Salivary functions in mastication, taste and textural perception, swallowing and initial digestion.

Saliva exerts multiple functions in relation to the initial digestive processes taking place in the upper parts of the gastrointestinal tract. Ingestion of food and beverages, in turn, is a strong stimulus for secretion of saliva with a differential composition depending on the neuronal stimulation pattern. This review paper provides insight into the mechanisms by which saliva acts in relation to taste, mastication, bolus formation, enzymatic digestion and swallowing. Also, the protective functions of saliva including maintenance of dental and mucosal integrity will be discussed as they indirectly influence the digestive process. The final part of this study focuses on the implications of xerostomia and salivary gland dysfunction on gastrointestinal functions.

Salivary secretion in health and disease.

Saliva is a complex fluid produced by 3 pairs of major salivary glands and by hundreds of minor salivary glands. It comprises a large variety of constituents and physicochemical properties, which are important for the maintenance of oral health. Saliva not only protects the teeth and the oropharyngeal mucosa, it also facilitates articulation of speech, and is imperative for mastication and swallowing. Furthermore, saliva plays an important role in maintaining a balanced microbiota. Thus, the multiple functions provided by saliva are essential for proper protection and functioning of the body as a whole and for the general health. A large number of diseases and medications can affect salivary secretion through different mechanisms, leading to salivary gland dysfunction and associated oral problems, including xerostomia, dental caries and fungal infections. The first part of this review article provides an updated insight into our understanding of salivary gland structure, the neural regulation of salivary gland secretion, the mechanisms underlying the formation of saliva, the various functions of saliva and factors that influence salivary secretion under normal physiological conditions. The second part focuses on how various diseases and medical treatment including commonly prescribed medications and cancer therapies can affect salivary gland structure and function. We also provide a brief insight into how to diagnose salivary gland dysfunction.

The effect of parotid gland-sparing intensity-modulated radiotherapy on salivary composition, flow rate and xerostomia measures.

OBJECTIVES: To describe parotid gland (PG) saliva organic and inorganic composition and flow rate changes, after curative intensity-modulated radiotherapy (IMRT) for head and neck cancer (HNC), and analyse the relationship between PG saliva analytes and xerostomia measures. METHODS AND MATERIALS: Twenty-six patients recruited to five prospective phase 2 or 3 trials which assessed toxicity and efficacy of IMRT by HNC subsite, provided longitudinal PG saliva. Salivary flow rate, and subjective and objective xerostomia measures were prospectively collected and saliva tested for inorganic and organic analytes. Statistical comparisons of longitudinal analyte changes and analysis for a relationship between dichotomized xerostomia score and saliva analytes were performed. RESULTS: One hundred and forty-two PG saliva samples from 26 patients were analysed. At 3-6 months after IMRT, stimulated and unstimulated saliva showed significantly decreased flow rate, total protein (TP) secretion rate, phosphate concentration and increased lactoferrin (LF) concentration. Stimulated saliva alone had elevated LF secretion rate and beta-2-microglobulin (B2 M) concentration with decreased calcium (Ca2+ ) and magnesium (Mg2+ ) concentrations and Ca2+ secretion rate. At >12 months, under stimulated and unstimulated conditions, increased LF concentration and decreased Mg2+ and phosphate concentration persisted and, in stimulated saliva, there was decreased potassium (K+ ) and Mg2+ concentration. Unstimulated TP secretion rate was lower in the presence of high-grade xerostomia. Otherwise, no relationship between xerostomia grade and PG salivary flow rate, TP and Ca2+ secretion rate was found. CONCLUSION: Fewer significant differences in PG saliva analytes >12 months after IMRT indicate good functional recovery. Residual xerostomia after IMRT will only be further reduced by addressing the sparing of subsites of the PG or other salivary gland tissues, in addition to the PG.

Concentration of salivary protective proteins within the bound oral mucosal pellicle.

