Apoptosis and mitosis of parenchymal cells in the duct-ligated rat submandibular gland

Apoptosis and proliferation of parenchymal cells during atrophy of rat submandibular gland induced by double duct ligation were investigated using immunohistochemistry for proliferating cell nuclear antigen (PCNA), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end labelling (TUNEL) and transmission electron microscopy (TEM). At 2 and 3 days after ligation, increased PCNA positive cells and mitoses were seen in ducts; thereafter PCNA positive cells decreased in number. At 3 and 4 days, the acinar cell population rapidly decreased, with many remaining TUNEL positive acinar cells. During this period, TEM showed typical apoptotic acinar cells that were phagocytosed by adjacent acinar cells or intraepithelial macrophages. After 7 days, most acinar cells had disappeared, leaving prominent residual ducts; a few acinar cells remained, especially at the lobule periphery. Submandibular gland duct ligation thus induced marked depletion of acinar cell by apoptosis and a concurrent short-lived cycle of duct cell proliferation.     

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.     

Tight junctions in the rat parotid gland

The aim of this study was to elucidate the distribution and morphological changes of tight junctions during secretion in parotid gland acinar cells. Localization of tight junction-associated polypeptide ZO-1, and of tight junction transmembrane protein Occludin, was examined in rat parotid gland by immunofluorescence and immunogold labelling of ultrathin sections. Adult male Sprague-Dawley rats were intraperitoneally injected with IPR and, after 10 and 30 minutes, parotid glands were extirpated. In control specimens, positive immunoreaction for ZO-1 and Occludin was observed on the adluminal side between adjacent cells in the form of narrow elongated profiles corresponding to intercellular canaliculi. After IPR injection, canaliculi became dilated and fluorescence was no longer seen as a continuous line but appeared as an aggregation of separate bright particles. ZO-1 was more widely distributed and was recognized in other areas of the cytoplasm as well. Concurrently, omega-shaped concavities, marked by actin fluorescence, appeared along the intercellular canaliculi. We concluded that, during exocytosis, the selective permeability barrier to the paracellular pathway, based on tight junctions, becomes more leaky, owing to segregation of Occludin caused by intracellular ZO-1 distributional changes associated with actin filaments.     

Quantitative immunocytochemistry of rat submandibular secretory proteins during chronic isoproterenol administration and recovery

We utilized quantitative electron microscopic immunogold labeling procedures to follow changes in the intragranular content of five secretory proteins of the rat submandibular gland (SMG) during and after chronic treatment with the beta-adrenergic agonist isoproterenol (IPR). Labeling intensities (gold particles/microns2) of acinar cell secretory granules for mucin and glutamine/glutamic acid-rich proteins, major secretory proteins of the normal SMG, showed opposite responses to IPR. Labeling intensities increased for mucin and decreased for glutamine/glutamic acid-rich proteins immediately after IPR injections began, then rapidly returned to control levels after cessation of IPR treatment. SMG Protein C immunoreactivity, found in both acinar and intercalated duct granules, was less affected by IPR. However, opposite changes in labeling intensity were observed between acinar and intercalated duct granules. Labeling intensities for proline-rich proteins, IPR-inducible secretory proteins, increased only after 10 days of stimulation and maintained a high level even after cessation of drug treatment. Type 2 cystatin, another IPR-inducible protein, increased gradually with chronic IPR treatment and decreased slowly during the recovery phase. These results suggest that chronic beta-adrenergic stimulation affects the expression of genes for several rat SMG secretory proteins in a different manner.     

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.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Roles of Munc18-3 in amylase release from rat parotid acinar cells

Several "soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor" (SNARE) proteins have been identified in rat parotid acinar cells, including VAMP-2, syntaxin 4, and SNAP-23. Furthermore, an association between Munc18c (Munc18-3) and syntaxin 4 has been reported. However, the role of Munc18-3 in secretory granule exocytosis on parotid acinar cells remains unclear. In the present study, we investigated the role of Munc18-3 in rat parotid acinar cells. Munc18-3 was localized on the apical plasma membrane where exocytosis occurs and interacted with syntaxin 4. Anti-Munc18-3 antibody dose-dependently decreased isoproterenol (IPR)-induced amylase release from SLO-permeabilized parotid acinar cells. Furthermore, stimulation of the acinar cells with IPR induced translocation of Munc18-3 from the plasma membrane to the cytosol. Munc-18-3 was not phosphorylated by a catalytic subunit of protein kinase (PK) A but phosphorylated by PKC. Treatment of the plasma membrane with PKC but not PKA induced displacement of Munc18-3 from the membrane. The results indicate that Munc18-3 regulates exocytosis in the acinar cells for IPR-induced amylase release and that phosphorylation of Munc18-3 by PKA is not involved in the mechanism.     

