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Submandibular gland transfer in the correction of dry eye.

Following rabbit experiments which showed that microvascular submandibular gland transfer could ameliorate the deleterious effects of lacrimal gland ablation, a clinical transfer of the submandibular salivary gland to the temple region was performed. The beneficial results from this initial operation have led to the procedure being used in a total of eight patients with 12 transfers. The procedure has led to increased moisture in the eye and decreased symptoms but the assessment of change in corneal function and pathology is difficult.

Adolescent↗

Expression and postnatal changes of adrenergic receptor subtype mRNA in rat submandibular glands.

Adrenergic receptors (ARs) are involved in regulating saliva secretion and composition in salivary glands. Nine AR subtypes, including three alpha1-ARs (alpha1a-, alpha1b- and alpha1d-ARs), three alpha2-ARs (alpha2A-, alpha2B- and alpha2C-ARs) and three beta-ARs (beta1,beta2- and beta3-ARs), have been identified through molecular cloning. The five subtype genes, alpha1a-, alpha1b-, alpha2A-, beta1-, and beta2-ARs, were expressed in rat submandibular glands. In contrast, the other four subtype mRNAs, alpha1d-, alpha2B-, alpha2C- and beta3-ARs, were not detected by reverse transcription-polymerase chain reaction (RT-PCR). The steady-state mRNA expression for the five AR subtypes in rat submandibular glands was measured by quantitative competitive RT-PCR using synthetic DNA as internal standard at different stages of postnatal development. The relative rank order of AR subtype mRNA expression was alpha1a>beta2>beta1>alpha2A>alpha1b at all stages except that beta1- and alpha2A-subtypes were reversed at 2 weeks of age. The gene expression of alpha1a-AR subtype relative to total AR was low at 2 weeks of age and increased and reached a maximum at 6 weeks of age, whereas those patterns of alpha2A-, beta1- and beta2-AR subtypes were similar to each other and their gene expressions were high at 2 weeks of age and then decreased. On the other hand, the gene expression of alpha1b-AR subtype did not change over the different stages in relation to that of a housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase, and to total AR. Although rat submandibular glands contain the five AR subtype mRNAs, distinct subtype-specific expression is evident.

Age Factors↗

The effects of p-octopamine on salivary flow rates and protein secretion by rat submandibular glands.

The effects of p-octopamine injected i.v. and i.p on salivary flow rates and proteins secreted by the submandibular glands of rats were studied with and without various types of autonomic blockers at different doses, and with two enzyme inhibitors. The salivary flow rates and the amounts of protein secreted progressively increased with increasing doses injected both i.v. and i.p., whereas they were dramatically reduced with almost all autonomic blockers and disulfiram, a dopamine-beta-hydroxylase inhibitor. Salivation was completely abolished in response to p-octopamine in combination with metoprolol or phenoxybenzamine at high doses, and simultaneous injections of prazosin and propranolol. The concentration of protein in submandibular saliva in response to p-octopamine injected i.v. and i.p. was not dose-dependent and significantly increased with all of the alpha-blockers except yohimbine, and with atropine and disulfiram. The protease activity was dose-dependent but was reduced significantly with alpha-blockers except yohimbine and with two enzyme inhibitors. The alpha-type of protein was secreted in response to p-octopamine injected i.v. and i.p. at all doses except with the lowest dose i.p., which caused the beta-type to be secreted. The alpha-type was completely replaced by the beta-type with all alpha-blockers at all doses, except with yohimbine, but no change was observed with various types of beta-blockers, yohimbine, atropine, and two enzyme inhibitors. Thus, p-octopamine could stimulate both the alpha- and beta-adrenoceptors in the submandibular glands of rats.

Adrenergic alpha-Antagonists↗

Tissue hyperosmolality as a causal factor in vasodilatation following sympathetic stimulation of the submandibular gland.

