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Tlx1 encodes a transcription factor expressed in several craniofacial structures of developing mice. The role of Tlx1 in salivary gland development was examined using morphological and immunohistochemical analyses of Tlx1 null mice. Tlx1 is expressed in submandibular and sublingual glands but not parotid glands of neonatal and adult male and female C57Bl/6J (Tlx1 +/+ ) mice. TLX1 protein was localized to the nuclei of terminal tubule cells, developing duct cells and mesenchymal cells in neonatal submandibular and sublingual glands, and to nuclei of duct cells and connective tissue cells in adult glands. Occasionally, TLX1 was observed in nuclei of epithelial cells in or adjacent to the acini. Submandibular glands were smaller and sublingual glands were larger in size in mutant mice (Tlx1 -/- ) compared to wild-type mice. Differentiation of terminal tubule and proacinar cells of neonatal Tlx1 -/- submandibular glands was abnormal; expression of their characteristic products, submandibular gland protein C and parotid secretory protein, respectively, was reduced. At 3 weeks postnatally, terminal tubule cells at the acinar-intercalated duct junction were poorly developed or absent in Tlx1 -/- mice. Granular convoluted ducts in adult mutant mice were decreased, and epidermal growth factor and nerve growth factor expression were reduced. Along with normal acinar cell proteins, adult acinar cells of Tlx1 -/- mice continued to express neonatal proteins and expressed parotid proteins not normally present in submandibular glands. Sublingual gland mucous acinar and serous demilune cell differentiation were altered. Tlx1 is necessary for proper differentiation of submandibular and sublingual gland acinar cells, and granular convoluted ducts. The mechanism(s) underlying Tlx1 regulation of salivary gland development and differentiation remains unknown., (© 2023 Anatomical Society.)

Objective: This study aimed to demonstrate the immunohistochemical changes associated with MMP-2 and type 1 collagen separately for the first time in the major salivary glands (the parotid, submaxillary, and sublingual glands) that occur with aging in mice., Material and Methods: Fourteen Balb/c white mice (50-80 g) were used in this study. The animals were divided into two equal groups. Group I consisted of young animals (2-month-old) (n=7) and Group II consisted of older animals (18-month-old) (n=7). After routine histological follow-ups, Hematoxylin-eosin (H&E), Masson's Trichrome staining and immunohistochemical staining was performed for type I collagen and MMP-2., Results: We observed that there were age-related decreases in the number of acinar cells, increase in eosinophilic zymogen granules in cells, collagen accumulation in fibrotic areas and dilatation in interlobular ducts. Also, while type I collagen and MMP-2 immunoreactivity were moderate in the salivary glands of the young mice, they were high in the salivary glands of the old mice (p=0.001). In the H-score assessment, MMP-2 immunoreactivity was lower at a significant level in young mice than in old mice (p=0.001)., Conclusions: This study showed that anatomical, physiological and morphological abnormalities occur in all three major salivary glands as a natural consequence of aging.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva., (Copyright © 2015 the American Physiological Society.)

β-defensins are small, cationic peptides with broad-spectrum antimicrobial activity that are produced by mucosal epithelia. However, little is known about the expression of β-defensins in the major salivary glands. The purpose of this study was to characterize expression of rat β-defensin-1 (RBD-1) and -2 (RBD-2) mRNA within the major salivary glands together with the effect of injection of intraductal lipopolysaccharide (LPS) on that expression. β-defensin mRNA expression was quantitated by RT-PCR in salivary gland tissues and salivary acinar and striated duct cells collected by laser captured microdissection. RBD-1 and -2 were expressed in the parotid gland, the submandibular gland, and the sublingual gland. β-defensins were expressed in both the acinar and striated duct cells of the major salivary glands. Intraductal injection of LPS increased expression of RBD-1 and -2 mRNA, which peaked at 12 hrs. These results suggest that salivary cells (acinar and striated duct cells) have the potential to produce β-defensins.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

Electrophoretic polypeptide profiles of normal human saliva differ markedly between different reports. Since both methodological variations and polymorphism may explain these differences, in this study we aimed to establish whether or not the salivary electrophoretic polypeptide profiles of subjects from a healthy population share discrete molecular features. To this end, parotid, submandibular/sublingual and whole salivas were collected separately from each of 40 young and 34 elderly clinically healthy adults and processed for SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Each type of glandular saliva displayed a different group of invariant (i.e. present in every subject) electrophoretic polypeptide bands while whole saliva showed a profile that reflected mostly the combined contribution of the major salivary glands. Some minor variant (i.e. absent in some subjects) bands were identified in each type of saliva. Regarding those interindividual variations, no age- or sex-dependence was appreciated. Altogether, these results demonstrate the occurrence of distinctive electrophoretic polypeptide patterns, in addition to some minor variations, for each type of normal saliva, thus providing a background for further populational studies on salivary polypeptide profiles.

