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Intermittent fasting exerts a profound beneficial influence on a spectrum of diseases through various mechanisms including regulation of immune responses, elimination of senescent- and pathogenic cells and improvement of stem cell-based tissue regeneration in a disease- and tissue-dependent manner. Our previous study demonstrated that alternate-day fasting (ADF) led to alleviation of xerostomia and sialadenitis in non-obese diabetic (NOD) mice, a well-defined model of Sjögren's syndrome (SS). This present study delved into the previously unexplored impacts of ADF in this disease setting and revealed that ADF increases the proportion of salivary gland stem cells (SGSCs), defined as the EpCAM hi cell population among the lineage marker negative submandibular gland (SMG) cells. Furthermore, ADF downregulated the expression of p16 INK4a , a cellular senescence marker, which was concomitant with increased apoptosis and decreased expression and activity of NLRP3 inflammasomes in the SMGs, particularly in the SGSC-residing ductal compartments. RNA-sequencing analysis of purified SGSCs from NOD mice revealed that the significantly downregulated genes by ADF were mainly associated with sugar metabolism, amino acid biosynthetic process and MAPK signaling pathway, whereas the significantly upregulated genes related to fatty acid metabolic processes, among others. Collectively, these findings indicate that ADF increases the SGSC proportion, accompanied by a modulation of the SGSC property and a switch from sugar- to fatty acid-based metabolism. These findings lay the foundation for further investigation into the functionality of SGSCs influenced by ADF and shed light on the cellular and molecular mechanisms by which ADF exerts beneficial actions on salivary gland restoration in SS., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Salivary glands are physiologically orchestrated by the coordinated balance between cell differentiation, proliferation, apoptosis, and interactions between epithelial, mesenchymal endothelial, and neuronal cells, and they are frequent sites of manifestations of Sjögren's syndrome (SS) or IgG4-related disease (IgG4-RD). However, little is known about salivary gland homeostasis and its involvement in those diseases. Inhibitor of DNA binding/differentiation 4 (Id4) is an Id protein involved in the transcriptional control of many biological events, including differentiation. Studies of Id4-deficient mice revealed that Id4-deficient submandibular glands were smaller and exhibited accelerated differentiation, compared with those from wild-type littermates. In addition, dry mouth symptoms and Th17 expansion in splenocytes were also observed in the absence of Id4. Furthermore, Id4 levels in the salivary glands of patients with IgG4-RD, but not SS, were significantly decreased compared with those of healthy controls. miRNA-mRNA integrated analysis demonstrated that miR-486-5p was upregulated in IgG4-RD patients and that it might regulate Id4 in the lesion sites. Together, these results provide evidence for the inhibitory role of Id4 in salivary differentiation, and a critical association between Id4 downregulation and IgG4-RD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Radiotherapy has become a basic treatment modality for head and neck cancer. However, radiotherapy results in inevitable side effects, particularly radiation sialadenitis, that significantly impairs quality of life. A previous study indicated that nerve growth factor (NGF) has a radio-protective effect, but the mechanism was not determined in salivary glands. In this study, we explored the functional role and mechanism regarding how NGF protects salivary glands against IR-induced damage., Main Methods: Human salivary gland (HSG) cells and C57BL/6 mice were selected to establish an IR-induced salivary gland damage model in vitro and in vivo. Recombinant NGF protein and NGF siRNA and over-expression plasmids were applied to manipulate NGF expression in vitro. AAV-NGF was retrogradely perfused into the submandibular gland (SMG) through the SMG duct to manipulate NGF expression in vitro. Small-molecule inhibitors and siRNAs were applied to inhibit AKT and JNK. Western blotting, quantitative PCR, flow cytometry and histology assays were performed to analyse the functional role and mechanism of NGF., Key Findings: Our study demonstrated that NGF expression was upregulated following radiotherapy both in human HSG cells and mouse SMG tissues. NGF could reduce IR-induced HSG cell apoptosis, and AAV-mediated gene therapy could restore the salivary flow rate and protect the salivary gland against IR-induced apoptosis in vivo. Mechanistically, NGF protects salivary glands from IR-induced apoptosis by de-phosphorylating JNK kinase rather than promoting AKT phosphorylation., Significance: The current study findings indicated that the modulation of the NGF pathway might prevent IR-induced salivary hypo-function., (Copyright © 2020. Published by Elsevier Inc.)