In a previous investigation, parasympathetic activation of the submandibular gland in the cat was found to cause a considerable increase of regional tissue osmolality, the degree of which was related to the evoked functional hyperemia; intra-arterial hypertonic infusion to the resting gland producing tissue hyperosmolality of similar magnitudes caused graded and marked dilatations (Lundvall and Holmberg 1974). It was concluded that hyperosmolality contributes significantly to the functional hyperemia response. In the present study evidence is presented to indicate that tissue hyperosmolality is a mediator of the dilatation associated with sympathetic activation as well. An increase of tissue hyperosmolality, as traced in the venous effluent, was found at all frequencies of sympathetic stimulation (2-16 Hz). At high stimulation rates it sometimes exceeded the resting control level by more than 20 mOsm/kg H2O. There was a direct relation between the degree of venous hyperosmolality and the hyperemia response observed immediately after cessation of stimulation. Comparison of the dilator effects evoked by sympathetic stimulation and by hypertonic infusion to the resting gland indicated that tissue hyperosmolality is an important causal factor for the nerve induced dilatation, especially at low and moderate stimulation rates.

Animals↗

Intracellular accumulation of basement membrane components during morphogenesis of rat submandibular gland.

The distribution of two basement membrane (BM) components, laminin (LN) and type IV collagen (COLL IV), during acino-tubular morphogenesis of rat submandibular gland was examined immunohistochemically to determine the role of BM in the development of acino-tubular structures. On day 14 of gestation, LN could be found only in the BM separating an undifferentiated cell cluster of gland epithelium from surrounding mesenchyme. However, during a short period through days 15 to 17, LN was detected not only in the BM but also in intracellular vesicles of the cells of the terminal cluster. Immunoelectron microscopy showed the intracellular immunoreactive sites to be rough endoplasmic reticulum, indicating that active LN synthesis occurs in the cells of the terminal cluster. Intracellular immunostaining of LN disappeared completely on day 19 with the development of simple epithelium from the cell cluster, even though BM remained reactive. COLL IV also was accumulated in the intracellular vesicles of terminal cluster cells on day 16 of gestation but not on day 19. These results indicate that synthesis of certain BM components is transiently stimulated in gland epithelium before the formation of simple epithelial structure, and that these components are significantly involved in morphogenesis of the submandibular gland.

Animals↗

Early phenotypic and genotypic alterations in submandibular gland oncogenesis in rats.

The present study evaluates the phenotypic and genotypic changes that take place during early oncogenesis. The submandibular glands of male rats were injected with a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. Gland samples were taken at 0, 7, 30 and 150 days post-injection and submitted to histological, biochemical, immunocytochemical and PCR evaluation. Histopathological analysis was performed on hematoxylin-eosin stained slides. Total protein content was assessed by Lowry's method and the protein profile was analyzed by 12% SDS-PAGE. Bcl-2 was demonstrated by silver-enhanced gold immunolabeling. p53 immunolabeling was performed using the streptavidin-biotin system. All the treated animals developed carcinoma-like lesions at 30 and 150 days. Total protein concentration rose significantly (p < 0.05) above control values at 7, 30 and 150 days. The treated glands exhibited positive immunolabeling for p53 in the nuclei of neoplastic cells at 30 and 150 days. Treated glands also showed positive cytoplasmic immunolabeling for Bcl-2, exhibiting statistically significant differences between 7, 30 and 150 days (p = 0.0015), and with controls (p < 0.0001). No p53 mutations were observed whereas a point mutation, C-to-A, of the Bcl-2 gene was detected at 7, 30 and 150 days by PCR amplification. This mutation led to a single aminoacid change (thre --> asn) in the protein molecule. Our results suggest that the early histopathological changes correspond to quantitative and qualitative protein changes. The histopathological, biochemical, immunocytochemical and genetic alterations observed during the course of experimental carcinogenesis in the submandibular gland of the rat could constitute reproducible indices of malignant transformation applicable to human oncogenesis, given the high degree of homology between the oncogenes of mice, rats and human beings.

9,10-Dimethyl-1,2-benzanthracene↗

Further studies on protein secretion by rat submandibular glands in response to the beta 1- and alpha 1- adrenoceptor stimuli during postnatal development.

Developmental changes in salivary proteins secreted by the submandibular glands of male rats from 2 to 10 weeks of age in response to the beta 1- and alpha 1-adrenoceptor agonists, isoproterenol, methoxamine, dopamine, phenylephrine, epinephrine, norepinephrine and alpha-methylnoradrenaline, were studied by different electrophoreses and amino acid analysis. The electrophoretic patterns of proteins secreted by the glands in response to both stimuli changed dramatically every week up to 7 weeks of age, but thereafter appeared similar to those of adult submandibular salivary proteins. At 2 and 3 weeks of age, a few differences were found between the types of proteins secreted in response to two stimuli. These findings were also confirmed by an analysis of amino acid composition. The glandular levels of cGMP from 2 weeks and cAMP from 3 weeks of age were significantly elevated in response to methoxamine and isoproterenol. We conclude that functional maturation precedes morphological maturation in the submandibular glands of male rats.