OBJECTIVES: To study which salivary proteins form the protective bound mucosal pellicle and to determine the role of transglutaminase in pellicle development. MATERIALS AND METHODS: Oral epithelial cells were collected and underwent washes of different strengths, followed by homogenisation. SDS-PAGE, western blotting, IgA ELISAs and amylase activity assays were completed on cell homogenates and compared to saliva samples to confirm which salivary proteins were bound to cell surfaces. RESULTS: Salivary mucins, MUC5B and MUC7, were strongly retained on the oral epithelial cell surface. Other bound proteins including cystatin S, carbonic anhydrase VI, secretory component and IgA could be washed off. IgA was present in concentrated levels in the bound mucosal pellicle compared to amounts in saliva. Amylase, one of the most abundant proteins present in saliva, showed minimal levels of binding. Transglutaminase 3 presence was confirmed, but proteins that it catalyses cross-links between, statherin and proline-rich proteins, showed minimal presence. CONCLUSION: Some protective salivary proteins including mucins and IgA become concentrated on oral surfaces in the bound mucosal pellicle, through specific interactions. Concentration of mucins would contribute to lubrication to prevent abrasion damage to soft tissues, whilst increased IgA could create an 'immune reservoir' against mucosal infection.

Composition of enamel pellicle from dental erosion patients.

Oral health is dependent upon a thin mobile film of saliva on soft and hard tissues. Salivary proteins adhere to teeth to form the acquired enamel pellicle which is believed to protect teeth from acid erosion. This study investigated whether patients suffering diet-induced dental erosion had altered enamel pellicles. Thirty patients suffering erosion were compared to healthy age-matched controls. Subjects wore a maxillary splint holding hydroxyapatite and human enamel blocks for 1 h. The acquired enamel pellicle was removed from the blocks and compared to the natural incisor pellicle. Basic Erosive Wear Examination scores confirmed that dental erosion was present in erosion patients and absent from healthy age-matched controls. Erosion patients had half the amount of proteins (BCA assay) within the acquired pellicle forming on splint blocks compared to normal controls (p < 0.05). In particular, statherin, a calcium-binding protein, was 35% less abundant (p < 0.05). Calcium concentration within the acquired pellicle was also reduced by 50% in erosion patients (p < 0.001). In contrast, the natural pellicle on the incisor had similar amounts of total protein in erosion patients and healthy controls. In summary, the formation of new acquired pellicles on surfaces was reduced in erosion patients, which may explain their greater susceptibility to acid erosion of teeth.

Comparison of the possible protective effect of the salivary pellicle of individuals with and without erosion.

The acquired pellicle adheres to tooth surfaces and has been suggested to provide differing degrees of protection against acidic erosion. This study investigated whether pellicle formed on enamel blocks in patients suffering dietary dental erosion modified the effect of an in vitro simulated dietary challenge, in comparison with pellicle formed on enamel blocks in healthy subjects and to no-pellicle enamel samples. Sixty subjects recruited from dental erosion clinics were compared to healthy age-matched controls. Subjects wore a custom-made maxillary splint holding human enamel blocks for 1 h during which the acquired enamel pellicle was formed. Enamel blocks were removed from the splints and a simulated dietary erosive challenge of 10 min was performed. In addition the challenge was performed on 30 enamel samples without pellicle. Profilometry showed no statistical difference between samples from the erosion subjects with a mean step height of 1.74 µm (SD 0.88) and median roughness (Sa) of 0.39 µm (interquartile range, IQR 0.3-0.56) and the controls with 1.34 µm (SD 0.66) and 0.33 µm (IQR 0.27-0.38), respectively. The control samples without pellicle had Sa of 0.44 µm (IQR 0.36-0.69) and these differences were statistically significant compared to those from the healthy subjects (p = 0.002). Mean (SD) microhardness reduction with a 100-gram load for the erosion group was 113.5 (10) KHN, for healthy subjects was 93 (15.4) KHN and for the enamel samples without pellicle 139.6 (21.8) KHN and all groups were statistically different. The microhardness and roughness data suggested the pellicle influenced erosion under these study conditions.

