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Epidermal growth factor gene expression is regulated differently in mouse kidney and submandibular gland.

The concentration of the peptide mitogen epidermal growth factor (EGF) is hormonally and developmentally regulated in the granular convoluted tubule cells of the mouse submandibular gland. Using a labeled EGF nucleic acid probe, we have demonstrated that submandibular gland EGF mRNA concentrations increase during postnatal development of the gland and after the administration of testosterone or thyroid hormone. Recently, it was reported that EGF mRNA is present in kidney as well as a number of other mouse tissues. A comparison of EGF gene regulation in submandibular gland and kidney revealed that kidney EGF mRNA levels also increase during the postnatal period. Opposite sex differences were observed, with submandibular gland levels being about 16-fold higher in the male than in the female and kidney levels being 2- to 4-fold higher in the female than in the male. Renal EGF mRNA concentrations are less responsive to hormones than those in the submandibular gland. Renal EGF was localized immunocytochemically to the cells of distal convoluted tubules.

Adrenalectomy↗

Fluid and protein secretion by the submandibular glands of weanling rats in response to cholinergic and peptidergic agonists at various doses.

Fluid and protein secretion by the submandibular glands of 25-day-old rats were examined and compared in response to three cholinergic and four peptidergic sialogogues at various doses. All cholinergic and peptidergic agonists used were potent sialogogues for the submandibular glands of the weanling rats over the wide range of doses used. The cholinergic agonists, bethanechol and methacholine and the peptidergic agonists, substance P, substance PTyr8 and eledoisin-related peptide used intravenously, acted similarly to each other on the submandibular glands of the rats, late in the natural weaning period, but carbachol and physalaemin had slightly different effects. Of the peptidergic agonists, physalaemin was the most potent sialogogue among four tachykinins tested at the low dose. The types of protein secreted by the submandibular glands of the weanling rats in response to all sialogogues used here were typical of the beta-type. These results indicate that all agonists used could mainly stimulate the acinar cells of the submandibular glands of the weanling rats which have already fully developed functionally at this time.

Animals↗

Ultrastructure of chronically inflamed human submandibular glands.

The structure of chronically inflamed submandibular glands from four patients was examined by electron microscopy. The patients were free of the sicca syndrome and lacked other symptoms of Sjögren's syndrome or related autoimmune diseases. The affected glands were characterized by a reduction in acinar elements, by ductular proliferation and hyperplasia, and by large numbers of inflammatory cells in the stroma. This study indicates that many of the ultrastructural changes in salivary glands previously attributed to autoimmune diseases may also occur in patients who are free of such afflictions but whose salivary glands have undergone intermittent obstruction or infection.

Adult↗

An unusual sexually dimorphic mosaic distribution of a subset of kallikreins in the granular convoluted tubule of the mouse submandibular gland detected by an antibody with restricted immunoreactivity.

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

Animals↗

Immunohistopathological investigation on unspecific chronic sclerosing sialadenitis of the submandibular gland (Küttner tumor).

An immunohistochemical investigation was conducted on infiltrating lymphocytes in submandibular glands removed from three cases with unspecific chronic sclerosing sialadenitis of the submandibular gland (unspecific CSSS) which has been described for the first time by Küttner. A high degree of lymphocyte infiltration was recognized and formation of lymphatic follicles was observed. In particular, activated B cells were predominantly observed in that part of the lymphatic follicles which was equivalent to germinal centers. In the T cell subset, the helper/inducer T cells displayed significant dominance over suppressor/cytotoxic T cells, and were distributed in the dark peripheral zone of the follicles. The distribution pattern of these lymphocytes seemed to be similar to antigenically stimulated lymph nodes and it was recognized that the unspecific CSSS could be a site of exceedingly active local immune responses. The authors believe that these findings support the view that unspecific CSSS (Küttner tumor) is an immune disorder.

Antigens, CD↗

Autonomic nerve stimulation, kallikrein content anc acinar cell granules of the cat's submandibular gland.

The parasympathetic and sympathetic nerves to the submandibular gland of the anaesthetized cat were stimulated under specific conditions. 1. It was possible to decrease the kallikrein (kininogenase) content of the gland by as much as 90-95% by sympathetic nerve stimulation. In such a gland the appearance and concentration of secretory granules in the acinar cells were indistinguishable from an unstimulated gland. 2. Parasympathetic nerve stimulation, in contrast to sympathetic nerve stimulation, whilst having no significant effect on the kininogenase content of the gland, resulted in the disappearance of a great majority of the acinar granules. 3. These results demonstrate that the acinar granules in the submandibular gland of the cat are not a significant source of kallikrein. 4. Our experiments also failed to indicate any obvious correlation between the granules of the demilune cells and the kallikrein content of the gland. 5. The possibility is raised that kallikrein is located in the cells of the striated ducts.