Adrenergic Agonists↗

Role of nitric oxide in the regulation of blood flow in the rat submandibular gland during carotid artery occlusion.

The possible involvement of nitric oxide in the preservation of blood flow to the rat submandibular gland after uni- or bilateral occlusion of the common carotid was studied. Glandular blood flow and mean blood pressure were monitored before, during and after carotid occlusion in the presence and absence of the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine-methyl-ester (L-NAME). To calculate vascular resistance, the local perfusion pressure distal to the point of occlusion was also measured. In normal rats, uni- or bilateral carotid occlusion resulted in an immediate decrease in ipsilateral glandular blood flow. After the cessation of carotid occlusion, hyperaemia was observed in the submandibular gland. Both local perfusion pressure and vascular resistance decreased during carotid occlusion. In the group pretreated with L-NAME, trends in blood-flow responses to uni- or bilateral occlusion were identical to those registered in the control groups, though the magnitude of the alterations was significantly less. The well-maintained glandular blood flow was due to functioning vascular anastomoses and compensating dilatation of glandular blood vessels. Nitric oxide had only a restrained effect on this compensatory mechanism.

Analysis of Variance↗

Nodular hyperplasia of oncocytes in mouse submandibular glands.

Multinodular hyperplastic foci of oncocytes found in mouse submandibular glands were examined ultrastructurally, and their morphogenesis discussed. The oncocytic foci showed lobular architecture surrounded by myoepithelial cells, and tubular formations were frequently present. The cytoplasm of the oncocytes was distended by tightly-packed and non-oriented mitochondria which varied in size and shape. Furthermore, varying numbers of electron-dense fine granules, which were remnants of secretory granules of granular duct cells, were scattered in the cytoplasm. Within the salivary gland parenchyma neighboring the hyperplastic foci of oncocytes, intermediate-type cells between oncocytes and normal salivary gland epithelia were found. The result of the present study shows that the oncocytic change has a tendency to be more frequent in the granular ducts and less in the striated ducts, and that some oncocyte-transformed cells proliferate to form nodular hyperplastic foci.

Animals↗

Abundant androgen regulated mRNAs in mouse submandibular gland: cell-free translation of renin precursor mRNA.

Submandibular glands of male mice contain at least four abundant mRNAs that occur at low concentrations in glands of females. The male-specific mRNAs code for polypeptides of 48,000, 43,000, 29,000, and 27,000 MW. Androgenic regulation of these mRNAs is illustrated by their apparent absence in glands of castrate males and by their accumulation in glands of females treated with testosterone. Selective hybrid-arrested translation experiments also indicate reduced levels of these male-specific sequences in female gland cytoplasm. The 48,000 MW male-specific polypeptide is reduced in translation products directed by gland mRNA from C57BL10/J mice (variants deficient in salivary renin), suggesting the corresponding mRNA codes for a renin precursor. The identity of this polypeptide is confirmed by immune selection with renin-specific antibody.

Animals↗

Sialolith crystals localized intraglandularly and in the Wharton's duct of the human submandibular gland: an X-ray diffraction analysis.

The exact cause of the formation of sialoliths is unknown. Detailed knowledge of pathogenesis of sialolithiasis and composition of sialoliths is necessary to define new therapeutic procedures. The crystalline components of 23 sialoliths of human submandibular gland were investigated by X-ray powder diffraction analysis. All of the sialoliths localized in the ducts in the submandibular gland consisted of hydroxylapatite. However, in the sialoliths in the Wharton's duct, hydroxylapatite as well as whitlockite and brushite could be found in all except one case. Whitlockite was observed more often in the nucleus of the sialoliths and it was a common co-phase along with hydroxylapatite. The nucleus in one sialolith consisted of brushite and the cortex showed a co-phase of hydroxylapatite and brushite. The occurrence of whitlockite in the sialoliths in Wharton's duct may be due to a higher concentration of calcium and phosphate in saliva in this duct.