Bovine submaxillary mucin (BSM) is a heavily-glycosylated macromolecular (approximately 4 MDa) protein and is used in various biomaterial applications in light of its high viscosity and biocompatibility, in addition to use as a biochemical substrate or inhibitor as a result of its abundant O-glycans. Although it has been reported that N-glycosylation provides stability of human mucins, most BSM research has been focused on its O-glycans, while N-glycans have not been reported to date. In this study, a common N-glycan core component was detected by monosaccharide analysis of BSM, and the structures of the N-glycans and their relative quantities were determined by liquid chromatography-tandem mass spectrometry. Seventeen N-glycans comprising ten complex-type [Fucose 0~2 Hexose 3~4 N-acetylhexosamine 1~6 Sulfate 0~1 ; 61.1% (the sum of the relative quantities of each N-glycan out of the total N-glycans)], two high-mannose-type (Hexose 5~6 N-acetylhexosamine 2 ; 12.0%), and five paucimannose type (Fucose 0~1 Hexose 3~4 N-acetylhexosamine 2~3 ; 26.9%) were identified, but no hybrid-type or sialylated N-glycans were found. Additionally, these are less-branched structures compared to human mucins. Of these, ten glycans (77.2%), including two sulfated glycans (8.0%), were core fucosylated, which confer unique biological functions to glycoproteins. The N-glycosylation sites were identified from the analysis of glycopeptides from BSM. This study is the first confirmation of N-glycan attachment to BSM., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sialadenitis is an inflammatory condition affecting the salivary glands including the parotid, submandibular, and sublingual glands. There are several different types of sialadenitis, each with different sites of predilection. However, the pathogenic mechanism underlying the tissue specificity of sialadenitis is largely unknown. TRAF6 is a cytoplasmic adaptor protein that is necessary for the activation of dendritic cells in response to Toll-like receptor ligands, thereby regulating innate immune responses. We previously demonstrated that T cell-specific TRAF6-deficient mice (TRAF6ΔT mice) spontaneously develop systemic inflammatory disease. Here, we show that salivary secretion is reduced in TRAF6ΔT mice due to sialadenitis that occurs in the parotid and submandibular glands, but not the sublingual glands. Consistent with pathological findings, both CD4 + and CD8 + T cells predominantly infiltrated the submandibular glands; however, sublingual infiltration was rare in TRAF6ΔT mice. The TH1 cytokine IFN-γ, the TH1 cell attractant chemokine CCL2, and its cognate receptor CCR2 were upregulated concomitantly in both the submandibular and sublingual glands. Interestingly, the TH17 cell attractant chemokine CCL20 and its cognate receptor CCR6 were selectively increased in the submandibular glands, but not in the sublingual glands of TRAF6ΔT mice. Thus, the expression of TRAF6 in T cells might be implicated in tissue-specific sialadenitis by regulating the chemokine-chemokine receptor system., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Lipoxin receptor (ALX)/N-formyl peptide receptor (FPR)-2 is a G-protein-coupled receptor that has multiple binding partners, including the endogenous lipid mediators resolvin D1, lipoxin A 4 , and the Ca 2+ -dependent phospholipid-binding protein annexin A1. Previous studies have demonstrated that resolvin D1 activates ALX/Fpr2 to resolve salivary gland inflammation in the NOD/ShiLtJ mouse model of Sjögren syndrome. Moreover, mice lacking the ALX/Fpr2 display an exacerbated salivary gland inflammation in response to lipopolysaccharide. Additionally, activation of ALX/Fpr2 has been shown to be important for regulating antibody production in B cells. These previous studies indicate that ALX/Fpr2 promotes resolution of salivary gland inflammation while modulating adaptive immunity, suggesting the need for investigation of the role of ALX/Fpr2 in regulating antibody production and secretory function in mouse salivary glands. Our results indicate that aging female knockout mice lacking ALX/Fpr2 display a significant reduction in saliva flow rates and weight loss, an increased expression of autoimmune-associated genes, an up-regulation of autoantibody production, and increased CD20-positive B-cell population. Although not all effects were noted among the male knockout mice, the results nonetheless indicate that ALX/Fpr2 is clearly involved in the adaptive immunity and secretory function in salivary glands, with further investigation warranted to determine the cause(s) of these between-sex differences., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The hedgehog family includes Sonic hedgehog (Shh), Desert hedgehog, and Indian hedgehog, which are well known as a morphogens that play many important roles during development of numerous organs such as the tongue, pancreas, kidney, cartilage, teeth and salivary glands (SMG). In Shh null mice, abnormal development of the salivary gland is seen after embryonic day 14 (E14). Shh also induced lobule formation and lumen formation in acini-like structures in cultured E14 SMG. In this study, we investigated the relationship between Shh and epidermal growth factor (EGF)/ErbB signaling in developing fetal mouse SMG. Administration of Shh to cultured E13 SMG stimulated branching morphogenesis (BrM) and induced synthesis of mRNAs for EGF ligands and receptors of the ErbB family. Shh also stimulated activation of ErbB signaling system such as ERK1/2. AG1478, a specific inhibitor of ErbB receptors, completely suppressed BrM and activation of EGF/ErbB/ERK1/2 cascade in E13 SMGs cultured with Shh. The expressions of mRNA for Egf in mesenchyme and mRNA for Erbb1, Erbb2 and Erbb3 in epithelium of E13 SMG were specifically induced by administration of Shh. These results show that Shh stimulates BrM of fetal mouse SMG, at least in part, through activation of the EGF/ErbB/ERK1/2 signaling system., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aims: Calcineurin inhibitors are widely used for prevention of graft rejection and treatment of autoimmune disorders, which result in increased longevity and enhanced quality of life for patients. Unfortunately, the toxic side effects of these drugs (mainly renal, hepatic and cardiac) limit their use. In this work, we studied the effects of long-term treatment of rats with the immunosuppressant cyclosporin (CsA) or tacrolimus (Tac) on salivation, saliva composition and on the major salivary glands (parotid and submandibular) in terms of histological alterations and oxidative stress, evaluated as lipoperoxidation (thiobarbituric acid reactive species--TBARS) and antioxidant enzyme activity contents (superoxide dismutase--SOD, catalase--CAT and glutathione peroxidase--GPx)., Main Methods: Male adult rats were treated with either CsA (10 mg/kg/day) or Tac (1 mg/kg/day) subcutaneously for 30 or 60 days. At the end of the experimental periods, pilocarpine-stimulated salivary flow rate was measured, saliva samples were collected and the salivary glands were dissected for morphological and biochemical analyses., Key Findings: After a 60-day treatment with any of the immunosuppressants, the total protein, Ca(2+) and Na(+) saliva concentrations were decreased but salivary flow rates were unaffected. In addition, both parotid and submandibular glands showed decreased SOD, CAT and GPx activities, increased TBARS contents and histomorphological alterations involving the epithelium and acini., Significance: Based on these results, we suggest that the systemic long-term administration of the calcineurin inhibitor CsA or Tac induces an impairment of the antioxidant enzymatic defense in the rat major salivary glands, which may, in turn, lead to altered saliva composition., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Salivary glands develop as highly branched structures designed to produce and secrete saliva. Advances in mouse genetics, stem cell biology, and regenerative medicine are having a tremendous impact on our understanding of salivary gland organogenesis. Understanding how submandibular gland (SMG) initiation, branching morphogenesis, and cell differentiation occur, as well as defining the progenitor/stem cells and cell and tissue interactions that drive SMG development will help guide regenerative approaches for patients suffering from loss of salivary gland function. This review focuses on recent literature from the past 5 years investigating the regulatory mechanisms driving SMG organogenesis., (2015 Published by Elsevier Inc.)

