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Immunocytochemical localization of salivary peptide P-C in human submandibular gland.

Human saliva contains a proline-rich polypeptide, salivary peptide P-C, which potentiates insulin release and reduces glucagon release from perfused rat pancreas to decrease blood glucose level. To elucidate the process of secretion into humoral fluid of this peptide morphologically, we investigated ultrastructural localization of P-C in human submandibular gland by immunogold technique with anti-peptide P-C whose specificity to P-C was confirmed by immunoblotting. The labeling with gold particles which represents the distribution of P-C-like-immunoreactivity (P-C-LI) was detected in the secretory granules and rough endoplasmic reticula of the acinar serous cells and in few mucosa cells. P-C-LI was also observed in the lumen of striated duct but not intracellularly in the ductal cells themselves, indicating that P-C is not probably reabsorbed there. These results suggest that salivary peptide P-C is present in acinar serous cells, is secreted into the oral cavity, and may be reabsorbed through the digestive tract to modulate the blood glucose level after feeding.

Aged↗

In situ hybridization and monoclonal antibody analysis of plasma membrane Ca-pump mRNA and protein in submandibular glands of rabbit, rat and man.

The degree of supersaturation of saliva with calcium (Ca) is related to the mineral phase of enamel in erupted teeth, the incidence of caries, and the formation of calculus. The mechanisms for regulating salivary Ca concentration are therefore of relevance to dentistry. Sections of rabbit, rat and human submandibular gland (SMG) were processed for immuno-histochemistry with a specific anti-plasma membrane Ca-pump antibody, 5F10. Western blots confirm that the molecular weight of the proteins identified by our antibody (135 kDa) is consistent with an appropriate molecular weight for PMCA antigen (135-150 kDa). Tissue sections were also processed for in situ hybridization to study the distribution of the PMCA mRNA isoforms. In mammals, the PMCA1 gene is reported to code for a PMCA protein with a role in maintaining the intracellular Ca levels in both epithelial and non-epithelial cells. Other genes including the PMCA2 and PMCA4 genes may code for PMCA proteins specific to Ca transporting tissues. Our studies demonstrate cytoplasmic labeling of PMCA mRNA with hPMCA-1 and hPMCA-4 specific cDNA probes in humans, and rPMCA-1 and rPMCA-2 specific oligonucleotide probes in rats. Labeling of PMCA protein and all mRNA isoforms was found in the cytoplasm of the interlobular and intralobular ducts (except for intercalated ducts). The demonstrated presence of PMCA in SMGs of rabbit, rat, and man, may suggest a role for PMCA in the regulation of intracellular Ca and in a mechanism for regulating and maintaining the high concentration of Ca in salvia.

Animals↗

Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms.

Analyses of gene expression profiles at five different stages of mouse submandibular salivary gland development provide insight into gland organogenesis and identify genes that may be critical at different stages. Genes with similar expression profiles were clustered, and RT-PCR was used to confirm the developmental changes. We focused on fibroblast growth factor receptor 1 (FGFR1), as its expression is highest early in gland development. We extended our array results and analyzed the developmental expression patterns of other FGFR and FGF isoforms. The functional significance of FGFR1 was confirmed by submandibular gland organ culture. Antisense oligonucleotides decreased expression of FGFR1 and reduced branching morphogenesis of the glands. Inhibiting FGFR1 signaling with SU5402, a FGFR1 tyrosine kinase inhibitor, reduced branching morphogenesis. SU5402 treatment decreased cell proliferation but did not increase apoptosis. Fgfr, Fgf and Bmp gene expression was localized to either the mesenchyme or the epithelium by PCR, and then measured over time by real time PCR after SU5402 treatment. FGFR1 signaling regulates Fgfr1, Fgf1, Fgf3 and Bmp7 expression and indirectly regulates Fgf7, Fgf10 and Bmp4. Exogenous FGFs and BMPs added to glands in culture reveal distinct effects on gland morphology. Glands cultured with SU5402 were then rescued with exogenous BMP7, FGF7 or FGF10. Taken together, our results suggest specific FGFs and BMPs play reciprocal roles in regulating branching morphogenesis and FGFR1 signaling plays a central role by regulating both FGF and BMP expression.

Animals↗

Age-dependence of isoproterenol-induced DNA synthesis in submandibular glands of BALB/c mice.