A number of fibroblast growth factors (FGFs) are involved in regulatory mechanisms of the salivary gland development. However, the role of FGF-6 unique in myogenic cells has not been elucidated in the developing sublingual gland. In the present study, temporo-spatial expression of FGF-6 and its receptor (FGFR)-4, in conjunction with some related histo-chemical properties, were investigated in the sublingual gland of the prenatal and early postnatal mice. The earliest expression of both FGF-6 and FGFR-4 was detected in immature acinar cells at gestational day 17 (GD17). The staining intensity increased gradually and some acinar cells showed a distinct staining at postnatal day 0 (PD0). The immunopositive cells had a relatively round profile and were assumed to be acinar cells. The positive staining decreased thereafter and disappeared completely by PD11. To confirm the identity of cells positive for FGF-6, double immunolabeling with anti-alphasmooth muscle actin (alphaSMA) and anti-FGF-6 antibodies was performed. The positive staining of alphaSMA, a marker of myoepithelial cells, was detected in the flattened cells surrounding the acini but not in the cells positive for FGF-6. The staining properties of secretory granules in acinar cells were also examined with periodic acid-Shiff (PAS) and alcian blue (AB). PAS-positive granules abundant in the late gestational stages (GD17 to PD0) began to be replaced with AB-positive mucous granules at early neonatal days (PD0-3), when the FGF-6/FGFR-4 expression was the strongest. These findings suggest that FGF-6/FGFR-4 might be involved in the changes of secretory granule content of acinar cells in the sublingual gland during the late gestational and early neonatal stages.

The effect of triiodo-L-thyronine (T3) and propylthiouracil (PTU) on the initiation of epidermal growth factor (EGF) expression in the sublingual glands (SLGs) of postnatal mice was investigated by indirect enzyme-labeled and immunogold antibody methods for light and electron microscopy, respectively. In normal males, EGF immunoreactivity first appeared in a few scattered granular cells of striated ducts (SDs) at 5 weeks of age, and the immunoreactive cells had increased in number at 6 weeks of age. No EGF expression was observed in the glands of females at any ages examined. When T3 (1 mg/kg body weight) was given to males every other day for 2 weeks before examination, EGF expression began earlier; the immunoreactive granular cells were first detected at 4 weeks of age, and at later ages they were markedly increased in number compared to those of normal males. Moreover, T3 was capable of inducing EGF in the female glands. After T3 was administered to females in the same manner as in males, a few immunoreactive cells were first detected at 5 weeks of age, and increased numbers were detected at later ages. By contrast, when PTU (1 mg/kg body weight) was given to male mice every other day for 2 weeks before examination, the EGF-immunoreactive cells were markedly decreased in number compared to those of normal males of the same age. Electron microscopy revealed that many SD cells contained secretory granules, and that these cells constituted the granular striated tubule (GST) in a portion of SDs, but they were undetectable by light microscopy, because their secretory granules were minimal in size and few in number. Gold-labeling of EGF was confined to the secretory granules of scattered granular cells, whose secretory granules were far larger in size and more abundant than those of the GST cells. These results suggest that thyroid hormone is essential to differentiation of the cellular phenotype of GST precursor cells into typical granular cells (detectable by light microscopy) that express EGF in the mouse SLG, showing a close resemblance to the submandibular granular convoluted tubule cells.

NFS/N-sld mice harbor a spontaneous autosomal recessive mutation, sld (sublingual gland differentiation arrest) and histologically display attenuated mucous cell expression in sublingual glands (Hayashi et al. Am J Pathol 132: 187-191, 1988). Because altered serous demilune cell expression is unknown, we determined the phenotypic expression of this cell type in mutants. Moreover, we evaluated whether absence of glycoconjugate staining in 3-day-old mutant glands is related to disruption in apomucin gene expression and/or to posttranslational glycosylation events. Serous cell differentiation is unaffected, determined morphologically and by serous cell marker expression (PSP, parotid secretory protein; and Dcpp, demilune cell and parotid protein). Conversely, apical granules in "atypical" exocrine cells of mutant glands are PSP and mucin negative, but contain abundant SMGD (mucous granule marker). Age-related appearance of mucous cells is associated with expression of apomucin gene products, whereas SMGD expression is unaltered. "Atypical" cells thus appear specified to a mucous cell fate but do not synthesize mucin glycoproteins unless selectively induced postnatally, indicating the sld mutation disrupts apomucin transcriptional regulation and/or decreases apomucin mRNA stability.

EGFR activation has been related to an increase in synthesis and secretion of mucins in epithelial cells, so that the use of EGFR tyrosine kinase inhibitors has been proposed in the therapy of mucin hypersecretory diseases. In this paper, we describe the ultrastructural localisation of EGFR in the mucous elements of human major and minor salivary glands and relate it to mucin distribution. A post-embedding immunogold staining method has been applied to normal surgical samples of human submandibular, sublingual, and labial glands, using a mouse monoclonal antibody specific for the intracellular domain of human EGFR. In mucous cells of all the glands examined, specific reactivity was detected in the cytoplasmic basolateral portions and near the mucous droplets, but not on cell surfaces. Since this pattern of labelling must be related to the internalisation process of the ligand-GFR complex, our results support the hypothesis that EGFR activation takes place in mucous cells and affects mucin production in human salivary glands.

Background: The present study was aimed to determine the proliferation and distribution of myoepithelial cells during atrophy of rat sublingual glands., Methods: The excretory duct of the right sublingual gland of rats was doubly ligated with metal clips to induce atrophy in the gland. The atrophic sublingual glands were taken from 1 to 28 days after duct ligation and examined with single immunohistochemistry for actin as a marker of myoepithelial cells and with immunohistochemical double staining for actin and proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells., Results: In unligated sublingual glands, myoepithelial cells embraced acini and intercalated ducts, but not striated and interlobular excretory ducts. In the early stages of atrophy, myoepithelial cells surrounded small ducts but not large ones. However, in the later stages of atrophy, myoepithelial cells were also observed at the periphery of the large ducts. The immunohistochemical double staining showed that there were PCNA-positive myoepithelial cells in the normal as well as in the atrophic sublingual glands. However, the PCNA labeling indices of myoepithelial cells were low in the unligated and atrophic sublingual glands, and there were no statistically significant differences in these labeling indices., Conclusion: The observations suggest that the distribution of myoepithelial cells change during atrophy of rat sublingual glands and that myoepithelial cells have low proliferative activity in both the normal and atrophic condition of rat sublingual glands.

