Tyrosylprotein sulfotransferase in rat submandibular salivary glands.

1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu6, Ala3, Tyr1) (EAY; Mr 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2 mM 5'AMP, 20 mM MnCl2 and 50 mM NaF at pH 6.2. 2. The apparent Km values for EAY and PAPS were 1.6 x 10(-6) and 1.9 x 10(-6) M, respectively. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.     

Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat

Abstract. Although the rat salivary glands are deficient in acini at birth, acinar cells proliferate rapidly during the early post-natal period. The pattern of acinar cell proliferation was analysed in the parotid and submandibular glands of neonatal rats from day of birth until day 34. Mitotic and [³H]thymidine ([³H]TdR) labelling indices of the two glands show distinctly different patterns. Analysis of cell division in the rat parotid gland demonstrated a peak of mitotic index at 14 days (2.9 ± 0.4%) and labelling index at 16 days (25.2 ± 2.1%). Submandibular gland acinar cell proliferation reaches a zenith between 7–8 days; labelling index (14.2 ± 1.1%) and mitotic index (2.3 ± 0.3%). Cell proliferation decreases rapidly in both glands after reaching a peak in activity. Gland size increases more rapidly in the submandibular gland which correlates with the earlier shift from cell proliferation to differentiation which occurs in this organ. Circadian rhythms of [³H]TdR incorporation were also investigated in this study. A circadian rhythm of [³H]TdR incorporation into DNA occurs at 15 days after birth with a peak at 06.00 hours in both glands and a trough occurring at 15.00 hours in parotid gland and 18.00 hours in the submandibular gland. Determination of specific activity of DNA (ct/min per μg DNA) on days 8, 10, 12, 13, 14, 15, and 16 after birth at 06.00 and 15.00 hours indicated that a circadian rhythm in [³H]TdR incorporation into DNA began on day 14. The developmental switch from suckling to solid food may be an initiating factor in the sychronization of the circadian rhythm in cell proliferation.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.     

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.     

Androgen receptor in rat Harderian and submandibular glands

Summary Androgens regulate the development and sexual dimorphism of rodent Harderian and submandibular glands. This effect is believed to be mediated by the androgen receptor. Immunohistochemistry and immunoblotting were carried out to study the receptor in normal, castrated and dihydrotestosterone-supplemented rat Harderian and submandibular glands. Immunohistochemically, the most intense nuclear staining was observed in the acinar cells of the submandibular glands, followed by intercalated duct cells. The granular convoluted tubules showed weak immunostaining and the striated ducts were negative. In the Harderian gland, nuclear staining was seen in both type I and II secretory cells. Castration and treatment had no effect on the expression of the androgen receptor protein in either gland. A 110 K androgen receptor signal was detected by immunoblotting in the Harderian gland but not in the submandibular gland. An experiment was designed to explore the possible effect of proteinases on the receptor protein in the homogenate of submandibular gland. Our results demonstrate the cell-specific location of the receptor in Harderian and submandibular glands, and show that the expression of the receptor protein is androgen-independent.     

Secretion of saliva in X-irradiated rat submandibular glands

The mechanism of radiation-induced dysfunction in rat submandibular glands was investigated at the cellular level. After X irradiation (single dose, 15 Gy), a vacuolation in the acinar cells or an enlargement of the acinar lumen was observed as a typical morphological change for 2 weeks. As observed using a video-enhanced contrast differential interference contrast (VEC-DIC) microscope, exocytosis and shrinkage of the acinar cells induced by application of pilocarpine (100 microM) were markedly suppressed for 5 days and then recovered to 80% of the control levels. Using an immunohistochemical method, no significant change was observed in amylase distribution, but a marked loss of aquaporin 5 was found in the acinar cells after the irradiation. The extent and time course of pilocarpine-induced mobilization of intracellular Ca(2+) did not change after the irradiation. We conclude that radiation-induced dysfunction in the salivary glands is due to an impairment of exocytosis and a reduction of water secretion. The loss of aquaporin 5 and possibly other membrane-fusion proteins in acinar cells may be the major mechanism underlying such a dysfunction.     

Induction of cystatin S in rat submandibular glands by papain.

1. Papain (a cysteine proteinase) were administered into the oral cavity of rats twice daily for 5 days. This treatment caused a dramatic increase in the level of cystatin S (a cysteine proteinase inhibitor belonging to family 2 of cystatin superfamily) in enlarged submandibular glands. 2. Immunochemical analysis using antibody against rat cystatin S and electrophoretic analysis confirmed that the protein induced by papain was identical to that induced by isoproterenol. 3. Induction of the cystatin S in the submandibular glands by oral administration of papain suggested a biological response which plays a role in preventing injury exogenous proteinase.     

