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.     

Subcellular localization of blood group substances ABH in human salivary glands

We investigated the subcellular localization of ABH antigens in human submandibular, sublingual, and buccal glands by applying a post-embedding immunogold method using monoclonal antibodies specific for A, B, and H antigens. In most glands the immunoreactivity was usually restricted to mucous cells, in which only secretory granules and sometimes Golgi cisternae were specifically labeled. A and B antigens were demonstrated only in the glands of type A, B, and AB subjects, while H antigen was visualized in glands from individuals of all blood types. Moreover, differences were observed in the relative distribution of ABH antigens, depending on the type of gland.     

Adrenergic and cholinergic nerves of bovine,guinea pig and hamster salivary glands

Summary The distribution of formaldehyde-induced fluorescence and acetylcholine-esterase (AChE) activity was histochemically investigated in certain salivary glands of the cow (submandibular gland), guinea pig and hamster (submandibular and sublingual glands). Adrenergic nerves occur around the secretory acini of the bovine, guinea pig and hamster submandibular glands, as well as around those of the hamster sublingual gland. The mucous secretory acini of the guinea pig sublingual gland, however, seem to be devoid of adrenergic nerve supply. Except in the sublingual gland of the hamster, no adrenergic nerves occur in relation to duct cells.The pattern of AChE activity is similar to that of adrenergic nerves. Thus, AChE-positive nerves form a network around secretory acini of all the five glands examined. Furthermore, AChE activity was also observed in nerve fibres in close proximity to striated duct cells.Both adrenergic and AChE-containing fibres were observed around blood vessels of different sizes. Ganglionic cells are occasionally to be seen; they all display AChE-activity. No adrenergic ganglionic cells were observed in any of the glands examined.All glands were also studied in the electron microscope. Interest was focussed on the fine structure of the autonomic nerves with special reference to their contents and type of storage vesicles.The content of noradrenaline was chemically determined in each type of salivary gland studied.This work was supported by grants from the University of Umeå and from the Swedish Society for Medical Research and was also carried out within a research organization supported by the Swedish Medical Research Council (projects B73-04X-712-08C and B73-04X-56-09C). The authors are indebted to Miss Kristina Karlsson and Miss Marianne Borg for valuable technical assistance.     

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.     

Nerve growth factor-like immunoreactivities in rodent salivary glands and testis

Summary A series of polyclonal affinity-purified antibodies against mouse submandibular-gland nerve growth factor (NGF) are described. Using the submandibular gland of the male mouse and indirect immunofluorescence, the specificity and sensitivity of affinity-purified immunoglobulins and various other fractions from the immunized animals have been tested. It will be shown that affinity-purification schemes, including pre-purification of protein A-fractionated immunoglobulins to remove antibodies that bind to unrelated hydrophilic and hydrophobic proteins, significantly enhance the signal-to-noise ratio and specificity of the antibodies. The antibodies effectively detect NGF-like immunoreactivity in both fresh and fixed glandular tissue. Optimal fixation procedures are described. Fluorescence intensities are linearly correlated to log antibody concentration. By use of the best antibody fractions and optimal fixation protocols, the distribution of NGF-like immunoreactivity is described in eight different salivary glands (rat and mouse, male and female, submandibular and sublingual glands). In addition to the well-known large numbers of immunoreactive cells in the submandibular gland of the male mouse, immunoreactive cells were found in the sublingual gland of male mice and in the submandibular and sublingual glands of female mice. One antibody revealed a weak specific fluorescence also in the submandibular gland of the male mouse. In a survey of genital organs of male mice, one antibody revealed fluorescence in the germ cell line. We conclude that several polyclonal affinity-purified antibodies have been characterized that show a strong NGF-dependent binding to the secretory granules of tubular cells in the submandibular gland of male mice. These antibodies should make it possible to locate endogenous and perturbed NGF levels immunocytochemically, e.g., in the peripheral and central nervous system, where NGF concentrations may be several orders of magnitude lower than in the salivary glands.     