Light and electron microscopic immunolocalization of rat submandibular gland mucin glycoprotein and glutamine/glutamic acid-rich proteins

We studied the subcellular localization of two major secretory products of adult rat submandibular gland (RSMG), blood group A-reactive mucin glycoprotein and glutamine/glutamic acid-rich protein (GRP), by light and electron microscopic immunocytochemistry. The structure of the major neutral oligosaccharide of the mucin was shown to be: GalNAc alpha 1,3(Fuc alpha 1,2)Gal beta 1,3GalNAc. A mouse monoclonal antibody (1F9) with specificity for blood group A determinants was prepared against the mucin. The antibody recognized a single band of approximately 114 KD on Western blots of RSMG extract. A previously characterized monoclonal antibody (59) against GRP (Mirels et al.: J Biol Chem 262: 7289, 1987) reacted with a doublet of 45-50 KD on Western blots of extraparotid saliva. Immunofluorescence and immunoperoxidase staining of cryostat sections of RSMG with anti-mucin antibodies and anti-GRP antibodies revealed reactivity in acinar cells of the gland. No specific labeling was seen in duct cells of RSMG or in mucous acinar cells of the adjacent sublingual gland. Post-embedding immunogold labeling of thin sections of glutaraldehyde-fixed RSMG with anti-mucin showed strong labeling of the Golgi apparatus and secretory granules of acinar cells. Gold particles were seen mainly over electron-lucent areas of the granules. No labeling occurred over the endoplasmic reticulum. The labeling pattern with the anti-GRP antibodies was similar, except that both electron-dense and -lucent areas of the granules were labeled, and the endoplasmic reticulum was reactive. Double labeling with two different sizes of gold particles showed that both mucin and GRP co-localized in the same granules. Pre-absorption of the antibodies with their respective antigens eliminated immunolabeling of the acinar cells. These antibodies will be useful in studies of cell differentiation in RSMG and of synthesis, processing, and packaging of RSMG secretory products.     

Subcellular localization of epidermal growth factor in human parotid gland

The intracellular distribution of epidermal growth factor was investigated in human parotid gland by immunogold cytochemistry at the electron-microscopy level. Epidermal growth factor immunoreactivity was demonstrated in both acini and ducts. In acinar cells, secretory granules appeared moderately stained, clearly indicating that parotid gland contributes to salivary epidermal growth factor through granule exocytosis. In ductal cells, gold particles were found to decorate numerous cytoplasmic vesicles, particularly abundant in striated duct cells. Since epidermal growth factor reactive vesicles were seen not only at the cellular apex, but nearby lateral plasma membranes as well, it leads to the hypothesis that epidermal growth factor may be discharged both apically into the saliva, and basally into the interstitium.     

The presence of carbonic anhydrase in orbital glands. An enzyme-histochemical study in cynomolgus monkeys and rabbits

The distribution of carbonic anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbit. In the lacrimal gland of the cynomolgus monkey, a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.     

The presence of carbonic anhydrase in orbital glands

Summary The distribution of carbone anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbil. In the lacrimal gland of the cynomolgus monkey a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND : Temporal Dissociation of the Maximal Response from Secretion

Administration of the β-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [¹⁴C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [¹⁴C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.     

Secretory proteins as markers for cellular phenotypes in rat salivary glands

The neonatal submandibular glands (SMG) of the rat contain two types of cells: Type III cells secrete a group of proteins in response to beta-adrenergic stimulation, and Type I cells secrete a different protein, called Protein C (89 kDa), in response to cholinergic stimuli (Ball and Redman, 1984). Polyclonal antibodies raised to Protein B1 (26 kDa) showed that the several proteins in the B1-Immunoreactive Protein (B1-IP) group are localized exclusively to Type III cells. Although we expected that antibodies to Protein B1 would label only the submandibular gland, we found instead that the serous demilunes of the sublingual gland (SLG) and the acinar cells and intercalated ducts of the parotid gland (PRG) were strongly reactive in both the neonate and the adult. Immunoelectrophoretic analysis of gland extracts showed the major reactive species in the sublingual gland to have different mobilities than the B1-IP. On the other hand, reactive species in the parotid gland had mobilities identical to those of two SMG proteins. In the adult SMG, the neonatal Type I and Type III cells are not present, and the acinar cells are devoid of B1-IP reactivity; however, the cells of the intercalated ducts have components reactive with anti-B1 antibodies, and these do not appear to be identical to any neonatal bands. In contrast to the submandibular gland, the adult parotid and sublingual glands retain the localization of B1-IP reactivity in PRG acinar and intercalated duct cells and in SLG demilunes, and they show the neonatal immunoelectrophoretic pattern. This raises the possibility that the major B1-IP species in the adult PRG may be identical to transient proteins of the neonatal SMG.     