Adult↗

Potassium uptake in the mouse submandibular gland is dependent on chloride and sodium and abolished by piretanide.

Nervous or hormonal stimulation of salivary secretion in vivo is associated with a pronounced efflux of K+ from the secretory, acinar cells into the blood. This K+ efflux is followed in the post-stimulus period by a reuptake of K+ into the glandular tissue. In the present study we monitor the changes in [K+] of physiological solutions perfusing a flow chamber containing isolated segments of mouse submandibular glands. Nervous stimulation or the application of exogenous acetylcholine (ACh, 10(-5) M) to the isolated glandular tissue results in characteristic changes in the [K+] of the superfusate, indicating net K+ release followed by K+ reuptake. The post-stimulus reuptake of K+ is shown to be susceptible to blockade by either ouabain (10(-3) M) or piretanide (10(-4) M). The reuptake was markedly attenuated if Cl- in the superfusate was replaced by either NO3- or SO4(2-). The K+ uptake was, however, unaffected when Br- replaced Cl- in the superfusate. Similar effects were observed in the unstimulated glandular tissues. The introduction of Cl-(-)free media containing either NO3- or SO4(2-) resulted in a loss of K+ from the tissue which was followed, upon reintroduction of Cl-, by a pronounced uptake of K+. When Br- was substituted for Cl- there was very little change in [K+] upon removal or reintroduction of Cl-. The uptake of K+ induced by reintroduction of Cl- after a period of NO3- or SO4(2-) superfusion was blocked by both ouabain and piretanide. This uptake of K+ was also dependent on the presence of extracellular Na+. Both Cl- and Na+ had to be present in the superfusing medium for K+ uptake to be fully manifest. These findings indicate that the K+ uptake observed in both the resting and stimulated submandibular gland cannot be explained as solely due to the activity of the Na+-K+-adenosine triphosphatase (Na+-K+-ATPase). The demonstrated anionic selectivity, dependence on extracellular Na+ and susceptibility to blockade by the diuretic piretanide would strongly suggest that a coupled Na+-K+-Cl- co-transport system operates in submandibular glands as it does in other transporting epithelia to achieve K+ uptake.

Acetylcholine↗

Chronic sclerosing sialadenitis of the submandibular gland is mainly due to a T lymphocyte immune reaction.

The aim of our study was to investigate the role of immunopathological processes in the pathogenesis of chronic sclerosing sialadenitis of submandibular glands (Küttner tumor). For this purpose, biopsy specimens from submandibular glands of 22 patients with the histological diagnosis of chronic sclerosing sialadenitis were analyzed. Paraffin-embedded tissues were immunostained for T-lymphocyte subsets (CD3, CD4, CD8), cytotoxic T cells (granzyme B), B cells (CD20, Ki-B3), and macrophages (Ki-M1P). Polymerase chain reaction and capillary electrophoresis were used to detect rearrangements of the T-cell receptor gamma chain and the CDRIII region of the immunoglobulin heavy chain. In all cases, abundant cytotoxic T cells were found, especially in close association with ducts and acini. T-cell receptor gamma chain rearrangements showed a monoclonal pattern in 6 cases (27.3%), an oligoclonal pattern in 8 (36.4%), and a polyclonal pattern in 8 (36.4%). The B-cell reaction was less pronounced and largely restricted to lymph follicles. Molecular analysis of immunoglobulin heavy chain revealed a polyclonal rearrangement in 17 cases (77.3%). In conclusion, there is an intimate relationship between the T-cell-dominated inflammatory infiltrate and acinar and duct cells. This, together with the frequent demonstration of monoclonal and oligoclonal populations of cytotoxic T cells and their histopathological behavior, suggests that chronic sclerosing sialadenitis may be the result of an immune process triggered by intraductal agents.

Adult↗

The influence of aging on lysosomal acid DNase of the rat submandibular gland.

Lysosomal and cytoplasmic fractions were prepared from rat submandibular glands for investigation of the release of lysosomal acid DNase in relation to aging. It was found that the acid DNase activity ratio for cytoplasmic/lysosomal fractions in rats aged 27 months was higher than that in three-month-old rats. The release of acid DNase from the lysosomal fraction by shaking was markedly increased in the fraction from the older animals.