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.

Sublingual tissue PCO2 (PSLCO2) was continuously monitored with an ISFET-based PCO2 sensor during and after the open-heart surgery under cardiopulmonary bypass (CPB) in order to study the effect of CPB on the peripheral blood perfusion. In addition, PSLCO2 monitoring was carried out in several cases of off-pump CABG. In the cases of open-heart surgery with CPB, PSLCO2 increased from 35.0 +/- 5.6 mmHg at the induction of anesthesia to the maximum value of 55.7 +/- 6.0 mmHg during CPB. After declamping of the aorta, PSLCO2 decreased gradually to 49.0 +/- 4.0 mm Hg 6 hr after the admission to ICU. The value of arterial lactate as another index of peripheral blood perfusion also increased gradually after the start of CPB, reaching to the maximum value of 8.8 +/- 1.1 mmol.l-1 just after being admitted into ICU. In the case of off-pump CABG, PSLCO2 and arterial lactate showed a slight increase during the later part of the surgery, but the change was not so significant as in the case of open-heart surgery under CPB. Through this study, typical changing pattern of PSLCO2 during the open-heart surgery was recognized. The change of PSLCO2 always preceded that of arterial lactate. We also experienced one case in which early stage of hypoperfusion was detected through the monitoring of PSLCO2. These results suggest clinical advantages of PSLCO2 monitoring.

This study examined the occurrence and morphological features of serous-type cells in human sublingual gland, using immunocytochemistry for lysozyme. Lysozyme-positive cells usually formed demilunes and occasionally their own acini. They were also found among cells of an intercalated duct and in its immature acinus consisting of a small number of secretory cells. All these serous cells could be classified as seromucous cells because they simultaneously revealed reactivity for mucus, i.e., a periodic acid-Schiff (PAS) and a periodic acid-thiocarbohydrazide-silver proteinate (PA-TCHSP) reaction under the light- and electron-microscope, respectively. Immunogold labeling of lysozyme in the seromucous cells was distributed on variously sized secretory granules. These usually possessed a single electron-dense spherule in an electron-lucent matrix, while granules of a homogenous structure were also present. Lysozyme-positive cells filled with large, lucent secretory granules could hardly be morphologically distinguished from the lysozyme-negative mucous cells; they corresponded to "intermediate" cells designated under the light microscope. All "immature" secretory cells with only a few secretory granules were also lysozyme-positive seromucous cells. The present study demonstrated that the seromucous cells in the human sublingual glands conform closely with those in the human labial glands (MIYAZAKI et. al., 1998).

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 S10100A6 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.

Double lectin labeling for confocal microscopy and lectin-protein A-gold binding for electron microscopy were applied to the mouse sublingual gland in order to study surface and cytoplasmic sialoglycoconjugates. For this purpose, serially cut sections were submitted to sialidase followed by incubation with lectins recognizing usually acceptor sugars for terminal sialic acids. At the electron microscope level, the residues subtended to sialic acid were individually identified on adjacent sections by an indirect technique of labeling, whereas with confocal microscopy the above sugars were simultaneously visualized on the same section by a double staining method using fluorescein isothiocyanate (FITC)- and tetramethylrhodamine isothiocyanate (TRITC)-conjugated lectins. Acinar cells were found to contain the terminal sequence sialic acid-beta-galactose in abundance while the sequence sialic acid-alpha-N-acetylgalactosamine appeared to be present in modest amounts. Both sialoglycoconjugates were homogeneously codistributed inside acinar cells. The combination with a saponification method also allowed the occurrence of C4 acetylated sialic acids linked to beta-galactose to be discovered, at the electron microscope level, on acinar cell secretory products.

Our laboratory is concerned with elucidation of mechanisms regulating the exocrine secretion of mucin glycoproteins by mucous cells of salivary mucous glands. As a model system for our studies we use short-term cultures of acinar structures isolated from rat sublingual glands. Recent results are discussed from an on-going study of cross-talk between the two primary signaling pathways regulating exocrine secretion from isolated sublingual acini: the muscarinic cholinergic and vasoactive intestinal peptide (VIP) pathways. The combination of muscarinic agonist (carbachol) and VIP elicits a secretion equivalent to about 150% of the additive sum of the secretory responses to each agonist alone. This synergistic secretory response is only observed at submaximal concentrations of carbachol. VIP thus serves to decrease the EC50 for carbachol nearly three-fold. Results are thus far consistent with the hypothesis that a sustained rise in the intracellular concentration of calcium ions induced by VIP accounts for synergistic secretion and the heightened sensitivity to carbachol.