Animals↗

Sialic acid in the submandibular glands of suckling rodents and carnivores.

In the submandibular gland of suckling rats, the presence of sialic acid can be demonstrated only after the second week of life. In the submandibular glands of suckling rabbits, mice and rats, on the contrary, the determination of sialic acid indicated that in all subjects this component was already present at this stage and that the sialic acid content increased with growth. The presence of sialic acid, which was found at on earlier stage in rabbits, mice and cats than in rats was explained on the basis of some data relevant to the position and function of sialic acid in glycoprotein molecules.

Animals↗

Cytosol androgen binding in submandibular gland and kidney of the normal mouse and the mouse with testicular feminization.

Androgen binding was investigated in the cytosol of the submandibular gland and the kidney from normal mice, mice with testicular feminization (Tfm), and carriers of the Tfm gene. In both tissues from normal mice an androgen-binding complex was observed from birth throughout life that could not be detected in Tfm animals at any age. This complex was precipitated at 35 and 40% saturation with ammonium sulfate in the kidney and the submandibular gland, respectively, and had an approximate sedimentation coefficient of 8S in the submandibular gland and 10S in the kidney. In both tissues of the normal female, the apparent dissociation constants (Kd) for testosterone and dihydrotestosterone were about 1 nM, and the binding capacity averaged 20 fmol-mg protein-1. The number of binding sites in preparations from Tfm carriers was intermediate between that in the Tfm and the normal female . In 0.5M KCl the 8S receptor of normal submandibular gland sediments at about 3S, and the 10S receptor of normal kidney sediments at about 4.5S. In the submandibular gland, binding of approximate 3S size was also observed. The latter was present in Tfm animals as well as in normal mice, it precipitated at 70% saturation with ammonium sulfate, and it was detectable from birth. In the kidney the only low molecular weight binding for androgen was about 4.5S in size.

Androgen-Insensitivity Syndrome↗

Epidermal growth factor, renin, and peptidase in cultured tumor cells of submandibular gland origin.

Dimethylbenz[a]anthracene was injected into the submandibular glands of male Swiss-Webster mice. From tumors obtained, three cell lines were established. Immunocytochemical stainings revealed epidermal growth factor, renin, and peptidase in a significant portion of cultured tumor cells. In addition, the presence of epidermal growth factor was demonstrated by radioimmunoassays. Since epidermal growth factor, renin, and peptidase are localized in the granular convoluted tubules in mouse submandibular gland, the data suggest that the granular convoluted tubule cells are the targets of chemical carcinogens.

Animals↗

Processing of epidermal growth factor in the rat submandibular gland by Kallikrein-like enzymes.

Epidermal growth factor (EGF) is synthesized as a precursor which is processed intracellularly to a 6 kDa EGF in the rat submandibular gland. This gland contains very high amounts of kallikrein-like enzymes, and the purpose of the present study was to examine whether any of five such enzymes, rK1, rK2, rK7, rK9 or rK10, can process the rat EGF precursor. Molecular weight forms of EGF, that were N- or C-terminally extended compared to submandibular gland EGF were obtained from rat urine. These extended forms of EGF were incubated with each of the enzymes for 24 h at 37 degrees C. Two enzymes, rK7 and rK10, were able to cleave N- and C-terminally extended EGF, releasing a form of EGF which eluted similarly to submandibular gland EGF upon gel filtration, and which was recognized both by antibodies against rat EGF and by the EGF receptor. One enzyme, rK1, cleaved C- but not N-terminally extended EGF. Neither rK2, nor rK9 cleaved the extended forms of EGF. In previous immunohistochemical studies rK1, rK7 and rK10 have all been demonstrated in the EGF containing cells of the rat submandibular gland. EGF and rK1 are also synthesized in the rat kidney but the present study demonstrated that EGF and rK1 are not colocalized in this organ. Based on the cleavage of the extended forms of rat EGF by rK1, rK7 and rK10 and on the fact that the enzymes are abundant and colocalized with EGF in the rat submandibular gland, we suggest that rK1, rK7 and rK10 can be involved in the processing of the EGF precursor in the rat submandibular gland.

