyde and was confined to myoepithelial cells.Electron microscopical observations revealed the presence of acid phosphatase reaction product in lysosomes, immature granules and in GERL-like structures, the last being much more conspicuous in the granular tubule cells. ATPase reaction product was localized to the basal and luminal plasma membranes and lumina of both acinar and granular tubule cells. The Golgi complex of these two types of cells exhibited only moderate amounts of reaction product for thiamine pyrophosphatase. Alkaline phosphatase activity, on the other hand, was exclusively localized to myoepithelial cells in their plasma membranes and sometimes in the nuclear envelope.     

Cellular compartmentation of lysozyme and alpha-amylase in the mouse salivary glands. Immunogold approaches at light and electron microscopy level

The research was planned to study the subcellular distribution of enzymatic secretory products within the secretory structures of the mouse major salivary glands at light and electron microscopy level by immunogold silver stain (IGSS) technique and double-sided post-embedding immunogold binding and silver amplification in order to speculate about their compartmentation. In particular, we experimented the above immunogold labeling approaches to localize the lysozyme and to verify its distribution patterns in relation to another secretion enzyme, alpha-amylase. Co-presence of lysozyme and alpha-amylase was observed in the convoluted granular tubule cells of the submandibular gland and in the demilunar cells of the sublingual gland as well as in the electron-dense regions of the mottled secretory granules in the parotid gland. Exclusive binding patterns of lysozyme were observed in the acinar cells of the submandibular and sublingual glands where alpha-amylase did not occur.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Immunocytochemical localization of a developmentally regulated, isoproterenol-inducible protein (LM protein) in rat submandibular gland

Administration of the beta-adrenergic drug isoproterenol (IPR) produces hyperplastic and hypertrophic enlargements of the submandibular gland of the rat and induces the synthesis of specific proteins in this organ. One of these proteins, the LM (large mobile) protein, was demonstrated immunocytochemically in the submandibular glands of developing untreated and IPR-treated rats. Immunoreactive LM protein was absent in the glands of 20-day-old fetuses and 1- and 2-day-old rats. It was localized in the proacinar and immature acinar cells in the glands of 6- to 21-day-old animals, but it was undetectable at 28 days of age. In the glands of adult rats, secretory granules of the granular convoluted tubule cells showed immunostaining for the LM protein which was also present in trace amounts in the acinar cells. Daily administration of IPR for 5 days to newborn or 8- or 15-day-old rats caused an apparent acceleration of proacinar/acinar cell differentiation, and consequently it increased the frequency of cells immunostained for the LM protein as well as the amount of immunoreactive material in these cells. Thus, the expression of LM protein in the submandibular gland is developmentally regulated, and it is restricted to the stage of differentiation of proacinar cells from terminal tubule cells. IPR is capable of inducing this protein in fully differentiated acinar cells in 3-week-old or older animals.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

A comparative histochemical study of peroxidase activity in the submandibular glands of five mammalian species including man

Summary A light microscopic histochemical investigation of endogenous peroxidase activity in specimens of the submandibular salivary glands of man, hamster, rabbit, dog and guinea pig was carried out. A modification of the original Graham and Karnovsky diaminobenzidine (DAB)-hydrogen peroxide method was employed at different pH's.At all pH's (6.0, 7.6, and 9.0) a positive DAB reaction was found: in serous acinar cells in four of seven human submandibular glands, in convoluted tubule cells of the hamster, in acinar tissue, in secretory granular tubule cells and in the saliva of the guinea pig. This staining pattern was not markedly affected by KCN or 2,4-dichlorophenol (DCP). Furthermore, small cytoplasmic granules in collecting ducts of the dog displayed positive, KCN- and DCP-resistant DAB staining at all pH's tested. No reaction was observed in the acinar cells of the dog and rabbit glands.Mitochondrial oxidation of DAB in the striated duct cells occurred in all of the glands examined. Optimal staining of these cells was obtained at pH 6.0, but there was also strong positive staining at pH 7.6. At pH 9.0, however, the staining of the striated duct cells was very faint. The positive reaction in the striated duct cells was completely abolished by KCN.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

Sex-related expression of sialic acid acceptor sugars in the mouse submandibular gland. Simultaneous visualization by confocal laser scanning microscopy.

The novel combination of sialidase digestion with simultaneous PNA and DBA binding yielded marked differences on sialoglycoconjugate occurrence and distribution in the mouse submandibular gland acinar cells of the two sexes. Striking differences in the structure of terminal disaccharides within stored secretory sialoglycoconjugates were also found. High content of sialic acid, characterized by the terminal sequence sialic acid-alpha-N-acetylgalactosamine, was established to only occur in the male acini where secretory cells appeared to be differently stained; indeed, some cells exhibited codistribution of sialic acid-alpha-N-acetylgalactosamine and sialic acid-beta-galactose terminal disaccharides, whereas other ones exclusively contained one of the two kinds of terminal sequences. In the female acinar cells, the secretory products were found to be almost exclusively composed by glycoconjugates having sialic acid subtended to beta-galactose without appreciable differences between acinar cells. Our finding of such extensive differences in the acinar cells of male and female mice adds new insights into the submandibular gland sexual dimorphism, commonly attributed to the androgen responsiveness of the granular convoluted tubule portion of the gland.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

Transient appearance of substance P-like immunoreactivity in the granular convoluted tubules of the male mouse submandibular gland: light- and electron-microscopic studies

The time of appearance and distribution of substance P (SP)-like immunoreactivity in the granular convoluted tubule cells of the developing male mouse submandibular glands were examined, and the subcellular localization of SP-like immunoreactivity was investiagted by electron microscopy. At 25 days of age, SP-like immunoreactivity was first detected in the supranuclear cytoplasm of the granular convoluted tubule cells, which occurred either singly or in small clusters. At 30 and 35 days of age, granular convoluted tubule cells with SP-like immunoreactivity were more numerous than in the earlier stages, as the volume ratio of the cells increased. Not all granular convoluted tubule cells demonstrated SP-like immunoreactivity. The number of cells with SP-like immunoreactivity decreased at 60 days of age, and these cells had completely disappeared at 90 days of age. Most, but not all, secretory granules in the granular convoluted tubule cells were strongly labeled with gold particles, indicating that the subcellular site of SP-like substance is in the secretory granules within the cells. The findings suggest that the physiological role of the SP-like substance secreted from the GCT cells is restricted to the early postnatal stages, and that it may be involved in the development of the oral mucosa or digestive tract as a trophic factor.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.