A growing body of information now supports the suggestion that P2 receptors for extracellular nucleotides (primarily ATP) have a role in regulating salivary gland function. There is solid pharmacological and molecular evidence for the presence of P2X ligand-gated ion channel nucleotide receptors (P2X4 and P2X7/P2Z). More recently, our group and others have obtained evidence that multiple P2Y G protein-coupled nucleotide receptors (P2Y1 and P2Y2) are also expressed. Our studies have focused on defining the conditions under which P2Y receptors are expressed, the functional consequences of their activation, and the importance of co-expression of P2X and P2Y receptors. Functional and molecular approaches have been used to identify the P2 subtypes in salivary glands and in salivary cell lines. Assays include measurement of changes in [Ca2+]i, changes in transcellular short circuit current in monolayers, and RT-PCR to assess changes in receptor mRNA levels. The main observations are: (1) P2Y1 receptor activity is present in the submandibular gland (SMG) of immature rats but decreases over the first four weeks following birth, although mRNA levels remain relatively constant; (2) P2Y2 receptors are present in the cell lines and are up-regulated during short-term culture of normal parotid, sublingual, and SMG cells and following ligation of the main excretory duct of SMG; and (3) the P2X subtypes, P2X4 and P2X7, and the P2Y subtypes, P2Y1 and P2Y2, are co-expressed in salivary glands and salivary cell lines, and exhibit distinct basolateral versus apical localization in polarized cell monolayers as well as discrete patterns of intracellular signaling.

The B1-immunoreactive proteins (B1-IPs) are major secretory products of rat submandibular gland acinar-cell progenitors, and are also produced by neonatal and adult rat sublingual and parotid glands. In order to characterize the B1-IPs, we have previously isolated cDNA clones encoding rat parotid secretory protein (PSP; the predominant parotid B1-IP) and the related clone ZZ3, which is developmentally regulated in the neonatal submandibular gland. The remainder of the B1-IPs were uncharacterized. This report demonstrates that all of the B1-IPs are derived from the PSP and ZZ3 transcripts. Molecular cloning and Western-blot analyses using PSP- and ZZ3-specific antisera show that, of the B1-IPs, only PSP and neonatal submandibular gland protein A (SMGA) are products of the Psp gene. This finding corrects our previous assertion that SMGA is derived from ZZ3. Neonatal submandibular gland proteins B1 and B2, as well as apparent Mr 26000-28000 and Mr 18000-20000 forms in submandibular, sublingual and parotid glands, are derived from the gene encoding ZZ3 by differential N-glycosylation and by proteolytic cleavage. The apparent Mr 18000-20000 proteolytic products are significant in secretion product collected in vitro, but rare in gland homogenate and submandibular/sublingual saliva. The gene encoding ZZ3 has been named Smgb. Psp and Smgb are regulated similarly in the developing submandibular gland, but differently in the sublingual and parotid glands. The expression pattern of Psp is conserved between rat and mouse. However, no evidence for proteins derived from an Smgb-like gene was observed in neonatal mouse submandibular or sublingual glands.

The concentrations of principal neurotransmitters in the submandibular, parotid and sublingual glands were compared between two pairs of age-matched male and female ddY mice, one pair consisting of 4-week-old and the other 8-week-old animals. Sex-dependent differences in both noradrenaline and acetylcholine concentrations were observed only in the submandibular gland, although each neurotransmitter showed distinct features. The acetylcholine concentration in the submandibular gland was higher in the female at both ages, whereas the noradrenaline concentration was higher in the female at the age of 4 weeks but became higher in the male by the age of 8 weeks. On the other hand, the total amounts of noradrenaline and acetylcholine per submandibular gland were already greater in the male at 4 weeks, and the male parotid and sublingual glands also had a greater noradrenaline content by 4 weeks and 8 weeks, respectively. Each type of gland had similar growth rates over the 4-week period, and the male submandibular and parotid glands were heavier than the female. In addition, each type of gland had its characteristic ratio of noradrenaline to acetylcholine concentration, which did not differ between the sexes and remained in similar basic patterns during the period examined, except for the submandibular gland of 8-week-old male mice, which developed greater amounts of the sympathetic neurotransmitter noradrenaline.

The main purpose was to compare the changes in fatty acid composition of lipids induced by essential fatty acid (EFA) deficiency in the rat submandibular, parotid and sublingual glands. Three groups of rats were fed for 28 weeks (1 week gestation, 3 weeks lactation and 24 weeks thereafter) diets containing 7% hydrogenated coconut oil (HCO) (EFA-deficient in both n-6 and n-3), 7% soybean oil (SBO) (control) and 7% safflower oil (SFO) (deficient in n-3). Rats were killed and salivary glands were dissected out. Lipids were extracted and the fatty acid composition of total lipids and phospholipids was determined by gas chromatography-mass spectrometry. The fatty-acid compositional changes indicative of an EFA deficiency, such as decreases in the levels of 18:2 n-6, along with an accumulation of 20:3 n-9, were generally observed in all the salivary glands of rats fed 7% HCO diet. In the submandibular glands, the proportions of 16:1, 18:1 n-9 and 18:1 n-7 were also higher in the HCO-fed group than in the other two groups. There were some differences in the fatty acid composition of the three glands. Total lipids of parotid gland had higher levels of 12:0 and 18:1 n-9 as compared to the other two glands. The levels of 18:0, 20:3 n-9, 20:3 n-6 and 20:4 n-6 were, however, lower in the parotid gland as compared with the other glands. In total phospholipids of rats fed SBO- and SFO-containing diets, the sublingual gland had lower levels of 18:2 n-6 and higher levels of 20:4 n-6 than the parotid or the submandibular. These differences in fatty acid composition may be related to possible differences in chain elongation/desaturation. The changes in fatty acid composition were also reflected in total lipids of plasma, liver and whole saliva of rats fed the various diets. A number of fatty acids were identified in saliva by gas chromatography-mass spectrometry.