Animals↗

Expression of phosphodiesterase 3 in rat submandibular gland cell lines.

A recent preliminary (unpublished) study showed that phosphodiesterase (PDE) 3A and 3B are expressed in rat submandibular glands. Here, PDE3 activity was detected in homogenates of rat submandibular gland acinar epithelial (SMIE) cells, but not rat A5 (epithelial duct) cells. Most of the PDE3 activity in SMIE cells was recovered in the particulate fraction. Only PDE3B mRNA was detected by reverse transcription-polymerase chain reaction in RNA from SMIE cells. The nucleotide sequence of the fragment was identical to the sequence of rat PDE3B. The PDE3 specific inhibitor, OPC3689 (10 and 50 microM), inhibited the growth of SMIE cells (19 and 63%), but not A5 cells. As the submandibular gland contains many types of cells, these results indicate that PDE3B may regulate a cAMP pool that is important in submandibular gland acinar epithelial cell function.

3',5'-Cyclic-AMP Phosphodiesterases↗

Submandibular gland transfer: a new method of preventing radiation-induced xerostomia.

OBJECTIVE: Radiation-induced xerostomia is a significant morbidity of radiation therapy in the management of patients with head and neck cancers. We have recently reported a method of transfer of one submandibular gland to the submental space in a small pilot series of eligible surgical patients. The submental space was shielded during postoperative radiation therapy. The transferred gland continued to function after the completion of radiation therapy and none of the patients developed xerostomia. The purpose of this article is to present the technique of submandibular gland transfer in detail and to evaluate the postoperative survival and function of the transferred submandibular glands. DESIGN: Prospective clinical trial. METHODS: The submandibular gland was transferred on eligible patients as part of their surgical intervention. The patients were followed clinically, with salivary flow and radioisotope studies. RESULTS: We performed the surgical transfer of the submandibular salivary gland in 24 of 25 patients placed on the protocol. All the glands survived transfer and functioned well postoperatively as demonstrated on the salivary flow and the radioisotope studies. The surgical transfer was relatively simple and added 45 minutes to the surgical procedure. There were no complications attributed to the submandibular gland transfer. CONCLUSIONS: We have successfully demonstrated that the submandibular gland can be surgically transferred to the submental space with its function preserved. The gland seems to continue functioning even after radiation therapy with the appropriate shielding. This surgical transfer procedure has the potential to change the way we currently manage patients with head and neck cancer.

Adult↗

Bilateral irradiation of head and neck induces an enhanced expression of substance P in the parasympathetic innervation of the submandibular gland.

Substance P and calcitonin gene-related peptide (CGRP) are present in nerve fibers innervating the submandibular gland. Radiotherapy of tumors in the head and neck region usually embraces the salivary glands in the irradiated field and consequently a dramatic decrease in salivary function is seen. In this study, the submandibular glands and ganglia of rats subjected to fractionated irradiation were examined by use of immunohistochemical techniques for demonstration of substance P and CGRP. Irradiation was given on five consecutive days (daily doses of 6-9 Gray) with unilateral or bilateral irradiation techniques. Specimens of control and experimental animals were processed in parallel. A marked increase in the expression of substance P in the ganglionic cells--presumably parasympathetic--and in the number of fibers showing substance P-like immunoreactivity in association with acini and small ducts was seen in response to bilateral irradiation. (Surprisingly, unilateral irradiation of the parotid area had no effect on peptide distribution in the irradiated gland and ganglion). No changes in the pattern of CGRP immunoreactivity occurred. In the trigeminal ganglion, which supplies the submandibular gland with the majority of the sensory substance P- and CGRP-containing nerve fibers, no changes in the expression of substance P or CGRP immunoreactivity were seen. The results suggest that bilateral irradiation leads to an increase in the synthesis of substance P-like substance in the parasympathetic ganglionic cells supplying the submandibular gland with secretory nerves, and can thus be an additional factor in explaining the altered secretory capacity of salivary glands.

Animals↗

Biologically active polypeptides in submandibular glands.

Since the discovery of kallikreins in the submandibular glands in 1963 by Werle and Roden, a great number of biologically active polypeptides has been purified from, or claimed to be present in, the submandibular of the mouse and of other species. In this review, available data on the occurrence, chemical properties, localization, hormonal control, synthesis, secretion, and possible physiologic roles of 25 biologically active factors in mouse submandibular gland are analyzed. In general, these factors are androgen dependent, but not affected by the sex genotype, and are localized in the granular convoluted tubule cells in the gland. They are secreted into the saliva, but are also found in the circulation. Their physiologic roles are largely unknown.