The age-dependency of isoproterenol (IPR) induced DNA synthesis was investigated in different organs of Balb/c mice. Although modifications of the physiological rate of DNA synthesis after IPR injection occurred also in liver and spleen, a quasi-linear decrease of the peak of IPR response with advancing age was observed only in submandibular glands. Such a decrease was observed when animals were injected with 10(-4) g IPR per g body weight, lower doses being unable to discriminate between young and old mice. In spite of some differences between mice and rats, the early appearance and linearity of the age-dependency remain common features of IPR response.

Age Factors↗

[Immunohistochemical study on the distribution and coexistence of SP, VIP and NPY in the rat submandibular gland].

The distribution and coexistence of SP, VIP and NPY in the submandibur gland of rat have been studied with immunohistochemical ABC methods. The results indicated that SP, VIP and NPY-IR nerve fibers were presented as varicose and linear profiles, which mainly travelled around acini, duct and blood vessels. The ganglion cells in submandibular gland showed SP, VIP and NPY-IR all positive immunocytochemically. The findings indicated that the neuropeptides were coexisted in the same neuron body and might play the role of regulation of the secretary activity and effective blood supply.

Animals↗

Effects of parasympathectomy on androgen responses of the rat submandibular gland.

The effects of androgen on the activities of esteropeptidase and delta 4-3-ketosteroid 5 alpha-reductase were investigated. Ratios of the denervated side to the non-operated side for gland weight and esteropeptidase activity were higher in androgenized females than in untreated females. The specific activities of 5 alpha-reductase were increased by denervation but the total activities were decreased. The results suggest that parasympathectomy leads to higher androgen responsiveness in the rat submandibular gland, which relates to the observed reduction in weight loss of the gland and the increases in its esteropeptidase activities.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗

Concurrent cytochemical localization of adenylate cyclase and peroxidase in the developing rat submandibular gland.

An electron microscopic histochemical technique for the concurrent localization of adenylate cyclase and endogenous peroxidase is described. The procedure involves incubation of glutaraldehyde fixed tissue in adenylate cyclase medium followed by washing and incubation in 3,3'-diaminobenzidine tetrahydrochloride medium to demonstrate peroxidase activity. Adenylate cyclase was localized at the cell surface of the tissue investigated (20 day fetal rat submandibular gland) while peroxidase was localized in the rough endoplasmic reticulum and secretory granules of some cells. Biochemical and histochemical controls indicate that the procedure is valid. The potential use of this procedure and variations of the procedure are discussed.

Adenylyl Cyclases↗

Transforming growth factor beta in chronic obstructive sialadenitis of human submandibular gland.

BACKGROUND: The exact pathomechanism of inflammation progress and fibrosis in chronic obstructive sialadenitis is unknown. The aim of the present study was to assess whether there is an association between transforming growth factor beta (TGF-beta) and fibrogenic process of chronic sialadenitis. METHODS: Tissue samples of 12 patients with chronic sialadenitis and 4 normal tissue samples of the submandibular gland were examined immunohistochemically for identification of TGF-beta. TGF-beta1 messenger RNA (mRNA) expression was analysed semiquantitatively using reverse transcription polymerase chain reaction and gel electrophoresis to correlate its expression levels with stages of the disease. RESULTS: TGF-beta positive cells could be found in the secretory duct system of all examined samples. However, an intense TGF-beta immunoreactivity was observed in inflamed salivary glands. With progress of disease TGF-beta1 mRNA expression increases significantly. CONCLUSION: Expression of TGF-beta in chronic sialadenitis and its apparent increase in advanced stages of the disease, suggests that this growth factor may play a role in glandular fibrosis.

Chronic Disease↗

Spinal metastasis of submandibular gland adenoid cystic carcinoma: a case report.

BACKGROUND: Adenoid cystic carcinoma is a rare malignant tumor of the salivary glands, with pulmonary metastasis being the most common site of distant metastasis. CASE DESCRIPTION: The author reports an unusual case of intradural adenoid cystic carcinoma metastasis of the lumbar spine in a patient with recent prostate carcinoma suffering from progressive back pain. The primary submandibular tumor was resected 7 years before the appearance of the spine metastasis, and a prostatic carcinoma had been detected 1 year before. The patient died of systemically advanced disease a few months after surgical decompression. CONCLUSION: Late submandibular gland adenoid cystic carcinoma recurrence may present as an intradural lumbar spine metastasis. The case further displays difficulty in diagnosis and differentiation of metastasis in case of metachronous tumors.