Localization of low molecular weight protease inhibitor in serous secretory cells of the respiratory tract

We prepared in rabbits an antiserum against low molecular weight protease inhibitor (LMI) purified from the sputum of patients with purulent bronchitis. Using this antiserum in an immunoperoxidase staining method we found that this inhibitor was located exclusively in the serous cells of the submucosal glands of human upper and lower airways. The inhibitor was localized also in serous cells of the sublingual and submandibular glands. In contrast, LMI could not be demonstrated in the serous cells of the parotid gland. In the tissues investigated a strong association between the localization of the protease inhibitor and lysozyme was observed. Our observations indicate that the inhibitor may be present together with lysozyme as a secretory product in the serous cell granules. The possible consequences of the coexistence of these two proteins in the defense mechanism of the respiratory tract is discussed.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Autoradiographic localization of transported neutral amino acids in epithelia of cat submandibular and sublingual salivary glands

Summary Light-microscopic autoradiography was used to localize the cellular sites for neutral amino acid uptake in submandibular and sublingual salivary gland epithelia. The vasculature of isolated glands was perfused for 3–5 min with either L-(3-³H)serine or L-(4-³H)phenylalanine and then fixed by perfusion with buffered glutaraldehyde. In the submandibular gland the small neutral amino acid L-serine and the aromatic amino acid L-phenylalanine were localized to central acinar cells, demilunar cells and ductal cells. In the sublingual gland silver grains associated with each of these tritiated amino acids were localized to central acinar and ductal cells. Perfusion of both submandibular and sublingual glands with unlabelled L-serine (25 mM) or L-phenylalanine (30 mM) resulted in a significant decrease in the silver grain density associated with each labelled amino acid. The absence of silver grains in the lumina of acinar and ductal cells and the presence of tight junctions near the apical surface of the epithelium strongly suggest that the initial uptake of these amino acids was mediated by basolateral plasma membrane carriers.     

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

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

Nitric oxide synthase immunoreactive nerves in rat and ferret salivary glands, and effects of denervation

Nitric oxide has been implicated in mechanisms mediating nerve-evoked vasodilatory and secretory responses in salivary glands. In the present study, the occurrence and distribution of nitric oxide synthase (NOS)-immunoreactive nerves in ferret and rat salivary glands were investigated using immunocytochemistry with rabbit and sheep NOS antisera, and using NADPH-diaphorase enzyme histochemistry. In the parotid, submandibular and sublingual glands of the rat and the ferret, NOS-immunoreactive varicose terminals encircled acini and arteries of various sizes. In the ferret, collecting ducts were also supplied with NOS-immunoreactive fibres. In the rat, only the granular ducts of the submandibular gland were supplied with such fibres. The NOS-immunoreactive innervation of acinar cells was more abundant in the rat than in the ferret, whereas the opposite was true for the innervation of blood vessels. No NOS immunoreactivity was observed in the vascular endothelium. In both species, NOS-positive ganglionic cell bodies were found in the hilar regions of the submandibular and sublingual glands, whereas none could be detected in the parotid glands. NADPH-diaphorase reactivity had the same neuronal distribution as NOS immunoreactivity and, in addition, NADPH-diaphorase reactivity was expressed in ductal epithelium. Neither sympathetic denervation (by removal of the superior cervical ganglion) nor treatment with the sensory neurotoxin capsaicin reduced the NOS-immunoreactive innervation of the parotid gland. However, parasympathetic denervation (by cutting the auriculo-temporal nerve) caused an almost total disappearance of the NOS-immunoreactive innervation. The present findings provide a morphological background to the suggested role of nitric oxide in parasympathetic secretory and vascular responses of salivary glands. This revised version was published online in November 2006 with corrections to the Cover Date.     

Immunofluorescence-microscopic demonstration of myosin and actin in salivary glands and exocrine pancreas of the rat

Summary Actin and myosin were localized in various salivary glands (parotid, submandibular, sublingual, lingual and Harderian gland) and the exocrine pancreas of rats by indirect immunofluorescence microscopy using specific rabbit antibodies against chicken gizzard myosin and actin. A bright immunofluorescent staining with both antibodies was observed at three main sites: (1) In myoepithelial cells of all salivary glands, (2) in secretory gland cells underneath the cell membrane bordering the acinar lumen (except Harderian and mucous lingual gland), and (3) in epithelial cells of the various secretory ducts (of all glands) in similar distribution as in acinar cells. The present immunohistochemical findings in acinar cells could lend further support to a concept suggesting that myosin and actin are involved in the process of transport and exocytosis of secretory granules.Supported by grants form Deutsche Forschungsgemeinschaft (Dr. 91/1, Ste. 105/19 and U. 34/4). We thank Mrs. Ursula König, Mrs. Christine Mahlmeister and Miss Renate Steffens for excellent technical assistance.     

Histochemical studies on irradiated and obstructive human submandibular glands. Protein distribution and lectin-binding in the degenerative glands.