One group of mice was injected subcutaneously with 20 mg/kg body wt isoprenaline each day for 10 days; another group (control) was injected with saline. Half the animals of each group were kept untreated for a further 10 days for restoration. Chronic administration of isoprenaline caused enlargement of parotid (4-fold) and submandibular glands (1.7-fold) but had no effect on sublingual glands. Concomitantly, noradrenaline and acetylcholine contents were, in parallel, increased in parotid, decreased in sublingual, and unchanged in submandibular glands. Under these conditions, pilocarpine- or isoprenaline-induced salivation was not affected but phenylephrine-induced salivation was augmented; the protein component patterns of saliva characteristic of the three sialogogues were also changed. In addition, secretory proteins whose synthesis was induced by isoprenaline were found to be secreted by stimulation with different types of sialogogues. Most changes were reversible. These results indicate that continued beta-adrenoceptor stimulation not only causes broadly altered forms of saliva, probably by involving, in part, alpha-adrenoceptor hypersensitivity, but also changes the activities of both sympathetic and parasympathetic nerves to salivary glands in parallel, though the extent differs among the three glands.

To investigate the ultrastructure of gap junctions and functional differences between connexin (gap junction structural protein) 32 and 43 in the rat submandibular and sublingual glands, tracer and freeze-fracture methods were carried out, and the expression of both connexins was examined. In both glands, western blot analysis with anti-connexin 32 and 43 antibodies revealed bands of about 27kD and 43kD, respectively. Immunofluorescence showed the presence of connexin32-positive spots between acinar cells in both glands. In contrast, connexin43-positive spots were observed at the periphery of the acinar structures in either gland. Positive spots for both connexins could not be detected between ductal cells in both glands. By immunocytochemistry, connexin32 was found on the gap junctional membranes of acinar cells and connexin43 on the gap junctional membranes of myoepithelial cells. It is surmised that connexin32 of the gap junction is related to the secretory function of acinar cells and that connexin43 is associated with the contraction of the myoepithelial cells.

We examined the expression and localization of the gap junction proteins connexin32 and 43 in rat submandibular and sublingual glands. Western blot analysis with anti-connexin32 and 43 antibodies showed bands of approximately 27 KD and 43 KD, respectively, in both glands. Immunofluorescence microscopy demonstrated the presence of reactive spots for connexin32 between acinar cells in both glands. The frequency of connexin32-positive spots in the submandibular glands was approximately equal to that in the sublingual glands. In contrast, reactive spots for connexin43 were observed at the periphery of the alveolar structures in both glands. The connexin43-positive spots in the sublingual glands were more frequent and larger than those in the submandibular glands. No positive spots for both connexins were detected between duct cells in either gland. Immunoelectron microscopy revealed that connexin32 was localized to the gap junctional membranes between acinar cells. Immunolabeling for connexin43 was located on the gap junctions between thin processes of myoepithelial cells. These results suggest that connexin32 of the gap junction is associated with regulation of the secretory function of acinar cells and that connexin43 is associated with that of contraction of the myoepithelial cells in rat salivary glands.

A human sublingual gland cDNA library was screened with a polyclonal antiserum against deglycosylated MG1 and a positive clone, pSM2-1, was isolated which codes for 196 amino acids in the carboxyl-terminal region of this mucin. This region is cysteine-rich and contains a C2-like domain upstream of the extreme carboxyl-terminal domain in which the arrangement of cysteines is nearly identical to that in human von Willebrand factor, human intestinal mucin MUC2, human tracheobronchial mucin MUC5 and porcine and bovine submaxillary gland mucins. Northern analyses with pSM2-1 showed MG1 transcripts are abundant in sublingual gland and barely detectable in submandibular gland. This study provides the first primary sequence data on human salivary mucin MG1 and the significance of the results is discussed with respect to the biosynthesis and differential expression of MG1 in human salivary glands.

Lectin and S-100 protein histochemistry during fetal growth and development (10-38th gestational weeks) of these glands was studied. The histological development of glandular structures followed the known pattern for other salivary glands. Using biotinylated lectins Ulex europeus-I, Dolichos biflorus, Glycine maximus (soyabean), Helix pomatia, Arachis hypogaea (peanut) and Triticum vulgare (wheatgerm), the binding level and, by implication, the concentration of associated specific oligosaccharide available for binding was low at 10 to 19 weeks and generally higher as maturity increased through the middle and late stages of development. S-100 protein reactivity was demonstrated in the cytoplasm of basophil acinar cells of the gland primordia from their origin. Stereological analysis of these developing salivary glands showed a highly significant progressive increase in proportional gland volume occupied by acini from 25% at 20 weeks to 60% at 38 weeks (p < 0.0001), and a comparable halving of the relative gland volume occupied by connective tissue in the same period (p < 0.0001). The extent of these changes depended upon the stage of differentiation and maturation of the glands but by the late stage of fetal development, histochemical reactions were similar to known adult patterns.

The salivary glands of 5-week-old mice and rats were divided into submandibular, parotid, and sublingual and analysed to determine basal levels of the neurotransmitters noradrenaline (NE) and acetylcholine (ACh) and the possible neurotransmitters dopamine (DA) and 5-hydroxytryptamine (5-HT), using a combination of high-performance liquid chromatography coupled with coulometric and amperometric detection and a direct injection technique employing crude homogenate supernatants. In both species, levels of NE were higher in the submandibular than in the other salivary glands, whereas levels of ACh were higher in the mouse submandibular and the rat sublingual glands than in other glands. In all the salivary glands, levels of DA were markedly lower than those of other target substances. Levels of 5-HT were similar in all salivary glands. These results show that in mouse and rat salivary glands, species differences in neurotransmitter distribution are relatively small, whereas there are considerable differences in distribution between the salivary glands.