In the present study, the conformational changes of bovine submaxillary mucin (BSM) adsorbed on a hydrophobic surface (polystyrene (PS)) as a function of concentration in bulk solution (up to 2mg/mL) have been investigated with biomolecular probe-based approaches, including bicinchoninic acid (BCA), enzyme-linked immunosorbent assay (EIA), and enzyme-linked lectin assay (ELLA). The conformation and hydrodynamic diameter of highly purified BSM molecules, as characterized by circular dichroism (CD) spectroscopy and dynamic light scattering (DLS), respectively, showed a slight, yet gradual coiling and compaction in response to the increase in BSM concentration in bulk solution. Adsorbed masses of BSM onto hydrophobic surface, as probe by BCA, showed a continuously increasing trend up to 2mg/mL. But, the signals from EIA and ELLA, which probe the concentration of available unglycosylated C-terminals and the central glycosylated regions, respectively, showed complicated non-linear responses with increasing surface concentration. The results from this study support the conventional amphiphilic, triblock model of BSM in the adsorption onto hydrophobic surface from aqueous solution. The biomolecular probe-based approaches employed in this study, however, provided further details on the conformational changes of BSM on surface, in particular the accessibility of glycosylated and unglycosylated domains with increasing surface concentration., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Neural crest cells in the embryo migrate to reach target sites as neural crest-derived cells (NCDCs) where they differentiate into a variety of derivatives. Some NCDCs are maintained in an undifferentiated state throughout the life of the animal and are considered to be a useful cell source for regenerative medicine. However, no established method to obtain NCDCs sufficient for regenerative medicine from adults with high purity has been presented, since their distribution in adult tissues is not fully understood. It is critical to identify reliable markers for NCDCs in adults, as the expressions of P0 and Wnt1, the most reliable NCDC markers, are shut off in the embryonic stage. To analyze the characteristics of NCDCs in adult tissues, we utilized a double transgenic mouse strain, P0-Cre/CAG-CAT-EGFP transgenic mice (P0 mice), in which NCDCs were shown to express EGFP and we were able to recognize GFP-positive cells in those. We focused on the submandibular glands (SMGs), which are known to be derived from the neural crest. GFP-positive cells were shown to be scattered like islands in the SMGs of adult P0 mice. We surgically removed SMGs from adult mice and digested samples into single cell suspensions. GFP-positive cells separated using flow cytometry expressed a high level of Sox10, a marker of embryonic neural crest cells, suggesting successful isolation of NCDCs. To identify candidate marker genes in isolated NCDCs, we performed DNA microarray analyses and real-time PCR analysis of GFP-positive and -negative cells isolated from P0 mice, then selected genes showing differential gene expression patterns. As compared to GFP-negative cells, GFP-positive cells expressed Gpr4 and Ednrb at higher levels, whereas Pdgfra and Pdgfrb were expressed at lower levels. Furthermore, DNA microarray analysis showed that GFP-positive cells were positive for aquaporin 5, a marker for acinar cells. Together, our results indicate that NCDCs in adult SMGs have characteristic gene expression profiles specially their cell surface molecules. Cell sorting using a combination of these specific cell surface proteins would be a useful strategy for isolation of NCDCs from SMGs with high purity., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Adenoviruses have been used for gene transfer to salivary gland cells in vivo. Their use to study the function of salivary acinar cells was limited by a severe inflammatory response and by the destruction of fluid-secreting acinar cells. In the present study, low doses of adenovirus were administered to express Stim1-mKO1 by retrograde ductal injection to submandibular glands. The approach succeeded in increasing muscarinic stimulation-induced Ca(2+) responses in acinar cells without inflammation or decreased salivary secretions. This increased Ca(2+) response was notable upon weak muscarinic stimulation and was attributed to increased Ca(2+) release from internal stores and increased Ca(2+) entry. The basal Ca(2+) level was higher in Stim1-mKO1-expressing cells than in mKO1-expressing and non-expressing cells. Exposure of permeabilized submandibular acinar cells, where Ca(2+) concentration was fixed at 50 nM, to inositol 1,4,5-trisphosphate (IP3) produced similar effects on the release of Ca(2+) from stores in Stim1-mKO1-expressing and non-expressing cells. The low toxicity and relative specificity to acinar cells of the mild gene transfer method described herein are particularly useful for studying the molecular functions of salivary acinar cells in vivo, and may be applied to increase salivary secretions in experimental animals and human in future., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Vampire bats are notorious for being the sole mammals that strictly feed on fresh blood for their survival. While their saliva has been historically associated with anticoagulants, only one antihemostatic (plasminogen activator) has been molecularly and functionally characterized. Here, RNAs from both principal and accessory submaxillary (submandibular) salivary glands of Desmodus rotundus were extracted, and ~200 million reads were sequenced by Illumina. The principal gland was enriched with plasminogen activators with fibrinolytic properties, members of lipocalin and secretoglobin families, which bind prohemostatic prostaglandins, and endonucleases, which cleave neutrophil-derived procoagulant NETs. Anticoagulant (tissue factor pathway inhibitor, TFPI), vasodilators (PACAP and C-natriuretic peptide), and metalloproteases (ADAMTS-1) were also abundantly expressed. Members of the TSG-6 (anti-inflammatory), antigen 5/CRISP, and CCL28-like (antimicrobial) protein families were also sequenced. Apyrases (which remove platelet agonist ADP), phosphatases (which degrade procoagulant polyphosphates), and sphingomyelinase were found at lower transcriptional levels. Accessory glands were enriched with antimicrobials (lysozyme, defensin, lactotransferrin) and protease inhibitors (TIL-domain, cystatin, Kazal). Mucins, heme-oxygenase, and IgG chains were present in both glands. Proteome analysis by nano LC-MS/MS confirmed that several transcripts are expressed in the glands. The database presented herein is accessible online at http://exon.niaid.nih.gov/transcriptome/D_rotundus/Supplemental-web.xlsx. These results reveal that bat saliva emerges as a novel source of modulators of vascular biology., Biological Significance: Vampire bat saliva emerges as a novel source of antihemostatics which modulate several aspects of vascular biology., (Published by Elsevier B.V.)

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor originating in major and minor salivary glands (SGs). Although the precise multifactorial etiology of human SG-MEC is largely unknown, we have recently shown that cytomegalovirus (CMV) is an important component of MEC tumorigenesis. Despite the well-documented overexpression of the EGFR → ERK signaling pathway in SG-MEC, there has been limited to no clinical success with inhibition of this pathway. Using our previously characterized mouse model of CMV-induced SG dysplasia/neoplasia, we report that inhibitors of the EGFR → ERK pathway do not ameliorate or rescue well-established pathology, either singly or in combination, but they do inhibit the evolution of progressive pathogenesis ("disease tolerance") in the face of mounting CMV burden. Failure to rescue SG pathology, suggested a possible increase in the ligand levels of alternative pathways that share cell proliferation and survival effectors (e.g. ERK and PI3K). Here we present evidence of a highly significant upregulation of ligands for the EGFR, FGFR, IL-6R, and TNFR signaling pathways, all of which converge upon the Raf/MEK/ERK amplifier module. This explains our finding that even in the presence of the highest nontoxic dose of an ERK phosphorylation inhibitor, pERK is undiminished. Given the considerable pathway crosstalk, a deep understanding of subversion and dysregulation of the SG interactome by CMV is a priori quite daunting. Circumventing this dilemma, we present evidence that concurrent inhibition of ERK phosphorylation (U0126) and CMV replication (acyclovir) obviates progressive pathogenesis and results in complete SG rescue (tumor regression). These findings provide a mechanistic foundation for potential clinical trials that utilize similar concurrent treatment with extant FDA-approved drugs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: We investigated salivary function using quantitative scintigraphy and sought to identify functional correlations between parotid dose and quality of life (QoL) for head and neck cancer (HNC) patients receiving intensity-modulated radiotherapy (IMRT)., Materials and Methods: Between August, 2007 and June, 2008, 31 patients treated IMRT for HNC were enrolled in this prospective study. Salivary excretion function (SEF) was previously measured by salivary scintigraphy at annual intervals for 2 years after IMRT. A dose-volume histogram of each parotid gland was calculated, and the normal tissue complication probability (NTCP) was used to determine the tolerance dose. QoL was longitudinally assessed by the EORTC QLQ-C30 and H&N35 questionnaires prior to RT, and at one, three, 12 and 24 months after RT., Results: A significant correlation was found between the reduction of SEF and the mean parotid dose measured at 1 year (correlation coefficient, R(2)=0.651) and 2 years (R(2)=0.310) after IMRT (p<0.001). The TD(50) of the parotid gland at 1 year after IMRT is 43.6 Gy, comparable to results from western countries. We further found that contralateral parotid and submandibular gland function preservation was correlated with reduced sticky saliva and a better QoL compared to the functional preservation of both parotid glands, as determined by the EORTC QLQ-H&N35 questionnaire., Conclusion: A significant correlation was found between the reduction of SEF and the mean parotid dose. Preservation of contralateral parotid and submandibular gland function predicts a better QoL compared to preservation of the function of both parotid glands., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

It is established that neuropeptide Y (NPY) is a transmitter of parasympathetic secretory impulses in submandibular gland. The neuropeptides substance P, vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) are likely mediators of secretory parasympathetic responses of the gland. Previously, we have shown that substance P, VIP and CGRP modulate voltage-dependent Ca(2+) channels (VDCCs) in hamster submandibular ganglion (SMG) neurons. In this study, we attempt to characterize the effect of NPY on VDCCs current using Ba(2+) (I(Ba)) in SMG neurons. Application of NPY caused both facilitation and inhibition of L-type and N/P/Q-type I(Ba), respectively. Intracellular dialysis of the Gα(s)-protein antibody attenuated the NPY-induced facilitation of I(Ba). The adenylate cyclase (AC) inhibitor, as well as protein kinase A (PKA) inhibitor attenuated the NPY-induced facilitation of I(Ba). Intracellular dialysis of the Gα(i)-protein antibody attenuated the NPY-induced inhibition of I(Ba). Application of a strong depolarizing voltage prepulse attenuated the NPY-induced inhibition of I(Ba). These results indicate that NPY facilitates L-type VDCCs via Gα(s)-protein involving AC and PKA. On the other hand, NPY also inhibits N/P/Q-type VDCCs via Gα(i)-protein βγ subunits in the SMG neurons., (Copyright © 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.)