Carcinoma, Adenoid Cystic↗

Phosphodiesterases 1 and 2 regulate cellular cGMP level in rabbit submandibular gland cells.

In rabbit salivary glands, stimulation of muscarinic cholinergic receptors causes production of cGMP through intracellular Ca2+ and nitric oxide. In this study, we investigated a role of cyclic nucleotide phosphodiesterase (PDE) in regulating the cellular cGMP level by using cells dispersed from the submandibular gland. Methacholine, a cholinergic agonist, rapidly elevated the cGMP level. The elevation was greatly enhanced by IBMX, a non-specific inhibitor for most isoforms of the 11 PDEs. The cGMP level was also elevated by MM-IBMX and EHNA, which inhibit the activities of PDE1 and PDE2, respectively. The elevation by the simultaneous application of the two drugs corresponded to 90% of that by IBMX. Therefore, PDE1 and PDE2 are the main PDEs that act to degrade cGMP in methacholine-stimulated cells. The presence of the two PDEs was confirmed by assaying their activities of the cell lysate. In unstimulated cells, the cGMP level was elevated by MM-IBMX and little elevated by EHNA. While the PDE2 activity was thus low, it was estimated that methacholine increases its activity approximately 50-fold. The strong activation can be explained by the elevation of the cGMP level because PDE2 is a cGMP-stimulated PDE. SNAP, a nitric oxide donor, causes production of cGMP without a receptor-operated increase in intracellular Ca2+ concentration. In SNAP-stimulated cells, MM-IBMX elevated the cGMP level higher than in methacholine-stimulated cells although the PDE1 activity is dependent on Ca2+/calmodulin. Besides Ca2+, other factors may regulate the PDE1 activity in living cells.

1-Methyl-3-isobutylxanthine↗

Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.

In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.

Animals↗

Submandibular gland squamous metaplasia mimicking recurrent squamous cell carcinoma: diagnostic dilemma.

New masses in the head and neck region of previously treated patients with cancer are considered suggestive of potential recurrence. Fine-needle aspiration is an excellent primary tool for evaluating these masses but may show atypical squamous cells that mislead clinicians to pursue aggressive treatment. We describe 3 patients in whom submandibular gland masses developed after radiation therapy for squamous cell carcinoma and for whom subsequent excisional biopsy findings showed benign squamous metaplasia. We review distinguishing clinical and histopathologic features that may allow these lesions to be identified. We also propose a treatment algorithm for this uncommon clinical scenario.

Aged↗

Mouse submandibular gland mucin: embryo-specific mRNA and protein species.

Mouse submandibular salivary gland (SMG) mucin is the primary histodifferentiation product of submandibular epithelia. We demonstrate marked differences between embryonic, neonatal, and adult SMG mucin mRNA and protein by Northern and Western blot analyses: E17 and 1-day-old neonates exhibit two unique mucin transcripts (1.20 and 0.85 kb) which are approximately 19% greater or smaller in size than the single (1.01 kb) adult transcript. Two embryonic protein isoforms (Mr approximately 110 and 152 kDa) are immunodetected compared to a single adult protein (Mr approximately 136 kDa), with the larger (approximately 152 kDa) embryonic isoform persisting in neonatal glands. Mucin transcripts are localized to the branching epithelia in E14 and older SMGs, with increased hybridization signal being seen in terminal bud and proacinar epithelial cells with age; a significant 26% increase in transcript levels is detected by RNase protection assay between E14 and E19. By contrast, submandibular mucin protein is not immunodetected until E17, being primarily immunolocalized to terminal bud and proacinar epithelial cell membranes. Our data clearly shows that substantial qualitative differences exist between embryonic and adult SMG mucin mRNA and protein.

Animals↗

Cultured tumor cells of murine submandibular gland origin: a model to investigate pHi regulation of salivary cells.