The binding properties of the rat sublingual glycoprotein (RSL) and its asialo product with lectins were characterized by quantitative precipitin(QPA) and precipitin inhibition(QPIA) assays. Among twenty lectins tested for QPA, native RSL reacted well only with Artocarpus integrifolia (jacalin), but weakly or not at all with the other lectins. However, its asialo product (asialo-RSL) reacted strongly with many Gal and GalNAc specific lectins-it bound best to three of the GalNAc alpha 1-->Ser/Thr (Tn) and/or Gal beta 1-->4GlcNAc (II) active lectins [jacalin, Wistaria floribunda and Ricinus communis agglutinins] and completely precipitated each of these three lectins. Asialo-RSL also reacted well with Abrus precatorius, Glycine max, Bauhinia purpurea alba, and Maclura pomifera agglutinins, and abrin-a, but not with Arachis hypogeae and Dolichos biflorus agglutinins. The interaction between asialo-RSL and lectins were inhibited by either Gal beta 1-->4GlcNAc, p-NO2-phenyl alpha-GalNAc or both. The mapping of the precipitation and inhibition profiles leads to the conclusion that the asialo rat sublingual glycoprotein provides important ligands for II (Gal beta 1-->4GlcNAc beta 1-->) and Tn (GalNAc alpha 1-->Ser/Thr) active lectins.

A highly selective, amiloride-sensitive, epithelial sodium channel from rat colon (rENaC), composed of three homologous subunits termed alpha, beta, and gamma rENaC, has been cloned by functional expression and was proposed to mediate electrogenic sodium reabsorption in aldosterone-responsive epithelia. To determine whether rENaC could account for sodium absorption in vivo, we studied the cellular localization of the sodium channel messenger RNA subunits by in situ hybridization and their cellular and subcellular distribution by immunocytochemistry in the kidney, colon, salivary, and sweat glands of the rat. In the kidney, we show that the three subunit mRNAs are specifically co-expressed in the renal distal convoluted tubules (DCT), connecting tubules (CNT), cortical collecting ducts (CCD), and outer medullary collecting ducts (OMCD), but not in the inner medullary collecting ducts (IMCD). We demonstrate co-localization of alpha, beta, and gamma subunit proteins in the apical membrane of a majority of cells of CCD and OMCD. Our data indicate that alpha, beta, and gamma subunit mRNAs and proteins are co-expressed in the distal nephron (excepting IMCD), a localization that correlates with the previously described physiological expression of amiloride-sensitive electrogenic sodium transport. Our data, however, suggest that another sodium transport protein mediates electrogenic amiloride-sensitive sodium reabsorption in IMCD. We also localized rENaC to the surface epithelial cells of the distal colon and to the secretory ducts of the salivary gland and sweat gland, providing further evidence consistent with the hypothesis that the highly selective, amiloride-sensitive sodium channel is physiologically expressed in aldosterone-responsive cells.

Simple mucin-type carbohydrate antigens Tn, sialosyl-Tn and T are often markers of neoplastic transformation and have very limited expression in normal tissues. We performed an immunohistological study of simple mucin-type carbohydrate antigens, including H and A variants, with well-defined monoclonal antibodies (MAb) on frozen and paraffin-embedded normal salivary gland tissue from 22 parotid, 14 submandibular, six sublingual, and 13 labial glands to elucidate the simple mucin-type glycosylation pattern in relation to cyto- and histodifferentiation. The investigated carbohydrate structures were predominantly observed in the cell cytoplasm, most often in the supranuclear area, suggesting localization to the Golgi region, whereas ductal contents were unstained. Mucous acinar cells expressed Tn, sialosyl-Tn, and H and A antigens, regardless of glandular location. Serous acinar cells, on the other hand, expressed A, H, and inconstantly sialosyl-T, Tn, and sialosyl-Tn antigens in major salivary glands, whereas serous cells of minor (labial) salivary glands expressed H exclusively, Tn and sialosyl-T antigens inconstantly, but never sialosyl-Tn and A antigens. The difference may be related to a more simple cytodifferentiation of serous cells of minor (labial) salivary glands as compared with major salivary glands. Duct cells in major salivary glands expressed A, H, and inconstantly T, sialosyl-T, and Tn antigens, whereas minor (labial) salivary glands ducts exclusively expressed H, T and sialosyl-T antigens, differences that may be related to dissimilarities in the duct system. Myoepithelial cells and basal cells exclusively expressed T and sialosyl-T antigens, which may prove useful in studies of salivary gland tumors, since these cells are known to play a key role in the histological characteristics of some salivary gland tumors. The results indicate a similar glycosylation pattern in the different major salivary glands, whereas minor (labial) salivary gland differ slightly in serous and duct cells. The limited and exclusive intracellular expression of the immature Tn, sialosyl-Tn, and T antigens indicates that these structures may be of value as markers of salivary gland tumors.

The development of vasoactive intestinal peptide, substance P and calcitonin gene-related peptide in parotid, submandibular and sublingual glands of the male rat was followed by immunochemistry and immunocytochemistry. The total amounts of these peptides increased in surges during the first 8 weeks of the animal's life; one within 2-4 weeks and the other beginning 1-2 weeks later. Nerve fibres containing these peptides were present at birth showing a pattern of distribution similar to that in adults. During the first 4 weeks the nerve fibres increased in number.

The purpose of this study was to show how some aspects of the biological matrix water can be investigated by thermoanalytical methods when using thermogravimetry and differential scanning calorimetry. Such methods allow to investigate the water in the rabbit sublingual gland and to quantitate it. The results supported the view that the water contained in this secretory organ show a large number of energetic interactions established on the basis of differential steps of water release by thermal disruption.