The analysis of O-glycans is essential for better understanding their functions in biological processes. Although many techniques for O-glycan release have been developed, the hydrazinolysis release method is the best for producing O-glycans with free reducing termini in high yield. This release technique allows the glycans to be labeled with a fluorophore and analyzed by fluorescence detection. Under the hydrazinolysis release conditions, a side reaction is observed and causes the loss of monosaccharides from the reducing terminus of the glycans (known as peeling). Using bovine fetuin (because it contains the sialylated O-glycans most commonly found on biopharmaceuticals) and bovine submaxillary gland mucin (BSM), here we demonstrate that peeling can be greatly reduced when the sample is buffer exchanged prior to hydrazinolysis with solutions of either 0.1% trifluoroacetic acid (TFA) or low-molarity (100, 50, 20, and 5 mM) ethylenediaminetetraacetic acid (EDTA). The addition of calcium chloride to fetuin resulted in an increase in peeling, whereas subsequent washing with EDTA abolished this effect, suggesting a role of calcium and possibly other cations in causing peeling. The presented technique for sample preparation prior to hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Treatment for otosclerosis involves patients' undergoing stapedectomy. Inadvertent damage to the chorda tympani nerve's (CTN's) secretory fibers during stapedectomy can result in inadequate secretory stimulation of the submandibular salivary glands (SMSGs) and sublingual salivary glands (SLSGs). Because most saliva originates from these glands, hyposalivation and subjective xerostomia manifest during resting periods when parotid gland secretions are minimal. Stimulation with food increases parotid gland salivation enough to overcome the subjective sense of dryness., Case Description: The author examined a 52-year-old man who had undergone bilateral stapedectomy because of hearing loss; his rheumatologist referred him to the Salivary Gland Center (New York City) because of a complaint of dry mouth. After the author examined the patient, he concluded that the patient had decreased SMSG and SLSG secretion and recommended that the patient use sugarless chewing gum or sour candy frequently to stimulate his parotid glands and use oral lubricants and sip water as needed., Clinical Implications: Stimulation of parotid gland secretion is independent of SMSG and SLSG activation. Therefore, the dental practitioner must become aware of the innervation of the salivary glands and each gland's secretory production during periods of oral stimulation and of rest.

Branching morphogenesis in murine submandibular glands (SMG) is regulated by growth factors, extracellular matrix (ECM) and many biological processes through interactions between the epithelium and the mesenchyme. MicroRNAs (miRNAs) are a set of small, non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. We hypothesized that branching morphogenesis is partly regulated by miRNAs. Forty-four miRNAs and novel miRNA candidates were detected in SMG at embryonic day 13 by a cloning method combined with Argonaute-2 immunoprecipitation. MicroRNA21 (miR-21) expression in the mesenchyme was up-regulated and accelerated by epidermal growth factor, which is known to enhance branching morphogenesis in vitro. Down-regulation of miR-21 in the mesenchyme by locked nucleic acids was associated with a decrease in the number of epithelial buds. Relative quantification of candidates for target genes of miR-21 indicated that two messenger RNAs (for Reck and Pdcd4) were down-regulated in the mesenchyme, where miR-21 expression levels were up-regulated. These results suggest that branching morphogenesis is regulated by miR-21 through gene expression related to ECM degradation in the mesenchyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Sjogren's syndrome and radiotherapy for head and neck cancers result in irreversible damage to functional salivary tissue, for which no adequate treatment is available. The microenvironment for salivary gland cell cytodifferentiation is critical for the future development of salivary gland regeneration, repair and tissue engineering treatments. Results from this study indicate that human submandibular cell line (HSG) cultured on Matrigel (2mg/ml) could be induced to differentiate into polarized secretory acinar-like cells. The HSG cells grown on Matrigel were evaluated by physiological functional assays, molecular and immunohistochemistry, immunofluorescence, and morphological assessments. The results showed (1) a decrease in cell proliferation; (2) an increase in cell apoptosis; (3) cellular polarization evident by transepithelial electrical resistance (TER), expressions of tight junction proteins (claudin-1, -2, -3, -4, occludin, JAM-A, and ZO-1) and transmission electron microscopy (TEM); (4) an increase in the production and/or secretion of acinar cell proteins, i.e., alpha-amylase, aquaporin-5, cytokeratins, and mucin-1, that were not associated with increases in mRNA transcription; (5) a decrease in vimentin expression; and (6) expression of potential stem cell biomarkers CD44 and CD166. The data indicated that Matrigel provided a suitable microenvironment for morphological and functional differentiation of HSG cells into 3D acinar like cells. This study provides an in vitro model and baseline data on future developments of new strategies for salivary gland regeneration and replacement., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Objective: The aim of this study was to evaluate correlations between levels of cytokines in secreted stimulated saliva in patients with chronic kidney disease (CKD) and hyposalivation., Study Design: Seventy patients with clearance <20 mL/min/1.73 m(2) were evaluated; 40 were predialysis, 21 hemodialysis, and 9 peritoneal dialysis, and they were matched with 70 control subjects. Salivary flow rate was measured and submandibular/sublingual saliva collected. Analyses were performed for whole protein content using a protein assay, and levels of tumor necrosis factor (TNF) α, interleukin (IL) 1β, γ-interferon (γ-INF), IL-6, IL-8, IL-10, monocyte chemotactic protein (MCP) 1, and soluble intercellular adhesion molecule (sICAM) 1, by using Luminex technology., Results: Patients with CKD had lower (P = .03) stimulated salivary secretion rate and higher salivary whole protein concentration (P = .002) than control subjects. Concentrations of IL-8 (P = .03) and MCP-1 (P = .002) were decreased and TNF-α/IL-10 (P = .05) and IL-8/IL10 (P = .03) ratios were decreased in CKD patients. CKD patients with low secretion levels of stimulated saliva expressed decreased levels of TNF-α (P = .04), IL-1β (P = .02), γ-INF (P = .03), IL-6 (P = .003), IL-8 (P = .005), MCP-1 (P = .006), and sICAM-1 (P = .02)., Conclusions: Salivary cytokines and secretion rates are significantly decreased in CKD patients. Further research is necessary to understand operating mechanisms and clinical implications of the down-regulation of inflammatory markers in saliva., (Copyright © 2010 Mosby, Inc. All rights reserved.)

RP-HPLC-ESI-MS profile of saliva samples from human preterm newborn showed a protein peak in the elution range 26.6-27.6min. Deconvolution of ESI-MS spectra revealed the presence of two proteins with average molecular mass (Mav) values of 17,239+/-3Da and 18,065+/-3Da in 9 samples, with Mav value of 17,239+/-3Da in 4 samples and Mav value of 18,065+/-3Da in 2 samples. MALDI-TOF-MS analysis of tryptic digest allowed identifying the proteins as two isoforms of small proline-rich protein 3 and cDNA amplification of RNA extracts from oral mucosa, parotid and submandibular gland samples, obtained at fetal autopsy, provided two nucleotide sequences in agreement with those reported in the literature. The two proteins differ for an octapeptide repeat (GCTKVPEP) and the substitution Leu-->Val, at position 148 and 140 of the mature form of the 18,065 and 17,239Da protein, respectively. During maturation the two proteins undergo two post-translational modifications, corresponding to N-terminal acetylation and removal of the initiator methionine. cDNA amplification did not allow to clarify if the proteins found in saliva originated from cellular shedding of the epithelium and/or secretion., (Copyright 2010 Elsevier Inc. All rights reserved.)