Immunohistochemical protein distribution of alpha-amylase (Am), lysozyme (Ly), cytokeratinin (CK), S-100 protein (S-100) and secretory component (SC), and lectin-binding (SBA and UEA-I) profiles were studied in 10 obstructive and 20 irradiated human submandibular glands which were surgically extirpated. Degenerative intensity of the glands was graded as I, II and III based on the order of severity. All proteins generally existed in serous acinic cells of the intact glands. The proteins immunoreactivities became weak even in mildly inflamed glands (grade I), and nearly disappeared from the moderately damaged glands (grade II). Duct cells had clear CK and some cells reacted with the anti-SC antibody, but other proteins were not observed on the ducts. Mucous cells possessed none of the proteins, and their lectin-binding was only traceable in some glands. Compared with immunoreactivities in the proteins, lectin-binding profiles were different. SBA and UEA-I bound somewhat similarly to both acinic and duct cells, and the binding was hardly affected even by severe degeneration (grade III). Between obstructive and irradiated glands, no obvious difference was observed in either protein distribution or lectin-binding. From the above, it seems that some proteins are more affective to the degeneration and that lectin-binding sugar residues are non-affective against the degenerative changes of the tissues.     

Comparative histochemical study of glycoconjugates of the major salivary glands and pancreas in humans

The human parotid, submandibular, sublingual salivary glands and pancreas have been studied with lectin--horseradish peroxidase conjugates (con A, PNA, SBA, WGA, LAL), aldehyde fuchsin and Bismark brown. Intercalated duct cells produce a specific aldehyde-fuchsin-reactive substance. These cells are found only in the submandibular and parotid, but not in the sublingual glands. Similar reactivity is found in B-insulocytes of the pancreas. Aldehyde-fuchsin marks cytoplasmic granularity of the striated duct cells of all large salivary glands. This specific granularity is also selectively stained with Bismark brown and con A. Using fucose-specific lectin from Laburum anagyroides bark (LAL), granularity in serocytes of the submandibular gland is demonstrated. Some individual variations are observed in PNA binding to serocytes of the submandibular gland. It reveals that thyroglobulin-peroxidase conjugate (previously reported as an available second-step reagent for indirect lectin histochemical methods) non-specifically binds to the striated duct cells of the submandibular gland. During control staining it is also found, that DAB-reaction for endogenous peroxidase can be used as a test-system for a selective histochemical exposure of nuclear regions of endotheliocytes, pericytes and striated duct epitheliocytes of the human salivary glands. Possible significance of the phenomena observed is discussed.     

Basic fibroblast growth factor in rat salivary glands

We studied the occurrence and localization of basic fibroblast growth factor (bFGF) in rat salivary glands using a specific monoclonal antibody. It was shown that the extract of rat salivary glands has a pronounced stimulatory activity on the growth of bovine capillary endothelial cells, which is blocked by the addition of an antibody against bFGF. The concentration of bFGF in the submandibular/sublingual gland, as determined by radioimmunoassay, was 80% that in the brain. Immunocytochemistry revealed bFGF-immunoreactivity localized primarily in the epithelial cells lining the striated ducts and excretory ducts of the parotid, sublingual and submandibular glands. In addition, intense bFGF-immunoreactivity was observed in the granular convoluted tubule of the submandibular gland, localized predominantly in the agranular pillar cells, which lay in small numbers among the majority of weakly immunostained cells containing many apical secretory granules. At the electron-microscopic level, the immunoreactive material was distributed diffusely in the cytoplasmic matrix and nuclei of all immunoreactive cells, whereas it was absent from all cytoplasmic organelles including the secretory granules. These results indicate that bFGF is localized in different cellular and subcellular compartments from those of other growth factors in the duct system of rat salivary glands.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

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.     

Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation

Aquaporin-5 (AQP5) is a water channel protein and is considered to play an important role in water movement across the plasma membrane. We raised anti-AQP5 antibody and examined the localization of AQP5 protein in rat salivary and lacrimal glands by immunofluorescence microscopy. AQP5 was found in secretory acinar cells of submandibular, parotid, and sublingual glands, where it was restricted to apical membranes including intercellular secretory canaliculi. In the submandibular gland, abundant AQP5 was also found additionally at the apical membrane of intercalated duct cells. Upon stimulation by isoproterenol, apical staining for AQP5 in parotid acinar cells tended to appear as clusters of dots. These results suggest that AQP5 is one of the candidate molecules responsible for the water movement in the salivary glands.     

Immunohistochemical localization of S100A1 and S100A6 in postnatally developing salivary glands of rats

 S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0–5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands. Accepted: 14 July 1998     

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.