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Insulin-like growth factor-I (IGF-I) is expressed in salivary glands. We examined the effects of IGF-I on cell number, the expression and distribution of tight junction proteins and the paracellular barrier function in cells derived from rat submandibular glands. When those cells were cultured in medium containing 10% foetal bovine serum (FBS) or IGF-I, the number of cells was comparable at 10 days. However, in the presence of inhibitor of IGF-I receptors, the number of cells cultured with FBS only was clearly reduced. The tight junction proteins occludin and claudin-3 were similarly detected by Western blotting in cells cultured with IGF-I or FBS. Immunostaining revealed that occludin and another tight junction protein (ZO-1) were similarly localized at intracellular junctions of cells cultured with IGF-I or FBS. The barrier functions were evaluated by transepithelial resistance (TER) and by FITC-dextran permeability. The TER values and FITC-dextran permeability of cells cultured with IGF-I or FBS were comparable. These observations suggest that IGF-I contributes to the maintenance not only of the cell number of salivary gland cells but also of their paracellular barrier function via the expression and distribution of tight junction proteins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

The heterogeneity of salivary ductal cells, with regard to their sensitivity to Ca(2+)-mobilizing agonists, was visualized by multi-photon microscopy. Stimulation of isolated parotid ducts with 0.1 and 1 microM epinephrine (Epi) elevated the intracellular Ca(2+) levels ([Ca(2+)](i)) in approximately 30% and >90% of the ductal cells, respectively. Of the 0.1 microM Epi-responsive cells, 80% responded rapidly to subsequent stimulation with 1 microM Epi. Similarly, threshold concentrations (0.5 or 1 microM) of phenylephrine (PhL), carbachol (CCh) or ATP, induced responses in approximately 20% of the ductal cells, and subsequent stimulations with 10 microM of the same agonist activated approximately 80% of ductal cells. These observations indicate that parotid ducts contain a certain subpopulation of cells, which exhibits particularly high sensitivity to these Ca(2+)-mobilizing agonists, compared to the remaining ductal cells. Sequential stimulation with threshold concentrations of PhL, CCh, and ATP induced Ca(2+) responses in approximately 33% of ductal cells. Of these responsive cells, the majority (69%) could only respond to one of the three agonists; while a small minority (9%) were capable of responding to all three agonists. These results indicate that low concentrations of PhL, CCh, and ATP activate different subpopulations of parotid ductal cells.

Objective: Immunoglobulin A (IgA) is transported across glandular epithelial cells by polymeric immunoglobulin receptor (plgR), with each receptor molecule participating in only one round of transcytosis. Nerve-related stimuli rapidly increase salivary secretion of IgA, while concentrations are increased in the autoimmune disease Sjögren's syndrome. Our aim here was to determine whether autonomic agonists and cytokines present in Sjögren's-affected glands can up-regulate salivary cell plgR expression., Methods: Cultures of rat parotid acinar cells (PAR C5) and human submandibular gland ductal cells (HSG) were exposed to carbachol or adrenaline for 24 h and to interleukin-4 and/or interferon-gamma for 48 h. The human colonic cell line HT-29 served as a positive control for cytokine response. plgR mRNA was quantified by reverse transcription and real-time PCR and protein expression was examined by immunoblotting., Results: Carbachol increased plgR mRNA levels significantly in all cells but adrenaline did so only with PAR cells (P<0.05). HSG and HT-29 cells both up-regulated plgR gene transcription on exposure to interleukin-4 and interferon-gamma either alone or in combination (P<0.05). By contrast, production of plgR mRNA in PAR cells tended to decrease in response to all cytokine treatments. plgR protein levels rose in line with mRNA expression in cytokine-treated HT-29 cultures (P<0.05)., Conclusions: Autonomimetics can up-regulate plgR transcription in transformed and neoplastic salivary and colonic cells, although intracellular coupling mechanisms require further investigation. Immunomodulatory cytokines increased plgR expression in one of the salivary cell lines, but additional work is needed to establish whether this occurs in Sjögren's patients.