There are few data available regarding the differences in intracellular Ca2+ responses of parotid acinar and ductal cells. This study investigated the Ca2+ mobilization that was induced by the chemical stimulation of acinar and ductal cells from rat parotid glands. In fura-2 loaded parotid cells, carbachol increased the intracellular Ca2+ concentration ([Ca2+](i)) to a greater extent in the acinar cells than in the ductal cells, but noradrenaline increased the [Ca2+](i) in the ductal cells more than in the acinar cells. Although there was no difference in the alpha1-adrenergic receptor agonist phenylephrine-induced Ca2+ mobilization between acini and ducts, the beta-adrenergic receptor agonist isoproterenol increased the [Ca2+](i) in only the ductal cells. Additionally, the effects of non-adrenergic, non-cholinergic neurotransmitters were investigated. Substance P and ATP increased the [Ca2+](i) in parotid acini and/or ducts. A substance P-induced Ca2+ response was observed in only acini, while the ATP-induced Ca2+ response was significantly higher in ducts than in acini. These results suggest that parotid acini have a greater sensitivity to cholinergic and substance P stimulation and a lesser sensitivity to beta-adrenergic and ATP stimulation than the ductal cells. In light of these results, substance P and isoproterenol will be useful for identifying parotid acini and ducts, respectively., (2009 Elsevier B.V. All rights reserved.)

Coordination of Ca(2+) signaling among cells contributes to synchronization of salivary gland cell function. However, mechanisms that underlie this signaling remain elusive. Here, intercellular Ca(2+) waves (ICW) in submandibular gland cells were investigated using Fura-2 fluorescence imaging. Mechanical stimulation of single cells induced ICW propagation from the stimulated cells through approximately 7 layers of cells or approximately 120microm. Our findings indicate that an extracellular ATP-dependent pathway is involved because the purinergic receptor antagonist suramin and the ATP hydrolyzing enzyme apyrase blocked ICW propagation. However, the gap junction uncoupler oleamide had no effect. ATP is released from mechanically stimulated cells possibly through opening of mechanosensitive maxi-anion channels, and does not appear to be directly linked to cytosolic Ca(2+). The ICW is propagated by diffusing ATP, which activates purinergic receptors in neighboring cells. This purinergic signaling induces a Ca(2+) transient that is dependent on Ca(2+) release via IP(3) receptors in the ER and store operated Ca(2+) entry (SOCE). Finally, inhibition of mitochondrial Ca(2+) uptake modified ICW indicating an important role of these organelles in this phenomenon. These studies increase our understanding of purinergic receptor signaling in salivary gland cells, and its role as a coordination mechanism of Ca(2+) signals induced by mechanical stimulation., (2009 Elsevier Ltd. All rights reserved.)

The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.

The peripheral projections of neurons expressing neuroendocrine secretory protein 55 (NESP55), a novel member of the chromogranin family, were studied by retrograde tracing technique. It was found that NESP55 positive neurons in the rat superior cervical ganglion projected to a number of targets including the submandibular gland, the cervical lymph nodes, the forehead skin, the iris, but not to the thyroid. Among these NESP55 positive target-projecting neurons, a subpopulation contained neuropeptide Y (NPY), a vasoconstrictor. Forepaw pad projecting neurons were found exclusively in the stellate ganglion, almost all of which (approximately 90%) were immunoreactive to NESP55. Colocalization of NESP55 and calcitonin gene-related peptide (CGRP), a peptide involved in sudomotor effects, was observed in a subpopulation of these paw pad projecting neurons, as was colocalization of NESP55 and NPY. The data suggest that NESP55 may have a functional role in some populations of sympathetic neurons.

Objective: To investigate the association of salivary flow rates with oral symptoms and oral mucosal status., Study Design: The study population included 462 Israeli subjects attending a xerostomia clinic. After patient history and oral mucosal examination, major gland sialometry, and complementary tests, patients were divided into 6 groups: drug-induced salivary gland hypofunction (SGH), Sjögren syndrome (SS), radiation-induced SGH, idiopathic SGH, xerostomia without SGH, and control., Results: Oral mucosal alterations were more prevalent in all SGH groups than in the control group. Oral symptoms (except speech impairment) were more frequent in all SGH groups. The postradiation group showed the highest frequency of oral mucosal alterations and of swallowing and mastication complaints. Individuals complaining of xerostomia (compared with those who did not) displayed lower major salivary gland flow rates and a higher frequency of oral mucosal alterations, Conclusions: Presence of oral mucosal alterations may help but are not enough to identify patients for further evaluation of SGH. Difficulties in mastication and swallowing are most specifically related to advanced SGH.

Agonist stimulation of exocrine cells leads to the generation of intracellular Ca(2+) signals driven by inositol 1,4,5-trisphosphate receptors (IP(3)Rs) that rapidly become global due to propagation throughout the cell. In many types of excitable cells the intracellular Ca(2+) signal is propagated by a mechanism of Ca(2+)-induced Ca(2+) release (CICR), mediated by ryanodine receptors (RyRs). Expression of RyRs in salivary gland cells has been demonstrated immunocytochemically although their functional role is not clear. We used microfluorimetry to measure Ca(2+) signals in the cytoplasm, in the endoplasmic reticulum (ER) and in mitochondria. In permeabilized acinar cells caffeine induced a dose-dependent, transient decrease of Ca(2+) concentration in the endoplasmic reticulum ([Ca(2+)](ER)). This decrease was inhibited by ryanodine but was insensitive to heparin. Application of caffeine, however, did not elevate cytosolic Ca(2+) concentration ([Ca(2+)](i)) suggesting fast local buffering of Ca(2+) released through RyRs. Indeed, activation of RyRs produced a robust mitochondrial Ca(2+) transient that was prevented by addition of Ca(2+) chelator BAPTA but not EGTA. When mitochondrial Ca(2+) uptake was blocked, activation of RyRs evoked only a non-transient increase in [Ca(2+)](i) and substantially smaller Ca(2+) release from the ER. Upon simultaneous inhibition of mitochondrial Ca(2+) uptake and either plasmalemmal or ER Ca(2+) ATPase, activation of RyRs caused a transient rise in [Ca(2+)](i). Collectively, our data suggest that Ca(2+) released through RyRs is mostly "tunnelled" to mitochondria, while Ca(2+) ATPases are responsible for the fast initial sequestration of Ca(2+). Ca(2+) uptake by mitochondria is critical for maintaining continuous CICR. A complex interplay between RyRs, mitochondria and Ca(2+) ATPases is accomplished through strategic positioning of mitochondria close to both Ca(2+) release sites in the ER and Ca(2+) pumping sites of the plasmalemma and the ER.