Angiotensins↗

The alpha and gamma subunits of 7S nerve growth factor are present in excess concentrations in the mouse submandibular gland.

The 7S nerve growth factor molecule, found in the mouse submandibular gland, is comprised of three distinct protein subunits named alpha, gamma and beta-NGF. In this paper, radioimmunoassays specific for each subunit were used to measure the concentrations of these subunits in homogenates of mouse submandibular gland. It was determined that there were excess concentrations of both the alpha and gamma subunits, more than enough to bind all of the beta-NGF in the gland to form 7S-NGF. The radioimmunoassay data was confirmed by gel filtration experiments. In the gel filtration experiments, the excess alpha and gamma subunits eluted at positions which would indicate that these excess subunits were free and not bound in the 7S-NGF complex. The identity of the excess alpha and gamma subunits was substantiated by ion exchange chromatography, isoelectric focusing polyacrylamide gels and immunoblotting experiments. In conclusion, there are considerable quantities of alpha and gamma subunits present in the submandibular gland which are not bound to beta-NGF. The functional significance of these excess concentrations of the alpha and gamma subunits is not known.

Animals↗

Contribution of confocal laser scanning microscopy to glycochemistry of mouse and rat submandibular glands by single and double lectin staining.

The localization of individual glycosidic residues in the mouse and rat submandibular glands was examined using confocal laser scanning microscopy (CLSM). For these organs we tested some procedures of fixation and embedding to better understand the distribution of some lectin-probes inside well preserved secretory cells and observed that fixation and inclusion steps did not influence appreciably the location and intensity of the reactive sites. The fixation mixture of 4% paraformaldehyde, 1% glutaraldehyde and 0.2% picric acid produced the most satisfactory results. In specimens labeled with PNA-, Con A-, LTA-FITC and WGA-, DBA-TRITC lectins, the convoluted granular tubules (CGT) proved to be composed of secretory granules with high-density lectin labeling. The complex organization of secretory glycocomponents within the granule matrix was further resolved by double labeling and dual scanning experiments. Some lectins exhibited colocalization while others displayed differential localization providing information about the occurrence of O- and N-linked glycoconjugates. The CLSM technique applied to fluorochrome-conjugated lectins also revealed a more marked dimorphism in the rat rather than in the mouse submandibular gland. In particular, the male rat submandibular gland was found to consist of CGT heterogeneous cell populations, while the mouse submandibular gland did not show glycochemical differences between cells. Female rats exhibited a lectin profile very different from that of female mouse.

Animals↗

Changes in phosphodiesterase activity in the developing rat submandibular gland.

Developmental changes (from 2 to 26 weeks) in phosphodiesterase (PDE) activity in the rat submandibular gland were investigated. Major activities for both cAMP- and cGMP-PDE were present in the 100000 x g supernatant fractions (70-90% of total activities), but not in the pellet fractions, during development. The effects of stimulators (Ca(2+)/calmodulin and cGMP) and inhibitors (cGMP, cilostamide, rolipram and zaprinast) were investigated in the supernatant fractions. During development, PDE4 (cAMP-specific PDE) was a major PDE, indicating that the majority of cAMP is hydrolysed by PDE4. In the young rat, PDE1 hydrolysed cGMP three-fold more than the control, and PDE2 (cGMP-stimulated PDE) was present, indicating that the concentration of intracellular cGMP may be enhanced, and cGMP may function in the growth pathway in the submandibular gland. Chromatograms eluted on a Mono Q HR5/5 ion-exchange column supported the results of the inhibition studies: PDE1, PDE2, PDE3, PDE4 and PDE5 were present in the young submandibular gland, and PDE1, PDE3, PDE4 and PDE5 in the adult gland. Expression of PDE5 was detected by inhibition studies, reverse transcriptase-polymerase chain reaction and Western blotting in the submandibular gland.

3',5'-Cyclic-AMP Phosphodiesterases↗

The localization of kallikrein in the dog and guinea-pig submandibular glands.

In the dog and guinea-pig submandibular glands kallikrein seems to be present in the striated duct cells. Following sympathetic nerve and in vivo isoproterenol stimulation of the dog and guinea-pig submandibular gland respectively, there is a reduction of kallikrein concentration. Ultrastructurally this reduction corresponds to the decrease of straited duct secretory granules in both species. Parasympathetic stimulation also causes some release of kallikrein from both species.

Animals↗