Differentiation of the myoepithelial cells of the rat submandibular gland in vivo and in vitro: an ultrastructural study

Cytodifferentiation of the myoepithelial cells (MEC) of the rat submandibular gland (SMG) was observed by studying the prenatal and postnatal development of the gland in vivo and in vitro by light and electron microscopy. The anlage of the SMG first appeared on the fourteenth day of gestation and, from its earliest inception, was surrounded by an intact basal lamina. Presumptive myoepithelial cells were first seen at 18 days of gestation coinciding with the onset of secretion in the rudiment. These cells were flattened, peripherally located and subjacent to the epithelial basal lamina. Initial deposition of cytofilaments in the MEC's was observed during the first three days following birth and fully matured cells were seen as early as one week after birth. Presumptive and immature MEC's were observed undergoing mitosis, but once cytofilament deposition had begun in the cells they did not divide. Myoepithelium developed in relation to embryonic secretory structures and were only observed surounding acini and intercalated ducts in the adult gland. New myoepithelial cells were formed as long as new acinar-intercalated duct units were formed. Myoepithelial cells did not produce secretory type granules at any time during their development or in their mature state. Development of the MEC's in vitro paralleled that in vivo and supported the above observations.     

Purification and characterization of kallikrein-like serine proteases from rat submandibular glands.

The purification and characterization of kallikrein-like proteases from rat submandibular glands is described. The proteolytic activity of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME). The purification scheme involved ammonium sulfate precipitation, chromatography on columns of DEAE-Sepharose and Sephadex G-100 and chromatofocusing. Three TAME-hydrolytic activity peaks were eluted from DEAE-Sepharose as unbound fraction (Pool 1), at 125 mM NaCl (Pool 2) and at 250 mM NaCl concentration (Pool 4). Pool 1 further resolved into two protease fractions (1A1 and 1A2), pool 2 into three protease fractions (2A1, 2A2 and 2A3) and pool 4 gave a single major protease peak (4A1) by chromatofocusing on PBE-94. Protease pools 2A2, 2A3, and 4A1 each gave a single band on SDS-polyacrylamide gel electrophoresis with an estimated molecular weight of 34 kDa, 46 kDa and 46 kDa respectively. Pools 1A1, 1A2, 2A1 and 2a2 gave a single precipitin line with anti-rat glandular kallikrein antibodies. 2A3 and 4A1 did not react with these antibodies. Synthetic substrates DL-val-leu-arg-pNA and Bz-pro-phe-arg-pNA, specific for kallikrein-like proteases, were hydrolyzed preferentially by 2A3 and 4A1 but were poor substrates for 1A1, 1A2, 2A1 and 2A2.     

Isoprenaline-induced changes in rat parotid and submandibular glands are age- and dosage-dependent.

Neonatal rats treated with chronic injections of isoprenaline (isoproterenol) for 10 days revealed differential induction of proline-rich proteins and glycoprotein synthesis between the parotid and submandibular glands. Biosynthesis of proline-rich proteins (Mr 17000-35000) and a Mr-220000 glycoprotein were detectable by solubilization in 10%-trichloroacetic acid extracts from parotid glands 14 days after birth. The enzyme lactose synthase (UDP-galactose: 2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase) (EC 2.4.1.22) is also induced 4-7-fold in specific activity compared with control neonatal rats, but again only after 14 days post partum, with isoprenaline treatment. This is in accord with the ability of the parotid gland to respond to beta-receptor stimulation and subsequent increases in intracellular cyclic AMP necessary for induction of protein synthesis [Grand, Chong & Ryan (1975) Am. J. Physiol. 228, 608-612]. Induction of the proline-rich proteins and a Mr-190000 glycoprotein in the soluble fraction from the submandibular gland were not detected until 49 days after birth under identical conditions in the same animal. Cyclic AMP in the submandibular gland undergoes increases on beta-receptor stimulation similar to those achieved in the adult animal, 1 day after birth (Grand et al., 1975). This same differential induction between parotid and submandibular gland was obtained with a range of isoprenaline dosages in adult animals. Trichloroacetic acid-soluble proline-rich proteins were isolated from parotid glands at a dosage of 4.0 mg of isoprenaline/kg body wt., but 7.0 mg/kg was required to induce also biosynthesis of these proteins in the submandibular gland. Gland hypertrophy showed the same differential dosage kinetics, based on gland weight, between the two glands; however, hypertrophy could be accomplished at a lower dosage of isoprenaline than that used to induce proline-rich-protein biosynthesis.     

Label-retaining cells in the rat submandibular gland.