The three major salivary glands of normal male and female Fischer 344 rats of different ages were examined for the localization of epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) by immunohistochemical staining. EGF was demonstrated only in the granulated convoluted tubule (GCT) cells of the submandibular gland, the results confirming the previous reports, and most abundantly in adult males and pregnant females. TGF alpha stain was localized in all three glands and was found throughout the entire duct system, excluding acinar cells. The myoepithelial cells of the sublingual gland were also reactive with the TGF alpha antibody. The specificity of the staining was confirmed by negative staining reaction with the absorbed antibody and by radio-immunoassay and Western blot methods. This is the first report describing the presence of TGF alpha in the rat salivary glands.

Exocrine glands including the submandibular gland, sublingual gland, exocrine pancreas and exorbital lacrimal gland of newborn rats aged 0 to 14 days were examined morphologically, and the following results on the accumulation of lipid droplets were obtained. Lipid droplets tended to localize in secretory cells, especially in their basal cytoplasm. The degree of lipid droplet accumulation varied with the type of exocrine gland. There were large accumulations in the sublingual gland, submandibular gland, and exocrine pancreas, but accumulations were small in the exorbital lacrimal gland. No difference in lipid droplet accumulation was recognized between the 2 types of secretory cells in the sublingual gland. The accumulation of lipid droplets peaked 24-48 hours after birth in the sublingual gland, submandibular gland and exocrine pancreas, but this peak was not clearly observed in the exorbital lacrimal gland. In the group of newborn rats separated from their mothers and therefore not suckled, no lipid droplets were observed in any gland, suggesting a close relationship between lipid droplet accumulation and suckling.

Microliths in Araldite-embedded pieces of submandibular and sublingual glands of cat were stained in semithin sections by Methylene Blue and Azure II followed by Basic Fuchsin, and were examined in ultrathin sections by electron-microscopical X-ray microanalysis. Calcium and phosphorus were detected in substantial aggregates of crystals that were stained by Basic Fuchsin and appeared to be hydroxyapatite, but were not detected in granular material that was stained by Methylene Blue and Azure II and appeared to be organic. The polychromatic stain thus appears to be a useful indicator of calcified material. The majority of microliths in acini contained substantial aggregates of crystals, whereas the majority of those in ducts did not. This corresponds to the distribution of the glandular calcium, and suggests that microliths are variously enriched with calcium according to its local level.

The B1-immunoreactive proteins of type III cells of the perinatal rat submandibular gland are immunologically cross-reactive with proteins of both the sublingual and parotid glands; in particular, protein SMG-A appears similar to a major parotid protein. We isolated SMG-A and the parotid protein (known as M1 or leucine-rich protein), prepared polyclonal antibodies to them, and compared their biochemical properties and immunological reactivities. They were identical in their molecular weight on SDS-PAGE (23.5 kDa), tenacious binding to Affi-gel Blue, isoelectric point (pH 4.53), and proteolysis to a 14 kDa peptide: Antibodies to SMG-A showed reactivity with protein SMG-C, a product of the neonatal type I cells, as well as with proteins SMG-B1 and SMG-B2, contrasted with the absence of reactivity of anti-M1 IgG with these proteins. Anti-M1 reacted with the "parotid secretory protein" (PSP) of the mouse, and M1 appears to be the homologue, in the rat, of mouse PSP.

Duct-ligated submandibular and sublingual glands of rats were evaluated immunohistochemically for changes in keratin (MoAb 1164), actin, S-100 protein and rat-EGF (rEGF). Normal salivary glands were reactive for keratin, S-100 protein and rEGF in the granular convoluted tubule (GCT) and duct cells, and for actin in the myoepithelium. Submandibular glands showed a marked reduction of S-100 protein and rEGF staining following duct ligation, and no increased staining of proliferating epithelial cells of the late stage in duct ligated glands. Sublingual glands revealed no marked changes for actin staining in myoepithelial cells, irrespective of atrophic changes occurring in acinar and duct cells after duct ligation. Immunohistochemical patterns differed for each type of gland; changes associated with the obstructive lesion were more prominent in the submandibular gland.

Epidermal growth factor (EGF) is a biologically active peptide involved in differentiation, growth, regeneration and repair of human and animal tissues. Quantitative biochemical studies showed in man the highest concentration of EGF in the parotid gland. The aim of the present study was to define EGF immunolocalization in the individual segments of the human major salivary glands (salivon). The material consisted of sections obtained from the surgically removed salivary glands: parotid, submaxillary and sublingual. Immunohistochemical studies were performed by PAP method using monoclonal antibody against human epidermal growth factor. EGF expression was found almost exclusively in the efferent pathways of the salivary glands, mostly in the intercalated ducts and Pflüger salivary tubules. These segments of the salivon are most developed in the parotid gland in which the staining was stronger than in other salivary glands.

Two separate sets of experiments were performed on female Wistar rats made diabetic with streptozotocin: (1) a time-course study where groups of three animals were removed at weekly intervals, up to 4 weeks after induction of diabetes, with an age-matched group of control (normal) animals kept for 4 weeks; (2) six further animals were made diabetic and kept for 7 weeks; three of these were given insulin in the final week. At the required time the animals were anaesthetized and the salivary glands removed and preserved by fixation or freezing. The frozen tissues were later homogenized and the protein and lipid content analysed. Histologically, intracellular lipid droplets had accumulated in the majority of the diabetic salivary glands. In the time-course experiment, the visible amount of intracellular lipid reached a maximum after 2 weeks and then decreased, with a concomitant disappearance of interstitial lipid. The increased lipid content was not attributable to any one class. The fatty acid profiles of the glands showed an increase in the percentages of C18:0 (stearic acid) and C18:2w6 (linoleic acid) and a decrease in the percentages of C18:1w9 (oleic acid) and C20:4w6 (arachidonic acid). After 1 week of insulin treatment the lipid content and the fatty acid profiles returned to normal. Thus the effect of insulin on salivary gland lipid metabolism is rapid both in its occurrence and reversibility. The effects seen in the diabetic rats are considered to be due to a lack of insulin and not to the presence of streptozotocin.