The postnatal development of granular convoluted tubules (GCT) in the duct system of the rodent submandibular gland is known to be androgen-dependent, but the underlying molecular mechanism is unclear. To test the possible role of the transcription factor, cyclic AMP response element-binding protein (CREB), in the androgen-induced differentiation of GCT, the effect of testosterone on the expression and localization of epidermal growth factor (EGF), a marker of GCT cells, and of CREB was examined in the submandibular glands of immature 3-week-old rats. Northern blotting demonstrated increases in both EGF and CREB mRNA 1-4 days after testosterone administration. Immunoprecipitation also indicated that CREB protein was increased in amount with testosterone administration, and that induced CREB was phosphorylated at the serine residue as in the active form of CREB. In situ hybridization demonstrated that cells with CREB mRNA signal first appeared in the distal portions of striated ducts at 1 day and had increased in number by 4 days after giving testosterone, when cells with EGF mRNA signal became evident in the same duct portions. Immunohistochemistry also showed the occurrence of CREB protein in the nuclei of duct epithelial cells before their differentiation into EGF-positive GCT cells. Finally, pieces of submandibular gland from immature rats were cultured in vitro and their expression of EGF mRNA analysed by the reverse transcriptase-polymerase chain reaction. Testosterone in the medium caused a marked enhancement of EGF expression in the gland in 1-4 days, which was attenuated by simultaneous administration of the antisense oligonucleotide for CREB as well as that for the androgen receptor. These results suggest the CREB is upregulated by androgen and has a crucial role in androgen-induced differentiation of GCT in the duct system of the rat submandibular gland.

Sodium pump and carbonic anhydrase activity have been described in the salivary glands. However, it remains to be elucidated whether these energy sources are used for secretion, excretion or both. In addition, the differences in the function of excretion and the role of the excretory duct cells are currently unknown in salivary glands. Expression of P-glycoprotein (P-gp), which is an ATPase-binding efflux pump, was tested in normal major and minor salivary glands from humans. P-gp was distributed on the basolateral membrane of serous acinar cells in the major salivary glands and the minor salivary glands. In particular, it was found to be present on the basolateral membrane and cytoplasm of acinar demilunar cells in the anterior lingual gland. Intense expression was identified in the basolateral membrane of the striated duct cells of the major salivary glands. P-gp was distinctly positive in the basolateral and/or luminal membranes of the initial part and in the luminal membrane of the terminal part of the excretory duct cells of the major salivary glands, whereas it was positive in the luminal membranes of both the initial part and the terminal part of the excretory duct cells of the minor salivary glands. These disparate distributions between the major and the minor salivary glands suggest different physiological excretions in the striated duct. P-gp may be physiologically involved in an important part of the transporter system, not only in the acinar serous cells and the striated duct cells, but also in the excretory duct cells in the salivary glands.

The minor salivary glands are important components of the oral cavity, present in most parts of the mouth, and their secretions directly bathe the tissues. Individual glands are usually in the submucosa between muscle fibres, and consist of groups of secretory endpieces made up of mucous acinar cells and serous or seromucous demilune cells. The ductal systems comprise intercalated ducts, intralobular ducts usually lacking basal striations, and excretory ducts opening directly through the mucosa Minor glands secrete highly glycosylated mucins, containing blood group determinants, and probably active in tissue lubrication and bacterial aggregation. They also secrete several antimicrobial proteins and immunoglobulins, and the lingual serous (von Ebner's) glands secrete digestive enzymes and proteins with possible taste perception functions. Minor gland morphology and function can conveniently be studied in the rat. There are substantial differences between major and minor salivary glands, as well as among the minor glands, in the nature and composition of their mucous and serous secretory products. The role of minor salivary glands in the function and defence of the oral cavity may be better understood as a result of new physiological and molecular methods applicable to samples of limited size and availability.

A major obstacle in studying the physiological and biochemical processes of salivary secretion is the lack of a good ductal cell line model. HSY, an immortalised cell line originating from human parotid gland intercalated ducts, provides a possible model for purinergic mechanisms in ductal cells. Unlike the biphasic dose response to ATP of isolated submandibular ductal cells, the rise in [Ca2+]i in HSY cells shows single Michaelis-Menten kinetics with an apparent Ka of 0.8 microM. Pre-incubation with thapsigargin inhibited the ATP induced [Ca2+]i rise. Both ATP (10 microM) and carbachol (100 microM) increased IP3 production. Intercalated duct cells may differentiate into acinar or ductal cells in response to appropriate stimuli from extracellular matrix We therefore attempted to induce a duct-like phenotype in the striated duct-derived HSY cells by growing them on microcarrier beads coated with type I collagen. In Ca-containing medium cells grown on all substrates showed similar responses to ATP. In contrast, in Ca-free medium, [Ca2+]i rose only slightly in cells grown on beads relative to those on glass. This probably resulted from reduced IP3 production. Carbachol also induced a much smaller increase in [Ca2+]i and less IP3 production in cells grown on Cytodex-3. The HSY response to purinergic stimuli by an increase in [Ca2+]i and IP3 means that they can be used to study the metabotropic purinergic pathway. The impairment in the HSY responses grown on Cytodex-3 can be used to probe phosposinositol signal transduction in salivary cells.