EGF: new tricks for an old growth factor.

During the past year, the biology of epidermal growth factor (EGF) has been investigated in lower organisms (Caenorhabditis elegans, Drosophila and bacteria). These experiments have produced some surprising results: the identification of defects produced by mutation of EGF-like genes; the role of EGF receptors in bacterial invasion; and the role of EGF-like precursors as receptors for a bacteria toxin.

Salivary Factors that Maintain the Normal Oral Commensal Microflora.

The oral microbiome is one of the most stable ecosystems in the body and yet the reasons for this are still unclear. As well as being stable, it is also highly diverse which can be ascribed to the variety of niches available in the mouth. Previous studies have focused on the microflora in disease-either caries or periodontitis-and only recently have they considered factors that maintain the normal microflora. This has led to the perception that the microflora proliferate in nutrient-rich periods during oral processing of foods and drinks and starves in between times. In this review, evidence is presented which shows that the normal flora are maintained on a diet of salivary factors including urea, lactate, and salivary protein degradation. These factors are actively secreted by salivary glands which suggests these factors are important in maintaining normal commensals in the mouth. In addition, the immobilization of SIgA in the mucosal pellicle indicates a mechanism to retain certain bacteria that does not rely on the bacterial-centric mechanisms such as adhesins. By examining the salivary metabolome, it is clear that protein degradation is a key nutrient and the availability of free amino acids increases resistance to environmental stresses.

Intraoral Microbial Metabolism and Association with Host Taste Perception.

Metabolomics has been identified as a means of functionally assessing the net biological activity of a particular microbial community. Considering the oral microbiome, such an approach remains largely underused. While the current knowledge of the oral microbiome is constantly expanding, there are several deficits in knowledge particularly relating to their interactions with their host. This work uses nuclear magnetic resonance spectroscopy to investigate metabolic differences between oral microbial metabolism of endogenous (i.e., salivary protein) and exogenous (i.e., dietary carbohydrates) substrates. It also investigated whether microbial generation of different metabolites may be associated with host taste perception. This work found that in the absence of exogenous substrate, oral bacteria readily catabolize salivary protein and generate metabolic profiles similar to those seen in vivo. Important metabolites such as acetate, butyrate, and propionate are generated at relatively high concentrations. Higher concentrations of metabolites were generated by tongue biofilm compared to planktonic salivary bacteria. Thus, as has been postulated, metabolite production in proximity to taste receptors could reach relatively high concentrations. In the presence of 0.25 M exogenous sucrose, increased catabolism was observed with increased concentrations of a range of metabolites relating to glycolysis (lactate, pyruvate, succinate). Additional pyruvate-derived molecules such as acetoin and alanine were also increased. Furthermore, there was evidence that individual taste sensitivity to sucrose was related to differences in the metabolic fate of sucrose in the mouth. High-sensitivity perceivers appeared more inclined toward continual citric acid cycle activity postsucrose, whereas low-sensitivity perceivers had a more efficient conversion of pyruvate to lactate. This work collectively indicates that the oral microbiome exists in a complex balance with the host, with fluctuating metabolic activity depending on nutrient availability. There is preliminary evidence of an association between host behavior (sweet taste perception) and oral catabolism of sugar.

Influence of Chemical Species on Polyphenol-Protein Interactions Related to Wine Astringency.

One of the most accepted mechanisms of astringency consists of the interaction between polyphenols and some specific salivary proteins. This work aims to obtain further insights into the mechanisms leading to a modulation of astringency elicited by polyphenols. The effect of the presence of different chemical species (present in food and beverages as food additives) on the polyphenol-protein interaction has been evaluated by means of techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis and cell cultures using a cell-based model of the oral epithelium. Results obtained showed that several chemicals, particularly sodium carbonate, seem to inhibit polyphenol binding to salivary proteins and to oral epithelium. These results point out that polyphenol-saliva protein interactions can be affected by some food additives, which can help to better understand changes in astringency perception.