Morphological changes in the submandibular glands and in the X zone of the adrenal gland following ovariectomy in mice

Summary Morphological changes in submandibular glands of female mice following ovariectomy were studied morphometrically by light microscopy and ultrastructurally by electron microscopy. The X zone of the adrenal gland was examined in order to assess possible changes that might be expected to occur after ovariectomy.In submandibular glands, 1 to 4 weeks after ovariectomy, no changes were observed in percentages of the acinar, intercalated duct, and granular convoluted tubular areas occupying photomicrographs. However, an increase in the granular content of both intercalated duct and granular convoluted tubular cells was recognized. By contrast, the glandular picture 4 months after ovariectomy changed remarkably, resembling that of the male mouse both morphometrically and in terms of fine structure. In the adrenal cortex of control female mice, the X zone became thinner with aging. As compared with this, the X zone of ovariectomized mice at any time after the operation was thicker than that of controls.These observations suggest that the absence of ovarian hormones in the ovariectomized mouse may lead to prolonged functioning of X zone cells, which in turn may cause masculinization of the submandibular gland.     

Accumulation of Secretory Vesicles in the Lacrimal Gland Epithelia Is Related to Non-Sjögren's Type Dry Eye in Visual Display Terminal Users

Previous observations in a rat model of a non-Sjögren''s syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.     

Immunocytochemical localization of heparan sulphate proteoglycan in the rat submandibular gland

Summary Heparan sulphate proteoglycan is the predominant proteoglycan synthesized by the parenchymal cells of the rat submandibular gland. A polyclonal antibody was used to localize this proteoglycan in the adult rat submandibular gland. Localization was accomplished by indirect immunoperoxidase cytochemistry at the light and electron microscopic levels. Heparan sulphate proteoglycan was localized in a continuous, linear pattern in the lamina densa of the basement membrane surrounding all of the epithelial components of the gland as well as the basement membrane of the capillaries and small arterioles in the glandular stroma. In addition, heparan sulphate proteoglycan was seen in vesicles and pits along the acinar cell basal plasmalemma adjacent to the basement membrane and in the endoplasmic reticulum and Golgi apparatus of the acinar cells.     

Expression and induction by beta-adrenergic agonists of the cystatin S gene in submandibular glands of developing rats.

Repeated administration of the beta-adrenergic agonist isoprenaline (isoproterenol, IPR), which produces hypertrophic/hyperplastic enlargements of rat submandibular and parotid glands, induces synthesis of a secretory protein shown to be a cysteine proteinase inhibitor, rat cystatin S. In the current study, Northern blot and hybridizations in situ were carried out to establish the developmental and beta-adrenergic regulation of the expression of the cystatin S gene. Cystatin S mRNA was not detected in submandibular glands of 20-day-old fetuses, nor in the glands of newborn or 10-day-old rats. However, steady-state levels of cystatin S mRNA increased between 21 and 28 days, reaching a conspicuously high concentration at 28 days; cystatin S mRNA then declined rapidly to a barely detectable level in glands of 32-day-old rats. IPR administration for 4 days induced high levels of cystatin S mRNA in submandibular glands of developing and adult rats. In both prepubertal and mature animals, induction of cystatin S mRNA in submandibular glands was more pronounced in female than in male animals. Hybridizations in situ revealed cystatin S mRNA only in acinar but not in duct cells of the submandibular gland. Developmentally, expression of the cystatin S gene coincided with acinar cell differentiation. These data suggest a complex neural, hormonal and developmental regulation of salivary cystatin genes.     

Effect of methylcobalamine on the processes of posttraumatic regeneration of the salivary glands

Experiments on rats were made to study membrane potentials (MP) of secretory cells of the salivary glands, the content of biogenic amines and lactate dehydrogenase (LDH) isozymes of the salivary gland tissue in trauma after pretreatment with methylcobalamine. Twenty-four hours after trauma the salivary gland showed a decrease in the content of LDH aerobic fractions, the lowering of noradrenaline concentration with no changes in the MP of glandular cells outside the zone of injury. Administration of cobalamine did not cause any changes in the parameters under study. There was an increase in the polarization level of acinar and duct cells, normalization of noradrenaline content, and a rise of adrenalin concentration with persistent reduction in aerobic fractions of LDH in salivary gland trauma after pretreatment with methylcobalamine. It is concluded that methylcobalamine administration may have a therapeutic effect in salivary gland trauma.