Cell shape in salivary glands is affected by mechanical forces. In the acini and ducts cell shape is modified by the contractions of the myoepithelial cells in both the secretory and ductal portions of the glands. At the organ level shape changes are due to muscle contraction during mastication, food intake and speech. All these factors may cause some degree of stretching of salivary cell membranes. Recent studies suggest that physical forces influence signal transduction, gene expression, secretory function, cell differentiation and proliferation. Here we overview membrane stretch-activated cellular events. Evidence from a variety of tissues suggests that mechanical forces may alter the properties of acinar cells leading to cytoskeletal reorganisation, changes in ion fluxes, modulation of secretory activity and subsequent release of transmitters such as ATP. Transmitters released from acinar cells may modulate the secretory activity of salivary tissue, and interact with classical regulatory pathways.

Signal transduction via P2 purinergic receptors was investigated in HSG cells, a continuous cell line originally derived from an irradiated human salivary gland. Ligand specificity for nucleotide receptors in HSG cells was investigated with various nucleotides and their analogues. Inositol 1,4,5-trisphosphate (IP3) production was significantly increased by ATP, UTP and ATP gamma S. The ligand specificity of this effect agreed well with that of the P2U purinergic receptor. On the other hand, 45Ca2+ influx was stimulated by ATP, UTP > ATP gamma S, ADP, UDP > ADP beta S > AMPPNP, GTP, TTP > CTP, GDP, TDP, AMPPCP, AMPCPP. This ligand specificity of 45Ca2+ influx was much broader than IP3 production. Also pertussis and cholera toxin had no effect on both IP3 production and 45Ca2+ influx by ATP or UTP. 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) stimulates 45Ca2+ influx more effectively than IP3 formation. A 53-kDa membrane protein was photolabelled with [alpha-32P]Bz-ATP. This 53-kDa protein is a putative P2 purinergic receptor. In particular, the labelling was inhibited by a ligand profile that corresponded to that for 45Ca2+ influx. These findings suggest that nucleotides stimulate 45Ca2+ influx and IP3 formation by separate pathways via pertussis and cholera toxin-insensitive G proteins. Thus, in HSG cells, IP3 formation is coupled to the P2U subclass, while 45Ca2+ influx is coupled to another subclass, such as P2X, that regulates calcium channels.

The prevalence and cellular distribution of human herpesvirus 7 (HHV-7) in archival labial salivary glands was analysed for virus-specific DNA sequences by polymerase chain reaction (PCR) and in situ hybridization signals. In addition, the cellular expression of HHV-7-encoded protein was detected by immunohistochemical staining with a virus-specific monoclonal antibody. Eleven of 20 samples were positive for the HHV-7 DNA sequence by PCR. Eighteen of 20 tissues analysed by in situ hybridization showed signals in ductal, serous and mucous cells. Some nuclei of these cells and also the myoepithelial population were positive. In immunolocalization studies, all 20 salivary glands consistently showed HHV-7-expressed protein in the cytoplasm of ductal cuboidal and columnar cells. The protein was also found in the cytoplasm of mucous and serous acinar cells that were immunopositive for HHV-7. The observations are consistent with the suggestion that the labial salivary gland is a site for virus replication, potential persistence and a source of infective HHV-7 in saliva.

To understand the specific cell type responsible for the synthesis of basement membrane components of the salivary gland, the effects of transforming growth factor (TGF)-beta 1 on morphological change, cellular proliferation and collagen synthesis were examined in these immortalized duct and myoepithelial cell clones, and the expression forms of their TGF-beta receptors analysed. When TGF-beta 1 was added to the cell clones in vitro, it induced a morphological alteration, with flattening in myoepithelial but not in duct cells. Although the growth of Mv1Lu mink lung epithelial cells was almost completely inhibited by TGF-beta 1, the duct and myoepithelial cells were partially resistant to such inhibition. By immunoblot analysis of immunoprecipitates, p53 was found bound to the simian virus-40 large T antigen, suggesting a functional loss of p53 in regulation of cell-cycle arrest. In the cloned myoepithelial cells but not the duct cells, TGF-beta 1 stimulated the production of type IV collagen. To attempt to understand the distinct responsiveness of cell clones to TGF-beta 1, the expression forms of TGF-beta receptors were examined by affinity cross-linking. Although the intensities of the cross-linked bands of the TGF-beta type II and type III receptors, particularly the type II, were weaker in the duct than the myoepithelial cell clones, the expression of the type II receptor mRNA was consistently detected in both clones. Accordingly, the reduction of TGF-beta 1 binding may have occurred at the post-transcriptional level. These findings imply that the cloned myoepithelial cells but not the cloned duct cells produce type IV collagen in response to TGF-beta 1 through the receptor-mediated signal transduction pathway, which is presumably disrupted in the cloned duct cells.