To identify stem cells in salivary glands, label-retaining cells (LRCs) were established in rat submandibular glands. Developing and regenerating glands were labeled with bromodeoxyuridine (BrdU). To cause gland regeneration, the glands were injured by duct obstruction. BrdU LRCs were observed in all the parenchymal structures except for the acinus of the glands labeled during regeneration. Among these LRCs, a few, but not many, expressed neither keratin18 (K18; an acinar/duct cell marker) nor alpha-smooth muscle actin (alphaSMA; a myoepithelial cell marker), and thus were putative stem cells. These (K18 and alphaSMA)(neg) LRCs were invariably observed in the intercalated duct and the excretory duct. In the intercalated duct, they were at the proximal end bordering the acinus (the neck of the intercalated duct). Next, to test the above identification, gland extirpation experiments were performed. LRCs were established by labeling developing glands with iododeoxyuridine (IdU) in place of BrdU. Removal of one submandibular gland forced the IdU-LRCs in the remaining gland to divide. They were labeled with chlorodeoxyuridine (CldU). The (K18 and alphaSMA)(neg) LRCs in the neck of the intercalated duct and in the excretory duct did not change in number or in IdU label. The CldU label appeared in these cells and then disappeared. These results indicate that the (K18 and alphaSMA)(neg) LRCs have divided asymmetrically and are thus considered salivary gland stem cells.     

Immunohistochemical evidence for the presence of progesterone receptor in rat submandibular glands.

Immunohistochemical analysis of progesterone receptor was carried out in rat submandibular glands. Immunoreactivity to progesterone receptors was found in cell nuclei of the intralobular duct system within male and female rat submandibular glands. The female glands contained more immunoreactive cells than the male glands. In ovariectomized rats progesterone receptor-containing cells decreased in number while testectomized glands revealed an increase. When estradiol was administered to gonadectomized rats of both sexes, the immunoreactivity in cells of the intralobular duct system markedly increased. These results suggest the possibility that progesterone may modulate various metabolic functions in the rat submandibular glands.     

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.     

Isolation and characterization of a mucin-glycoprotein from rat submandibular glands

A blood group A+ mucin-glycoprotein was purified from aqueous extracts of rat submandibular glands by sequential chromatography on columns of Sepharose CL-6B and Sephacryl S-300 in urea-containing buffers. Final purification was facilitated by reductive methylation which appeared to release contaminating (hydrophobic) peptides. Homogeneity of the purified mucin was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at varying concentrations of acrylamide, lectin affinity chromatography, and Western blot analysis. In contrast to previously described preparations, the purified mucin contained only trace amounts of N-acetylglucosamine and aromatic amino acids. In addition, only low levels of basic amino acids were present.     

Morphometric and fine structural study of experimental autoallergic sialadenitis of rat submandibular glands.

To further our understanding of the immunopathologic mechanisms involved in experimental autoallergic sialadenitis of rat submandibular gland (EAS), histometric and fine structural studies were undertaken. Rats were immunized with allogeneic submandibular glands (SMG) emulsified in complete Freund's adjuvant. Control rats were not treated (C) or adjuvant treated (At). The rats were sacrificed 7, 14, 21 and 28 days after immunization and their SMG were processed for light and electron microscopy. Groups "C" and "at" showed normal acini and ducts. The SMG at 14 days showed significant loss of acini and granular ducts, severe lymphocytic infiltration and the appearance of undifferentiated ducts. The cells of the latter showed abundant free ribosomes, few profiles of rer, no secretory granules and in some cells autophagic vacuoles. Pseudopods of many lymphocytes were found in juxtaposition to degenerating parenchymal cells, mast cells and eosinophils. The extralobular ducts were significantly increased at 7, 14, and 21 days. The immunized glands showed evidence of regeneration at 21 and 28 days. Terminal tubule cells, proacinar cells and acinar cells, at various stages of maturation, were found in the regenerating glands.     

Biological behavior of myoepithelial cells in the regeneration of rat atrophied sublingual glands following release from duct ligation

Summary The present study aimed to clarify how myoepithelial cells behave during regeneration of an atrophied sublingual gland by investigating cell proliferation and ultrastructure. Atrophy of rat sublingual glands was induced by unilateral ligation of the excretory duct near the hilum with metal clips, which were then removed after one week of ligation for regeneration. The sublingual glands 0–14 days after unligation were examined with single immunohistochemistry for actin as a marker of myoepithelial cells, double immunohistochemistry for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells, and transmission electron microscopy (TEM). The single immunohistochemistry and TEM showed that myoepithelial cells surrounded residual ducts in the atrophied glands and immature and mature acini in the regenerating glands. Although PCNA-positive myoepithelial cells were identified during regeneration, PCNA labeling indices of myoepithelial cells were low at all time points except at day 7. Ultrastructurally, myoepithelial cells showing bizarre shaped structures in the atrophy changed with maturation of differentiating acinar cells and appeared normal in the regenerated glands. There was no differentiation of the remaining duct cells to myoepithelial cells. These observations suggest that proliferation of myoepithelial cells and differentiation to myoepithelial cells do not commonly participate in the regeneration of atrophied sublingual glands and that the bizarre shaped myoepithelial cells in the atrophied sublingual glands recover the original shapes with acinar cell regeneration.