Aging↗

Immunocytochemical localization of renin in the submandibular gland of the mouse.

Renin was localized in the submandibular gland of the adult mouse at light and electron microscopic levels by the unlabeled antibody enzyme method of Sternberger. At the light microscopic level, renin was confined to the granular convoluted tubule (GCT) segment of the gland with considerable variation among GCT cells in intensity of staining. Some GCT cells failed to stain for renin. The pattern of staining was the same in the gland of male and female mice, but in the glands of females GCT segments were smaller and less numerous. At the electron microscopic level, staining for renin was also confined to the GCT cells, and was localized exclusively to the secretory granules. The intensity of staining of the secretory granules within a given GCT cell varied; some cells contained only minimally reactive or negative secretory granules. All other organelles within the GCT cell, except condensing vacuoles, failed to stain.

Animals↗

Localization of neonatal secretory proteins in different cell types of the rat submandibular gland from embryogenesis to adulthood.

In the neonatal rat submandibular gland, Type III cells contain a group of related proteins that we call the B1-immunoreactive proteins (B1-IP; 23.5, 26, and 27.5 kDa). Type I cells lack these, but synthesize a different protein, Protein C (89 kDa). With maturation of the gland, these neonatal cell types are no longer seen in the seromucous acini, which are no longer reactive for the B1-IP. Here, we report the ultrastructural immunocytochemical localization of the B1-IP and Protein C over the course of development. From their first appearance in the embryo, the B1-IP and Protein C are present in different cells which become morphologically typical Type I and III cells prior to birth. At all stages, Type I cells have strong Protein C labeling and no B1 labeling. By 3 days postpartum, ultrastructurally atypical Type III cells are seen (Type IIIP); these label for the B1-IP, but also show labeling with antibody to Protein C. In the next week, as mucous cells appear in the acini, these show both B1-IP and C labeling; the B1 marker is lost by 30 days postpartum, but adult mucous acinar cells continue to show Protein C reactivity. In view of the appearance of Protein C reactivity in neonatal Type IIIP and then in mucous cells, and the presence of B1 reactivity in early but not mature mucous cells, we suggest that Type III cells differentiate into mucous cells and that Type IIIP cells are intermediates in this transformation. We see no evidence for the differentiation of either Type III or mucous cells from Type I cells, although our data cannot rule out this possibility. In adult glands, cells with B1 labeling are seen in intercalated ducts. Cells that appear to be Type I cells are also present in these ducts and label for Protein C. Double labeling for B1-IP and Protein C demonstrated that the two markers were exclusively present in different cells within intercalated ducts. This is of considerable interest, as intercalated ducts have been reported to be the stem cell population for normal and trauma-induced cellular replacement.

Aging↗

Effects of repeated androgen treatments on metabolism and nuclear binding of androgen in the infant murine submandibular gland.

1. Androgen responsiveness of esteropeptidase of the murine submandibular gland developed rapidly in normal males compared with in normal females and castrated males. 2. Repeated treatments of infant mice of both sexes with testosterone (T), 5 alpha-dihydrotestosterone (DHT) or 5 alpha-androstane-3 alpha, 17 beta-diol increased androgen responsiveness of this enzyme, but did not affect those of 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDase; androgen metabolizing enzymes) of the gland. 3. Exchange assay of nuclear androgen receptor using 3H-DHT showed that in both sexes, amounts of binding in animals pretreated with T were higher than those in animals pretreated with sesame oil. 4. These results suggest that there is parallelism between the androgen responses and amounts of nuclear androgen binding, not androgen responses of 5 alpha-reductase and 3 alpha-HSDase.

3-Hydroxysteroid Dehydrogenases↗

Co-localization of the nerve growth factor precursor protein and mRNA in the mouse submandibular gland.

Indirect immunofluorescence on frozen sections of the mouse submandibular gland (MSG) and affinity-purified sera directed against synthetic peptides that reproduce sequences of the nerve growth factor (NGF) precursor protein permitted its localization in the basal parts of the cells forming the secretory tubules. In situ hybridization experiments employing 35S-labeled NGF cDNA probe localized the NGF mRNA in the same region. Conversion of proNGF to mature NGF results in an altered localization of the cleaved peptide throughout the cytoplasm of the tubular cells with a preferential concentration at their apical pole.

Animals↗