Mineral reaction kinetics constrain the length scale of rock matrix diffusion.

Mass transport by aqueous fluids is a dynamic process in shallow crustal systems, redistributing nutrients as well as contaminants. Rock matrix diffusion into fractures (void space) within crystalline rock has been postulated to play an important role in the transient storage of solutes. The reacted volume of host rock involved, however, will be controlled by fluid-rock reactions. Here we present the results of a study which focusses on defining the length scale over which rock matrix diffusion operates within crystalline rock over timescales that are relevant to safety assessment of radioactive and other long-lived wastes. Through detailed chemical and structural analysis of natural specimens sampled at depth from an active system (Toki Granite, Japan), we show that, contrary to commonly proposed models, the length scale of rock matrix diffusion may be extremely small, on the order of centimetres, even over timescales of millions of years. This implies that in many cases the importance of rock matrix diffusion will be minimal. Additional analyses of a contrasting crystalline rock system (Carnmenellis Granite, UK) corroborate these results.

Constitutive proteolysis of the ErbB-4 receptor tyrosine kinase by a unique, sequential mechanism.

The heregulin receptor tyrosine kinase ErbB-4 is constitutively cleaved, in the presence or absence of ligand, by an exofacial proteolytic activity producing a membrane-anchored cytoplasmic domain fragment of 80 kD. Based on selective sensitivity to inhibitors, the proteolytic activity is identified as that of a metalloprotease. The 80-kD product is tyrosine phosphorylated and retains tyrosine kinase activity. Importantly, the levels of this fragment are controlled by proteasome function. When proteasome activity is inhibited for 6 h, the kinase-active 80-kD ErbB-4 fragment accumulates to a level equivalent to 60% of the initial amount of native ErbB-4 (approximately 10(6) receptors per cell). Hence, proteasome activity is essential to prevent the accumulation of a significant level of ligand-independent, active ErbB-4 tyrosine kinase generated by metalloprotease activity. Proteasome activity, however, does not act on the native ErbB-4 receptor before the metalloprotease-mediated cleavage, as no ErbB-4 fragments accumulate when metalloprotease activity is blocked. Although no ubiquitination of the native ErbB-4 is detected, the 80-kD fragment is polyubiquitinated. The data, therefore, describe a unique pathway for the processing of growth factor receptors, which involves the sequential function of an exofacial metalloprotease and the cytoplasmic proteasome.

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts.

125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.

Down regulation of epidermal growth factor receptors: direct demonstration of receptor degradation in human fibroblasts.

The metabolism of the receptor for epidermal growth factor (EGF) has been measured by labeling the receptor in vivo with radioactive amino acid precursors and then determining, by immunoprecipitation with specific anti-EGF receptor antisera, the rate of degradation of the receptor when the cells are placed in a nonradioactive medium. In human fibroblasts the rate of EGF receptor degradation (t1/2 = 10.1 h) was faster than the rate of degradation of total cell protein. When EGF was added to the nonradioactive medium, the half-life of prelabeled receptor was decreased to 1.2 h in human fibroblasts. These data demonstrate by direct analysis of receptor protein that during "down regulation" the EGF receptor is rapidly degraded. Enhanced receptor degradation was observed 5-10 min after the addition of EGF. The EGF-induced degradation of the receptor was blocked by methylamine, chloroquine, iodoacetate, or incubation at 25 degrees C. We have also shown that EGF-induced down regulation in human fibroblasts results in a decrease in the total amount of EGF receptor protein present. The amount of EGF receptor protein has been quantitated by radiolabeling cellular protein and immunoprecipitation of the receptor. The EGF receptor constitutes approximately 0.0035% of the cellular protein in human fibroblasts.

Altered plasticity of the parasympathetic innervation in the recovering rat submandibular gland following extensive atrophy.