We have studied the signaling pathways involved in pilocarpine-induced mucin release in rat submandibular slices. Pilocarpine produced a significant increment of PGE2 levels and a positive (r=0.8870) and significant (p=0.0077) correlation between PGE2 production and mucin released was determined. The participation of PGE2 was confirmed by the use of indomethacin (indo) and of acetyl salicylic acid (ASA), cyclooxygenase inhibitors, which inhibited pilocarpine-induced mucin release. The muscarinic receptors involved in the regulation of mucin release were identified as M1 and M4 by the use of the selective acetylcholine receptors (mAChR) antagonists, pirenzepine, AF-DX 116, 4-DAMP and tropicamide. The secretory process was dependent on both, intracellular and extracellular calcium pools since it was inhibited by thapsigargin and verapamil. Cyclic AMP, nitric oxide synthase and PKC also participated in pilocarpine-induced mucin release. It is concluded that pilocarpine, by activation the M1 and M4 mAChR subtypes induces an increase of intracellular Ca2+ concentration ([Ca2+]I) and elevates cAMP levels, which in turn stimulates COX, PKC and NOS and promotes mucin exocytosis. PGE2 released induces cAMP accumulation which, together with PKC are involved in the PGE2 increased Ca2+/cAMP-regulated exocytosis. Thus, cAMP accumulation induced by cholinergic stimulation is, in part, the result of PGE2 production.

A novel muscarinic receptor antagonist, darifenacin, inhibited specific binding of [N-methyl-(3)H]scopolamine ([(3)H]NMS) in the mouse bladder, submaxillary gland and heart in a concentration-dependent manner. The inhibitory effect was most potent in the submaxillary gland, followed by the bladder and heart. In addition, darifenacin inhibited specific [(3)H]NMS binding in the membranes of CHO-K1 cell lines expressing muscarinic M(2) and M(3) receptor subtypes, and the potency was significantly (22-fold) greater at the M(3) than at the M(2) subtype. At 0.5 to 12 h after oral administration of darifenacin, a significant increase in K(d) values for specific [(3)H]NMS binding was seen in the bladder, submaxillary gland and lung of mice, compared with control values. Also, there was a sustained decrease in the B(max) values in the submaxillary gland. These data suggest that muscarinic receptor binding of oral darifenacin is rapid in onset and of a long duration. On the other hand, oral darifenacin exerted only temporary or little binding of muscarinic receptors in the heart and colon. Pilocarpine-induced salivary secretion in mice was continuously suppressed by oral darifenacin. The time-course of suppression coincided well with that for the muscarinic receptor binding in the submaxillary gland. The antagonistic effect of darifenacin against the dose-response curves for pilocarpine appeared to be insurmountable. In conclusion, the present study has shown that oral darifenacin may exert a pronounced and long-lasting binding of muscarinic receptors in tissues expressing the M(3) subtype.

Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.

We examined the expression of Slc12a2 (NKCC1) transcripts in the developing mouse by Northern blot analysis and in situ hybridization (ISH) using riboprobes transcribed from a cDNA encoding the transmembrane domain of human Slc12a2. In developing kidney, the 7.5-kb Slc12a2 transcript was expressed at all stages examined (13.5 d.p.c. to adult) but was more abundant in immature metanephroi. ISH revealed that NKCC1 was expressed in both mesenchymal cells and early nephric structures, but not branching ureteric buds, of developing metanephroi. A marked increase in expression was observed in the endocapillary cells of capillary loop stage glomeruli, and high expression was observed in the glomeruli of more mature nephrons. This was in contrast to Slc12a1 (NKCC2), where expression was excluded from the glomerulus. Extra-renal expression of Slc12a2 was examined in 13.5, 15.5, and 16.5 d.p.c. mouse embryos. Slc12a2 was highly expressed in the developing lung, gut, submandibular gland, tooth bud, and nasal epithelium. Slc12a2 expression was also observed in the developing central and peripheral nervous systems, including choroid plexus and trigeminal and dorsal root ganglia.

Ruminants secrete a large quantity of saliva that is rich in electrolytes; however, it remains unclear whether their parotid saliva contains epidermal growth factor (EGF). The present study was set up to examine the distribution of EGF and transforming growth factor-alpha (TGF-alpha) in the ovine parotid and submandibular glands and the salivary secretion of EGF-like binding activity (EGF-LBA) as the sum of EGF and TGF-alpha in conscious sheep. We also measured changes in the intragastric concentration of EGF-LBA in the ovine rumen and abomasum, and examined the effect of bilateral diversion of parotid saliva on intragastric EGF-LBA concentration in sheep. Both the ovine parotid and, to a lesser extent, the submandibular glands contained EGF-LBA. Immunohistochemical study showed that EGF and TGF-alpha-immunoreactivities were localized in the ductal epithelium in both glands. Transcriptional expression of EGF and TGF-alpha mRNA was demonstrated in both glands by reverse transcription-polymerase chain reaction (RT-PCR). In conscious sheep, the parotid gland continuously secreted EGF-LBA in the saliva before feeding, and the secretion of parotid EGF-LBA was markedly increased during feeding. After diversion of the parotid saliva for 1 week, EGF-LBA concentration in the ruminal fluid, but not in the abomasal fluid, decreased in the postprandial period, indicating that parotid EGF-LBA is a primary source of EGF-LBA for the rumen fluid during the postprandial period in sheep. Moreover, RT-PCR detected the expression of TGF-alpha mRNA in the rumen and abomasum and that of EGF in the abomasum, implying that these stomachs possibly supply, in part, EGF-LBA to the luminal fluid.

Vanilloid receptor 1 (VR1) is a polymodal receptor originally found in sensory neurons of the central nervous system. Recent evidence indicates that VR1 is also expressed in non-neuronal tissues. We report here endogenous expression of VR1 in rabbit submandibular gland (SMG) and its possible role in regulating saliva secretion based on: (i) the expression of VR1 mRNA and protein detected in SMG; (ii) VR1 was mainly localized in the basolateral membrane of duct cells and the cytoplasm of acinar cells and also in cytoplasm of primary cultured neonatal rabbit SMG cells; (iii) stimulation of neonatal rabbit SMG cells with capsaicin induced a significant increase in intracellular calcium, and capsazepine, a VR1 antagonist, abolished this increase; (iv) infusion of capsaicin via the external carotid artery to isolated SMG increased saliva secretion of the gland. These findings indicated that VR1 was expressed in SMG and appeared to play an important role in regulating saliva secretion.

The plasma membrane of cells from rat submandibular glands was isolated and extensively sonicated. The homogenate was centrifuged at high speed in a discontinuous sucrose gradient. Light fractions contained vesicles analogous to rafts: they were rich in cholesterol, they contained GM1 and caveolin-1, and P2X7 receptors were detected in these fractions. The location of the P2X7 receptors in rafts was abolished when cellular cholesterol was removed by methyl-beta-cyclodextrin (MCD). ATP activated neutral sphingomyelinase (N-SMase), which provoked a decrease of the cellular content of sphingomyelin and an increase of ceramide levels in these cells and in the rafts. Treatment with MCD and filipin (but not with alpha-cyclodextrin) abolished the increase of the intracellular concentration of calcium ([Ca2+]i) in response to epinephrine but not to ATP. MCD and filipin also inhibited the activation by ATP of phospholipase A2 (PLA2). Inhibition of N-SMase with glutathione or GW4869 prevented the activation of PLA2 by P2X7 agonists without affecting [Ca2+]i levels. We conclude that P2X7 receptors are present in both raft and nonraft compartments of plasma membranes; the receptors forming a nonselective cation channel are located in the nonraft fraction. P2X7 receptors in the rafts are coupled to the activation of N-SMase, which increases the content of ceramides in rafts. This may contribute to the activation of PLA2 in response to P2X7 receptor occupancy.

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.