Intracellular pH (pHi) and several ion transport mechanisms in cultured murine salivary gland cells (SCA-9) were studied using a videomicrofluorometric method and the H+-specific probe C-SNARF-1. The aim of this study was to test SCA-9 cells' pHi regulation mechanisms and evaluate if this cell line is representative of submandibular gland cells. Resting pHi in unstimulated cells was estimated to be 7.17+/-0.07. To investigate the presence of Na+/H+ and Cl-/HCO3- antiports as well as Na+/K+/2Cl- symports in SCA-9 cells, we used different specific blockers, dimethyl-amiloride, disulfonic stilbene, bumetanide and furosemide. In order to study SCA-9 cell capacity to regulate their pHi in response to alkaline and acid loads, we applied the NH4Cl prepulse method to all these blockers. The results showed that SCA-9 cells possess both antiports and symports involved in pHi regulation, and that this cell line can be used as a convenient model to study pHi regulatory mechanisms in salivary cells.

Acid-Base Equilibrium↗

[Transplantation of autologous submandibular glands in very severe keratoconjunctivitis sicca. 2 year outcome].

INTRODUCTION: The conservative treatment of the most severe cases of keratoconjunctivitis sicca (KCS) can be sometimes frustrating. Especially with an underlying autoimmunologic disorder, even the application of artificial tears as often as every 5 min may not prevent further damage to the ocular surface. A microvascular transplantation of the autologous submandibular gland (SG) can be performed by a maxillo-facial surgeon as an alternative approach for those cases. We report 2 years of ophthalmological experience with the results of this procedure. MATERIAL AND METHODS: To date 27 operations have been performed in 23 patients. The SG was moved from its natural site into the temporal fossa. The secretory duct was implanted into the conjunctival fornix and the gland's vessels connected to the temporal artery and vein. A complete ophthalmological examination has been performed in 25 eyes of 21 patients up to 1 year and in 11 eyes of 9 patients 2 years after surgery. RESULTS: Three months and 1 year postoperatively 19 of 25, and 2 years postoperatively 8 of 11 transplants remained vital. The baseline secretion increased in patients with a vital transplant from an average of 1.6 +/- 1.3 mm before the operation to 16.2 +/- 11.3 mm after 3 months and 20.6 +/- 10.6 mm after 1 year. Ten of 19 vital grafts were reduced 1 year after transplantation in a minor second procedure to control an increasing epiphora. Subsequently baseline secretion was reduced to 13.6 +/- 8.2 mm 2 years after transplantation. Patients with a vital graft reported in 84% of cases (16 of 19) at 3 months, and 79% at 1 year (15 of 19) and 2 years (7 of 8), a strong relief of dry eye symptoms. In 58% (3 months), 79% (1 year) and 63% (2 years) of the eyes with a vital transplant all artificial tear substitution could be stopped. Break-up time increased significantly, resulting in reduced bengal rose staining. CONCLUSION: The transfer of the autologous SG into the temporal fossa can be used to provide patients with very severe KCS with a continuous, endogenous source of ocular lubrication. Despite surgical denervation the graft maintains a sufficient baseline secretion over a period of years. Subjective symptoms and the application of pharmaceutical lubricating substances are reduced to a large extent. If epiphora occurs, it can be controlled by surgically reducing the transplant. The influence of SG saliva on the ocular surface is the object of ongoing studies.

Adult↗

Epithelial morphogenesis in mouse embryonic submandibular gland: its relationships to the tissue organization of epithelium and mesenchyme.

Epithelial tissues in various organ rudiments undergo extensive shape changes during their development. The processes of epithelial shape change are controlled by tissue interactions with the surrounding mesenchyme which is kept in direct contact with the epithelium. One of the organs which has been extensively studied is the mouse embryonic submandibular gland, whose epithelium shows the characteristic branching morphogenesis beginning with the formation of narrow and deep clefts as well as changes in tissue organization. Various molecules in the mesenchyme, including growth factors and extracellular matrix components, affect changes of epithelial shape and tissue organization. Also, mesenchymal tissue exhibits dynamic properties such as directional movements in groups and rearrangement of collagen fibers coupled with force-generation by mesenchymal cells. The epithelium, during early branching morphogenesis, makes a cell mass where cell-cell adhesion systems are less developed. Such properties of both the mesenchyme and epithelium are significant for considering how clefts, which first appear as unstable tiny indentations on epithelial surfaces, are formed and stabilized.

Animals↗