The major components of human submandibular-sublingual saliva (HSMSL) are mucins, amylases, cystatins, proline-rich proteins and statherin. Structure-function studies of these molecules have been hampered by the small amounts of purified materials that can be isolated from human secretions. The present study describes an integrated purification protocol for the large-scale preparation of many of these molecules. To dissociate partially heterotypic complexes among salivary molecules, HSMSL was initially fractionated into four pools by gel filtration with 6 M-guanidine hydrochloride. Subsequent fractionation of these four pools by gel-filtration and ion-exchange chromatography resulted in the purification of high- and low-Mr mucins, neutral and acidic cystatins, acidic and basic proline-rich proteins and statherin. Many variants or isoforms of these salivary molecules have been identified and biochemically characterized. Biochemical studies indicated that the low-Mr mucin exists as two isoforms which vary in their sialic acid to fucose ratios. Three isoforms of acidic cystatin S were characterized which differ in their phosphate content. Two isoforms of a basic proline-rich peptide were identified; the smaller peptide was a truncated form missing the first seven amino acids.

The lubricating properties of human submandibular-sublingual salivary fractions were examined using a servohydraulic model of mandibular movement. Fractions containing statherin exhibited a strong tendency to boundary lubrication. The lubricity of purified statherin was confirmed and compared to the amphipathic molecules gramacidin S and sodium dodecyl sulfate. Contact angle measurements of statherin paralleled the other amphipathic molecules. The helical content of statherin increased in trifluoroethanol indicating the presence of amphipathic helical regions. CD studies and hydrophobic moment calculations indicated that statherin adopts an amphipathic helical conformation at the N-terminus. An energy-minimized model of the polar N-terminal residues 1-15 suggested that this domain could be positioned in space to interact with a hydroxyapatite substrate. These data imply that under appropriate conditions statherin may display an amphipathic nature which enables it to function as a boundary lubricant on enamel.

Transferrin receptor expression in oral tumors was examined by staining with monoclonal antibody against the human transferrin receptor. The cells with positive reaction were recognized in the basal and parabasal layers of the normal epithelium. The staining was found in all the malignant tumors but not in the benign tumors. These results suggest that the immunohistochemical analysis of the transferrin receptor is useful for the diagnosis of oral malignant tumor in addition to the clinical and pathological examinations.

Formalin-fixed tissue specimens containing glycoproteins with side chain O-acylated sialic acids were used to re-examine, compare and evaluate the usefulness of three methods based on the periodic acid-borohydride reduction-saponification-periodic acid-Schiff sequence (PA-Bh-KOH-PAS) for the histochemical identification of 9-O-acyl sialic acids (9-O-AcSA). Method I, modified from Veh et al. (1979), involved a comparison of the staining intensely obtained when both oxidation steps of the PA-Bh-KOH-PAS sequence were carried out with the selective oxidation technique of Volz et al. (1987) with that obtained when the initial oxidation step was carried out with 0.5 M periodic acid for 4 h at room temperature. Methods II and III, modified from Reid et al. (1978), involved an initial PA-Bh step under oxidation conditions that cleaved all the vicinal diols associated with neutral sugars and side chain unsubstituted and 7-O-acyl sialic acids. The Schiff staining obtained following subsequent re-oxidation with either 0.5M (method II) or 1% periodic acid (method III) for 4 h at room temperature (PA-Bh-PAS procedure) identifies 9-O-AcSa.(ABSTRACT TRUNCATED AT 250 WORDS)

Sublingual salivary acini and submandibular tissue were incubated in DMEM medium in the presence of various concentrations (0-5%) of ethanol and the content of the three major prostaglandins, PGE2, PGF2 alpha and 6-keto-PGF1 alpha, were determined by radioimmunoassay. In In the sublingual gland, ethanol caused a decrease in PGE2 and PGF2 alpha levels, but had no effect on 6-keto-PGF1 alpha, while all three prostaglandins were affected in the submandibular gland. At 2.5% ethanol, the production of PGF2 alpha and PGE2 in sublingual gland decreased by 10% and reached maximum inhibition at 5% ethanol, at which concentration there was a 20 and 30% decrease in their levels. In submandibular gland, 2.5% ethanol caused a 20% decrease in PGE2, 30% in PGF2 alpha and 50% in 6-keto-PGF1 alpha; 40% inhibition in PGE2, 57% in 6-keto-PGF1 alpha and 65% in PGF2 alpha occurred in the presence of 5% ethanol. These findings suggest that alcohol impairs the function of salivary glands by inhibiting prostaglandin production.

In this study we extracted glycoconjugates from bovine sublingual gland and attempted to investigate anticoagulant properties on blood coagulation of humans. To this purpose we performed the thromboelastographic technique and selective hemocoagulation screening tests. In particular, findings from screening tests indicated the occurrence of hemostatic effects on thrombin time (TT) and activated partial thromboplastin time (aPTT) with a response in a dose-related manner, whereas significant changes were not found on prothrombin time (PT).