Tube formation of the developing mouse submandibular salivary gland (SMG) begins at embryonic day (E) 14. The SMG of Sonic hedgehog (Shh) null mice was recently shown to fail to progress to stages beyond around E14. Here, we examined the effects of Shh peptide on tube formation of SMG explants. When the SMG rudiments from E14 mice were cultured, terminal buds of glands treated with Shh peptide formed the acini-like structure with a lumen whereas those of control glands remained as cell masses. In the acini-like terminal buds of the treated glands, tight junction proteins of ZO-1 and claudin-3 delineated the lumen and the apical membrane protein aquaporin-5 accumulated at the luminal cell surfaces. Moreover, laminin-5 deposition at the basal lamina region of terminal buds was accelerated in treated glands. It is suggested that hedgehog signaling promotes lumen formation and cell polarization of developing SMG epithelium.

We examined the expression of Centromere protein F (CENP-F) mRNA in 26 human salivary gland tumors (seven pleomorphic adenomas, three Warthin tumors, seven mucoepidermoid carcinomas, four adenoid cystic carcinomas, four acinic cell carcinomas and one malignant myoepithelioma) and four normal submandibular glands using the real time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean expression level of CENP-F mRNA was higher in malignant tumors (1.05+/-0.32) than normal submandibular glands (0.11+/-0.05) and benign tumors (0.46+/-0.16). We found a significant association between the level of CENP-F mRNA expression and clinical stage in 16 malignant tumors (Mann-Whitney U test, p=0.027). We also found a significant correlation between the Ki-67 labeling index and CENP-F expression in malignant tumors (Spearmans correlation coefficient by rank test, p=0.029). These results indicate that human CENP-F mRNA is closely linked to the increased or abnormal cell proliferation in malignant conditions.

The present study was undertaken to characterize in vivo muscarinic receptor binding of N-desethyl-oxybutynin (DEOB), active metabolite of oxybutynin (anticholinergic agent), in the bladder, submaxillary gland, heart and colon of rats, in relation to the plasma concentrations and inhibition of salivation. In the in vitro experiment, DEOB, as well as oxybutynin, inhibited the concentration-dependently specific [3H]N-methylscopolamine (NMS) binding in rat tissues and the affinity of DEOB in the rat bladder, submaxillary gland and colon was significantly (about 2 times) greater than that of oxybutynin. Following i.v. injection of DEOB (2.73-27.3 micromol/kg), there were dose- and time-dependent increases in the apparent dissociation constant (Kd) for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats, compared with control values, and the effect was similar to that by i.v. injection of oxybutynin (2.54-25.4 micromol/kg). Plasma concentrations of DEOB and oxybutynin in these rats showed dose- and time-dependent increases. The pilocarpine-induced salivary secretion in rats was equipotently reduced by the i.v. injection of DEOB and oxybutynin. In conclusion, it has been shown that intravenously injected DEOB, as well as oxybutynin, binds significantly to muscarinic receptors in rat tissues including the bladder and salivary gland and the receptor binding activity of DEOB is roughly similar to that of oxybutynin.

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.

Post-ganglionic sympathetic neurons express the p75 neurotrophin receptor (p75NTR) and brain-derived neurotrophic factor (BDNF), which together have been implicated in controlling the degree of efferent innervation of peripheral organs [Kohn, J., Aloyz, R.S., Toma, J.G., Haak-Frendscho, M., Miller, F.D. 1999. Functionally Antagonistic Interactions between the TrkA and p75 Neurotrophin Receptors Regulate Sympathetic Neuron Growth and Target Innervation. J. Neurosci. 19, 5393-5408]. To examine this concept further, we developed null mutant mice lacking both p75NTR and BDNF, and assessed whether the loss of this receptor-ligand interaction negatively impacts the degree of sympathetic innervation to various target tissues. Between postnatal days 10 and 14, hearts, urinary bladders, kidneys, and submandibular salivary glands were isolated from p75(-/-)/BDNF-/-, p75-/-, BDNF-/-, and wild type siblings. Sympathetic axons were visualized using tyrosine hydroxylase (TH) immunohistochemistry, and TH protein levels were quantified by immunoblotting. Concerning the sympathetic innervation of the heart, urinary bladder and kidneys, no differences were seen in single and double null mutant mice, as compared with their wild type siblings. Sympathetic innervation of the submandibular salivary gland was, however, increased in both p75-/- and p75(-/-)/BDNF-/- mice over control mice. These results reveal that an absence of p75NTR and/or BDNF expression does not perturb the degree of sympathetic innervation of many peripheral tissues during postnatal development, and that a lack of p75NTR expression may actually enhance the density of these efferent fibers in other target tissues, such as the salivary glands.

Introduction: To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an alpha1-adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo., Methods: Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 degrees C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay., Results: The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% (n = 10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% (n = 10) and 1.3% (n = 30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 micromol/g (eye ball) and 29 micromol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland., Discussion: Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.

Head and neck irradiation results in salivary dysfunction and subsequent xerostomia. Twenty two patients with squamous cancer of oropharynx or hypopharynx underwent contralateral submandibular salivary gland transfer (SMSGT) to submental triangle to shield it from subsequent radiotherapy. Resting salivary outputs of transferred and untransferred gland (control) were measured before and after SMSGT and following radiotherapy, by cannulating individual submandibular duct. They were compared by paired samples t-test. Following radiation therapy transferred gland retained 73% and untransferred gland (control) retained 27% of baseline salivary output. This significant difference in post-radiation salivary outputs suggests preservation of function of transferred salivary gland.

Free radicals are involved in several diseases, including cancer, central nervous system alterations and inflammatory pathologies. Peroxidase is an oral enzyme implicated in the defence of oral cavity. It has been determined that flavonoids and lignans possess antioxidant and free radical scavenging either directly or indirectly, usually by means of increasing the secretion of free radicals scavenger enzymes. Larrea divaricata Cav. is a plant used in folk Argentine medicine for the treatment of cancer and inflammatory ailments. In this study, we have determined the effect and mechanism of action of an aqueous extract of the leaves of L. divaricata and NDGA on peroxidase secretion in female rat submandibular glands. The extract significantly increased the secretion and total peroxidase. % of secreted peroxidase (X +/- S.E.M.): extract maximum response: 150 +/- 10; % of total peroxidase (X +/- S.E.M.): extract maximum response: 1000 +/- 90. The effect of the extract on peroxidase secretion was mediated by beta1 adrenoceptors (% of secreted peroxidase: extract + atenol maximum response: 50 +/- 4 ). Meanwhile, NDGA produced a decrease in peroxidase secretion (peroxidase secreted: basal: 0.44 +/- 0.03; NDGA 2.5 x 10(-6) M: 0.20 +/- 0.02; prostaglandins E2 (PGE2) 10(-7)M: 1.32 +/- 0.5; NDGA + PGE2: 0.46 +/- 0.035), an effect that was exerted by the inhibition on prostaglandins synthesis.