Adult rat submandibular glands have a rich autonomic innervation, with parasympathetic and sympathetic nerves working in synergy rather than antagonistically. Ligation of the secretory duct rapidly causes atrophy and the loss of most acini, which are the main target cell for parasympathetic nerves. Following deligation, there is a recovery of gland structure and function, as assessed by autonomimetic stimulation. This study examines whether the parasympathetic nerves reattach to new target cells to form functional neuro-effector junctions. Under recovery anaesthesia, the submandibular duct of adult male rats was ligated via an intra-oral approach to avoid damaging the chorda-lingual nerve. Four weeks later, rats were either killed or anaesthetized and the ligation clip removed. Following a further 8 weeks, both submandibular ducts were cannulated under terminal anaesthesia. Salivary flows were then stimulated electrically (chorda-lingual nerve at 2, 5 and 10 Hz) and subsequently by methacholine (whole-body infusion at two doses). Glands were excised, weighed and divided for further in vitro studies or fixed for histological examination. Ligation of ducts caused 75% loss of gland weight, with the loss of most acinar cells. Of the remaining acini, only 50% were innervated despite unchanged choline acetyltransferase activity, suggesting few parasympathetic nerves had died. Following deligation, submandibular glands recovered half their weight and had normal morphology. Salivary flows from both glands (per unit of gland tissue) were similar when evoked by methacholine but greater from the deligated glands when evoked by nerve stimulation. This suggests that parasympathetic nerves had reattached to new target cells in the recovered glands at a greater ratio than normal, confirming reinnervation of the regenerating gland.

Epidermal growth factor is a major growth-promoting agent in human milk.

Human milk stimulates DNA synthesis in cell cultures in which growth has been arrested. The mitogenic activity of milk is neutralized by the addition of antibody to human epidermal growth factor. The results identify epidermal growth factor as a major growth-promoting agent in breast milk.

Interaction of activated EGF receptors with coated pit adaptins.

The epidermal growth factor (EGF) receptor interacts with plasma membrane-associated adapter proteins during endocytosis through coated pits. Almost 50 percent of the total pool of alpha-adaptins was coimmunoprecipitated with the EGF receptor when A-431 cells were treated with EGF at 37 degrees C, but not at 4 degrees C. Partial proteolysis of alpha-adaptin suggested that the amino-terminal domain is the region that associates with the EGF receptor. The extent of receptor-adaptin association was increased in cells depleted of potassium to block endocytosis. These data suggest that receptor-adaptin association occurs in intact cells before coated pits are fully assembled.

Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells.

A tenfold increase in phospholipase C activity specific for phosphatidylinositol 4,5-bisphosphate (PIP2) was immunopurified from extracts of A-431 epidermoid carcinoma cells stimulated with epidermal growth factor. This finding suggests a biochemical link between growth factor-stimulated tyrosine kinase activity and PIP2 hydrolysis.

Which behavior does the lamprey central motor program mediate?

The isolated lamprey spinal cord, when bathed in 2 millimolar D-glutamic acid, will generate a pattern of motor neuron discharge that has generally been assumed to represent the central motor program for swimming. Motion pictures of behaving lampreys were analyzed by a computer algorithm to estimate undulatory movement parameters that could be directly compared with those generated during D-glutamate--induced undulations. The D-glutamate--induced movement parameters were significantly different from those observed during normal behaviors, including swimming, but accurately predicted the undulations produced by spinally transected adult lampreys.

Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor.

Epidermal growth factor (EGF) treatment of A-431 epidermoid carcinoma cells elicited a redistribution of phospholipase C-gamma 1 (PLC-gamma 1) from a predominantly cytosolic localization to membrane fractions. The temporal coincidence of this redistribution with EGF stimulation of inositol phosphate formation and EGF increased phosphorylation of PLC-gamma 1 suggests that the membrane association of PLC-gamma 1 is a significant event in second messenger transduction.