FGF8 has been shown to play important morphoregulatory roles during embryonic development. The observation that craniofacial, cardiovascular, pharyngeal, and neural phenotypes vary with Fgf8 gene dosage suggests that FGF8 signaling induces differences in downstream responses in a dose-dependent manner. In this study, we investigated if FGF8 plays a dose-dependent regulatory role during embryonic submandibular salivary gland (SMG) morphogenesis. We evaluated SMG phenotypes of Fgf8 hypomorphic mice, which have decreased Fgf8 gene function throughout embryogenesis. We also evaluated SMG phenotypes of Fgf8 conditional mutants in which Fgf8 function has been completely ablated in its expression domain in the first pharyngeal arch ectoderm from the time of arch formation. Fgf8 hypomorphs have hypoplastic SMGs, whereas conditional mutant SMGs exhibit ontogenic arrest followed by involution and are absent by E18.5. SMG aplasia in Fgf8 ectoderm conditional mutants indicates that FGF8 signaling is essential for the morphogenesis and survival of Pseudoglandular Stage and older SMGs. Equally important, the presence of an initial SMG bud in Fgf8 conditional mutants indicates that initial bud formation is FGF8 independent. Mice heterozygous for either the Fgf8 null allele (Fgf8(+/N)) or the hypomorphic allele (Fgf8(+/H)) have SMGs that are indistinguishable from wild-type (Fgf8(+/+)) mice which suggest that there is not only an FGF8 dose-dependent phenotypic response, but a nonlinear, threshold-like, epistatic response as well. We also found that enhanced FGF8 signaling induced, and abrogated FGF8 signaling decreased, SMG branching morphogenesis in vitro. Furthermore, since FGF10 and Shh expression is modulated by Fgf8 levels, we postulated that exogenous FGF10, Shh, or FGF10 + Shh peptide supplementation in vitro would largely "rescue" the abnormal SMG phenotype associated with decreased FGF8 signaling. This is as expected, though there is no synergistic effect with FGF10 + Shh peptide supplementation. These in vitro experiments model the principle that mutations have different effects in the context of different epigenotypes.

Blood pressure lowering kallikrein-kinin and blood pressure raising renin-angiotensin systems play a major role in the maintenance of normal blood pressure. In a previous study, we have shown that a kallikrein-like prorenin converting enzyme (PRCE C) is elevated in the submandibular gland tissue of a mouse line (BPH) that was genetically selected and inbred for high blood pressure in comparison to normotensive line (BPN) that was derived from the ancestors of BPH line. In the present investigation we wanted to find out if elevated levels of PRCE C were involved in the modulation of tissue (local) renin-angiotensin system in the submandibular gland tissue. Results indicate significantly high renin activity but low angiotensin I level in the tissue of BPH mouse model. These results tend to suggest PRCE C's involvement in tissue (local) renin-angiotensin system.

We investigated the effects of the new muscarinic receptor antagonist solifenacin succinate [YM905; (+)-(1S,3'R)-quinuclidin-3'-yl 1-phenyl-1,2,3,4-tetrahydroisoquinoline-2-carboxylate monosuccinate] and the current antimuscarinic drugs for the treatment of overactive bladder (oxybutynin, tolterodine and darifenacin) on intracellular Ca(2+) mobilization in response to M(3) muscarinic receptor activation in bladder smooth muscle and submandibular gland cells isolated from Cynomolgus monkeys. Solifenacin concentration-dependently inhibited carbachol-induced Ca(2+) mobilization, with affinity constant values (pKi) of 8.5 +/- 0.053 in bladder smooth muscle cells and 8.2 +/- 0.051 in submandibular gland cells (n = 5). The pKi value of solifenacin was almost equivalent to the values of oxybutynin, tolterodine and darifenacin in bladder smooth muscle cells (8.7, 8.5 and 8.4, respectively), while being lower than those in submandibular gland cells (9.0, 8.7 and 8.8, respectively). The bladder-selectivity index (Ki ratio: submandibular gland/bladder) for solifenacin (2.1) was statistically higher, moreover, than those for oxybutynin, tolterodine and darifenacin (0.51, 0.65 and 0.46, respectively). These findings consequently indicate solifenacin's unique profile in terms of its selectivity for bladder smooth muscle cells over salivary gland cells in non-human primates, relative to oxybutynin, tolterodine and darifenacin. Solifenacin may, therefore, confer a promising therapeutic advantage for reducing adverse effects, such as dry mouth, exhibited by current antimuscarinic therapy for overactive bladder.

Free D-aspartic acid (D-Asp) has been reported to occur in a wide variety of tissues and cells, exclusively in central nervous system and endocrine tissues. In this manuscript, we demonstrate that large amounts of D-Asp are present in the exocrine tissue, salivary glands. In adult male rats, D-Asp concentrations in parotid and submandibular gland were 212+/-68 and 233+/-34 nmol/g wet weight, respectively, and were low (38+/-20 nmol/g wet weight) in sublingual gland. This result indicates that substantial level of D-Asp exists not only in central nervous system and endocrine tissues but also in exocrine tissues. Furthermore, D-Asp concentration in parotid gland increased transiently at 3 weeks of age and decreased thereafter. In contrast, the D-Asp level in submandibular gland continued to increase gradually from 1 to 7 weeks of age and remained at an adult level after 7 weeks of age. Using anti-D-Asp antibody, immunohistochemical study was done against these glands and it showed that the predominant localization of D-Asp in acinar cells in parotid gland, while D-Asp is specifically located in striated duct cells in submandibular gland. These results suggest that D-Asp may play different roles between the two glands.

The effect of clozapine on the intracellular concentration of calcium ([Ca2+](i)) in rat submandibular acinar cells was tested. By itself clozapine had no effect on the mobilization of intracellular pools of calcium or on the uptake of extracellular calcium. It inhibited the increase of the [Ca2+](i) in response to carbachol (half-maximal inhibitory concentrations, IC(50)=100nM) and to norepinephrine and epinephrine (IC(50)=10nM) without affecting the response to substance P, extracellular ATP or thapsigargin. Clozapine inhibited the uptake of extracellular calcium in response to epinephrine but not to substance P, ATP or thapsigargin. It also decreased the production of inositol phosphates elicited by epinephrine but not by substance P or fluoride. It is concluded that, by itself, clozapine has no effect on the [Ca2+](i) in rat salivary acinar cells. It selectively inhibits muscarinic and adrenergic receptors in the acinar plasma membrane.

In the present study, the effects of cAMP- and cGMP-phosphodiesterase (PDE) inhibitors, theophylline, and sildenafil on secretion of protein, amylase and epidermal growth factor (EGF) and the flow rate from rat submandibular saliva were examined in an acute experiment. Theophylline at doses of 25, 50, 85mgkg(-1) and sildenafil at doses of 1, 2, 5mgkg(-1) were administered intraperitoneally 2h before saliva collection. Pure submandibular saliva was collected intraorally by microployethylene tubes under anesthesia using dissecting microscope. Theophylline at doses of 25, 50, 85mgkg(-1) increased salivary flow rate to 197% (P<0.01), 186% (P<0.01), and 209% of control, respectively. Sildenafil at doses of 1, 2, 5mgkg(-1) also increased flow rate to 232% (P<0.01), 182% (P<0.01), and 197% of control, respectively. Theophylline at doses of 25, 50, 85mgkg(-1) increased total protein concentration to 98% (P<0.01), 84% (P<0.01), and 210% of control, respectively. Sildenafil at doses of 2 and 5mgkg(-1) increased total protein concentration to 75% (P<0.01), and 240% of control, respectively. Theophylline at dose of 85mgkg(-1) increased EGF concentration to 60% (P<0.01) of control. Sildenafil at doses of 2 and 5mgkg(-1) increased EGF concentration to 44% (P<0.05) and 90% (P<0.01) of control, respectively. No statistically significant change was observed in amylase activity by administration of theophylline or sildenafil. The present results indicate that increasing intracellular levels of cAMP and cGMP have stimulatory effects on salivary functions. Regarding beneficiary effects of increased salivary flow rate and secretion of EGF in maintaining oral health, theophylline and sildenafil may find good places in some oral diseases.